Method for detection of drug-selected mutations in the HIV protease gene

1. FIELD OF THE INVENTION

The present invention relates to the field of HIV diagnosis. More particularly, the present invention relates to the field of diagnosing the susceptibility of an HIV sample to antiviral drugs used to treat HIV infection.

The present invention relates to a method for the rapid and reliable detection of drug-selected mutations in the HIV protease gene allowing the simultaneous characterization of a range of codons involved in drug resistance using specific sets of probes optimized to function together in a reverse-hybridization assay.

2. BACKGROUND OF THE INVENTION

The human immunodeficiency virus (HIV) is the ethiological agent for the acquired immunodeficiency syndrome (AIDS). HIV, like other retroviruses, encodes an aspartic protease that mediates the maturation of the newly produced viral particle by cleaving viral polypeptides into their functional forms (Hunter et al). The HIV protease is a dimeric molecule consisting of two identical subunits each contributing a catalytic aspartic residue (Navia et al, Whodawer et al, Meek et al). Inhibition of this enzyme gives rise to noninfectious viral particles that cannot establish new cycles of viral replication (Kohl et al, Peng et al).

Attempts to develop inhibitors of HIV-1 protease were initially based on designing peptide compounds that mimicked the natural substrate. The availability of the 3-dimensional structure of the enzyme have more recently allowed the rational design of protease inhibitors (PI) using computer modeling (Huff et al, Whodawer et al). A number of second generation PI that are partially peptidic or entirely nonpeptidic have proven to exhibit particularly potent antiviral effects in cell culture. Combinations of various protease inhibitors with nucleoside and non-nucleoside RT inhibitors have also been studied extensively in vitro. In every instance, the combinations have been at least additive and usually synergistic.

In spite of the antiviral potency of many recently developed HIV-1 PI, the emergence of virus variants with decreased sensitivity to these compounds has been described both in cell culture and in treated patients thereby escaping the inhibitory effect of the antiviral (Condra et al.). Emergence of resistant variants depends on the selective pressure applied to the viral population. In the case of a relatively ineffective drug, selective pressure is low because replication of both wild-type virus and any variants can continue. If a more effective drug suppresses replication of virus except for a resistant variant, then that variant will be selected. Virus variants that arise from selection by PI carry several distinct mutations in the protease coding sequence that appear to emerge sequentially. A number of these cluster near the active site of the enzyme while others are found at distant sites. This suggests conformational adaptation to primary changes in the active site and in this respect certain mutations that increase resistance to PI also decrease protease activity and virus replication.

Amongst the PI, the antiviral activity of the PI ritonavir (A-75925; ABT-538). nelfinavir (AG-1343), indinavir (MK-639; L735; L524) and saquinavir (Ro 31-8959) have been approved by the Food and Drug Administration and are currently under evaluation in clinical trials involving HIV-infected patients. The VX-487 (141W94) antiviral compound is not yet approved. The most important mutations selected for the above compounds and leading to gradually increasing resistance are found at amino acid (aa) positions 30 (D to N), 46 (M to I), 48 (G to V), 50 (I to V), 54 (I to A, I to V), 82 (V to A, or F, or I, or T), 84 (I to V) and 90 (L to M). Other mutations associated with drug resistance to the mentioned compounds have been described (Schinazi et al). Saquinavir-resistant variants, which usually carry mutations at amino acid positions 90 and/or 48, emerge in approximately 45% of patients after 1 year of monotherapy. Resistance appears to develop less frequently with higher doses of saquinavir. Resistance to indinavir and ritonavir requires multiple mutations; usually at greater than 3 and up to 11 sites, with more amino acid substitutions conferring higher levels of resistance. Resistant isolates usually carry mutations at codons 82, 84, or 90. In the case of ritonavir, the mutation at codon 82 appears fist in most patients. Although mutant virions resistant to saquinavir are not cross-resistant to indinavir or ritonavir, isolates resistant to indinavir are generally ritonavir resistant and visa versa. Resistance to either indinavir or ritonavir usually results in cross-resistance to saquinavir. Approximately one third of indinavir resistant isolates are cross-resistant to nelfinavir as well.

The regime for an efficient antiviral treatment is currently not clear at all. Patterns of reduced susceptibility to HIV protease inhibitors have been investigated in vitro by cultivating virus in the presence of PI. These data, however, do not completely predict the pattern of amino-acid changes actually seen in patients receiving PI. Knowledge of the resistance and cross-resistance patterns should facilitate selection of optimal drug combinations and selection of sequences with non-overlapping resistance patterns. This would delay the emergence of cross-resistant viral strains and prolong the duration of effective antiretroviral activity in patients. Therefore, there is need for methods and systems that detect these mutational events in order to give a better insight into the mechanisms of HIV resistance. Further, there is need for methods and systems which can provide data important for the antiviral therapy to follow in a more time-efficient and economical manner compared to the conventional cell-culture selection techniques.

3. AIMS OF THE INVENTION

It is an aim of the present invention to develop a rapid and reliable detection method for determination of the antiviral drug resistance of viruses, which contain protease genes such as HIV retroviruses present in a biological sample.

More particularly it is an aim of the present invention to provide a genotyping assay allowing the detection of the different HIV protease gene wild type and mutation codons involved in the antiviral resistance in one single experiment.

It is also an aim of the present invention to provide an HIV protease genotyping assay or method which allows to infer the nucleotide sequence at codons of interest and/or the amino acids at the codons of interest and/or the antiviral drug selected spectrum, and possibly also infer the HIV type or subtype isolate involved.

Even more particularly it is an aim of the present invention to provide a genotyping assay allowing the detection of the different HIV protease gene polymorphisms representing wild-type and mutation codons in one single experimental setup.

It is another aim of the present invention to select particular probes able to discriminate wild-type HIV protease sequences from mutated or polymorphic HIV protease sequences conferring resistance to one or more antiviral drugs, such as ritonavir (A-75925; ABT-538), nelfinavir (AG-1343), indinavir (MK-639; L735; L524), saquinavir (Ro 31-8959) and VX-478 (141W94) or others (Shinazi et al).

It is more particularly an aim of the present invention to select particular probes able to discriminate wild-type HIV protease sequences from mutated or polymorphic HIV protease sequences conferring resistance to ritonavir (A-75925; ABT-538).

It is more particularly an aim of the present invention to select particular probes able to discriminate wild-type HIV protease sequences from mutated HIV protease sequences conferring resistance to nelfinavir (AG-1343).

It is more particularly an aim of the present invention to select particular probes able to discriminate wild-type HIV protease sequences from mutated HIV protease sequences conferring resistance to indinavir (MK-639; L735; L524).

It is more particularly an aim of the present invention to select particular probes able to discriminate wild-type HIV protease sequences from mutated HIV protease sequences conferring resistance to saquinavir (Ro 31-8959).

It is more particularly an aim of the present invention to select particular probes able to discriminate wild-type HIV protease sequences from mutated HIV protease sequences conferring resistance to VX-478 (141W94).

It is also an aim of the present invention to select particular probes able to determine and/or infer cross-resistance to HIV protease inhibitors.

It is more particularly an aim of the present invention to select particular probes able to discriminate wild-type HIV protease from mutated HIV protease sequences involving at least one of amino acid positions 30 (D to N), 46 (M to I), 48 (G to V), 50 (I to V), 54 (I to A or V), 82 (V to A or F or I or T), 84(I to V) and 90 (L to M) of the viral protease gene.

It is particularly an aim of the present invention to select a particular set of probes, able to discriminate wild-type HIV protease sequences from mutated HIV protease sequences conferring resistance to any of the antiviral drugs defined above with this particular set of probes being used in a reverse hybridization assay.

It is moreover an aim of the present invention to combine a set of selected probes able to discriminate wild-type HIV protease sequences from mutated HIV protease sequences conferring resistance to antiviral drugs with another set of selected probes able to identify the HIV isolate, type or subtype present in the biological sample, whereby all probes can be used under the same hybridization and wash-conditions.

It is also an aim of the present invention to select primers enabling the amplification of the gene fragment(s) determining the antiviral drug resistance trait of interest.

It is also an aim of the present invention to select particular probes able to identify mutated HIV protease sequences resulting in cross-resistance to antiviral drugs.

The preset invention also aims at diagnostic kits comprising said probes useful for developing such a genotyping assay.

The present invention also aims at diagnostic kits comprising said primers useful for developing such a genotyping assay.

4. DETAILED DESCRIPTION OF THE INVENTION

All the aims of the present invention have been met by the following specific embodiments.

According to one embodiment, the present invention relates to a method for determining the susceptibility to antiviral drugs of HIV viruses in a biological sample, with said method comprising:

a) if need be, releasing, isolating or concentrating the polynucleic acids present in the sample;

b) if need be amplifying the relevant part of the protease gene of HIV with at least one suitable primer pair;

c) hybridizing the polynucleic acids of step a) or b) with at least one of the following probes:

probes specifically hybridizing to a target sequence comprising codon 30;

probes specifically hybridizing to a target sequence comprising codon 46 and/or 48;

probes specifically hybridizing to a target sequence comprising codon 50;

probes specifically hybridizing to a target sequence comprising codon 54;

probes specifically hybridizing to a target sequence comprising codon 82 and/or 84;

probes specifically hybridizing to a target sequence comprising codon 90;

or the complement of said probes,

further characterized in that said probes specifically hybridize to any of the target sequences presented in

FIG. 1

, or to the complement of said target sequences;

d) inferring from the result of step c) whether or not a mutation giving rise to drug resistance is present in any of said target sequences.

The numbering of HIV-1 protease gene encoded amino acids is as generally accepted in literature. Mutations that give rise to an amino acid change at position 48 or 90 are known to confer resistance to saquinavir (Eriebe et al; Tisdale et al). An amino acid change at codon 46 or 54 or 82 or 84 results in ritonavir and indinavir resistance (Kempf et al; Emini et al; Condra et al). Amino acid changes at positions 30 and 46 confer resistance to nelfinavir (Patick et al) and amino acid changes at position 50 confers resistance to VX-487 (Rao et al). Therefore, the method described above allows to determine whether a HIV strain is susceptible or resistant to any of the drugs mentioned above. This method can be used, for instance, to screen for mutations conferring resistance to any of the mentioned drugs before initiating therapy. This method may also be used to s for mutations that may arise during the course of therapy (i.e. monitoring of drug therapy). It is obvious that this method may also be used to determine resistance to drugs other than the above-mentioned drugs, provided that resistance to these other drugs is linked to mutations that can be detected by use of this method. This method may also be used for the specific detection of polymorphic nucleotides. It is to be understood that the said probes may only partly overlap with the targets sequences of

FIG. 1

, table 2 and table 3, as long as they allow for specific detection of the relevant polymorphic nucleotides as indicated above. The sequences of

FIG. 1

, table 2 and table 3 were derived from polynucleic acid fragments comprising the protease gene. These fragments were obtained by PCR amplification and were inserted into a cloning vector and sequence analyzed as described in example 1. It is to be noted that some polynucleic acid fragments comprised polymorphic nucleotides in their sequences, which have not been previously disclosed. These novel polymorphic nucleotide sequences are represented in table 4 below.

The present invention thus also relates to these novel sequences, or a fragment thereof, wherein said fragment consists of at least 10, preferably 15, even more preferably 20 contiguous nucleotides and contains at least one polymorphic nucleotide. It is furthermore to be understood that these new polymorphic nucleotides may also be expected to arise in another sequence context than in the mentioned sequences. For instance a G at the third position of codon 55 is shown in SEQ ID N

o

478 m combination with a T at the third position of codon 54, but a G at the third position of codon 55 may also be expected to occur in the context of a wild type sequence. It is also to be understood that the above mentioned specifications apply to the complement of the said target sequences as well. This applies also to FIG.

1

.

According to a preferred embodiment the present invention relates to a method as indicated above, further characterized in that said probes are capable of simultaneously hybridizing to their respective target regions under appropriate hybridization and wash conditions allowing the detection of the hybrids formed.

According to a preferred embodiment, step c is performed using a set of at least 2, preferably at least 3, more preferably at least 4 and most preferably at least 5 probes meticulously designed as such that they show the desired hybridization results. In general this method may be used for any purpose that relies on the presence or absence of mutations that can be detected by this method, e.g. for genotyping. The probes of table 1 have been optimized to give specific hybridization results when used in a LiPA assay (see below), as described in examples 2 and 3. These probes have thus also been optimized to simultaneously hybridize to their respective target regions under the same hybridization and wash conditions allowing the detection of hybrids. The sets of probes for each of the codons 30,46/48, 50, 54 and 82/84 have been tested experimentally as described in examples 2 and 3. The reactivity of the sets shown in table 1 with 856 serum samples from various geographic origins was evaluated. It was found that the sets of probes for codons 30, 46/48, 50, 54 and 82/84 reacted with 98.9%, 99.6%, 98.5%, 99.20%, 95.4% and 97.2% of the test samples, respectively. The present invention thus also relates to the sets of probes for codons 30, 46/48, 50, 54, 82/84 and 90, shown in table 1 and table 7.

According to another even more preferred embodiment, the present invention relates to a method as defined above, further characterized in that:

step b) comprises amplifying a fragment of the protease gene with at least one 5′-primer specifically hybridizing to a target sequence located between nucleotide position 210 and nucleotide position 260 (codon 87), more preferably between nucleotide position 220 and nucleotide position 260 (codon 87), more preferably between nucleotide position 230 and nucleotide position 260 (codon 87), even more preferably at nucleotide position 241 to nucleotide position 260 (codon 87) in combination with at least one suitable 3′-primer, and

step c) comprises hybridizing the polynucleic acids of step a) or b) with at least one of the probes specifically hybridizing to a target sequence comprising codon 90.

According to another even more preferred embodiment, the present invention relates to a method as defined above, further characterized in that:

step b) comprises amplifying a fragment of the protease gene with at least one 3′-primer specifically hybridizing to a target sequence located between nucleotide position 253 (codon 85) and nucleotide positions 300, more preferably between nucleotide position 253 (codon 85) and nucleotide positions 290, more preferably between nucleotide position 253 (codon 85) and nucleotide positions 280, even more preferably at nucleotide position 253 (codon 85) to nucleotide position 273 (codon 91), in combination with at least one suitable 5′-primer, and

step c) comprises hybridizing the polynucleic acids of step a) or b) with at least one of the probes specifically hybridizing to a target sequence comprising any of codons 30, 46, 48, 50, 52, 54, 82 and 84.

It has been found, unexpectedly, that an amplified nucleic acid fragment comprising all of the above-mentioned codons, does not hybridize optimally to probes comprising codon 82, 84 or 90. On the other hand, a shorter fragment, for instance the fragment which is amplified by use of the primers Prot41bio and Prot6bio with respectively seq id no 5 and seq id no 4; hybridizes better to probes comprising codon 90. Better hybridization is also obtained when the fragment is amplified with primer Prot41bio in combination with primers Prot6abio, Prot6bbio, Prot6cbio and Prot6dbio. The present invention thus also relates to a method as defined above, finder characterized in that the 5′-primer is seq id no 5 and at least one 3′ primer is chosen from seq id no 4, seq id no506, seq id no 507, seq id no 508, and seq id 509. Likewise, another shorter fragment, for instance the fragment which is amplified by use of the primers Prot2bio and Prot31bio with respectively seq id no 3 and seq id no 6, was found to hybridize better to probes comprising codon 82 and/or 84. Hence the present invention also relates to a method as defined above, further characterized in that the 5′-primer is seq id no 5 and at least one 3′-primer is chosen from seq id no 4, seq id no506, seq id no 507, seq id no 508, and seq id no 509.

New sets of amplification primers as mentioned in example 1 were selected. The present invention thus also relates to primers: prot16 (SEQ ID NO 501), prot5 (SEQ ID NO 5), prot2abio (SEQ ID NO 503), prot2bbio (SEQ ID NO 504), prot31bio (SEQ ID NO 6), prot41-bio (SEQ ID NO 505), prot6a (SEQ ID NO 506), prot6b (SEQ ID NO 507), prot6c (SEQ ID NO 508) and prot6d (SEQ DID NO 509). A number of these primers are chemically modified (biotinylated), others are not. The present invention relates to any of the primers mentioned, primers containing unmodified nucleotides, or primers containing modified nucleotides.

Different techniques can be applied to perform the sequence-specific hybridization methods of the present invention. These techniques may comprise immobilizing the amplified HIV polynucleic acids on a solid support and performing hybridization with labeled oligonucleotide probes. HIV polynucleic acids may also be immobilized on a solid support without prior amplification and subjected to hybridization. Alternatively, the probes may be immobilized on a solid support and hybridization may be performed with labeled HIV polynucleic acids, preferably after amplification. This technique is called reverse hybridization. A convenient reverse hybridization technique is the line probe assay (LiPA). This assay uses oligonucleotide probes immobilized as parallel lines on a solid support strip (Stuyver et al., 1993). It is to be understood that any other technique based on the above-mentioned methods is also covered by the present invention.

According to another preferred embodiment, the present invention relates to any of the probes mentioned above and/or to any of the primers mentioned above, with said primers and probes being designed for use in a method for determining the susceptibility to antiviral drugs of HIV viruses in a sample. According to an even more preferred embodiment, the present invention relates to the probes with seq id no 7 to seq id no 477 and seq id no510 to seq id no 519, more preferably to the seq id no mentioned in Table 1 and Table 7, and to the primers with seq id no 3, 4, 5 and 6, 501, 502, 503, 504, 505, 506, 507, 508 and 509. The skilled man will recognize that addition or deletion of one or more nucleotides at their extremities may adapt the said probes and primers. Such adaptations may be requited if the conditions of amplification or hybridization are changed, or if the amplified material is RNA instead of DNA, as is the case in the NASBA system

According to another preferred embodiment, the present invention relates to a diagnostic kit enabling a method for determining the susceptibility to antiviral drugs of HIV viruses in a biological sample, with said kit comprising:

a) when appropriate, a means for releasing, isolating or concentrating the polynucleic acids present in said sample;

b) when appropriate, at least one of the primers of any of claims

4

to

6

;

c) at least one of the probes of any of claims

1

to

3

, possibly fixed to a solid support;

d) a hybridization buffer, or components necessary for producing said buffer;

e) a wash solution, or components necessary for producing said solution;

f) when appropriate, a means for detecting the hybrids resulting from the preceding hybridization;

h) when appropriate, a means for attaching said probe to a solid support.

DEFINITIONS

The following definitions serve to illustrate the terms and expressions used in the present invention.

The term “antiviral drugs” refers particularly to any antiviral protease inhibitor. Examples of such antiviral drugs and the mutation they may cause in the HIV protease gene are disclosed in Schinazi et al., 1997. The contents of the latter two documents particularly are to be considered as forming part of the present invention. The most important antiviral drugs focussed at in the present invention are disclosed in Tables 1 to 2.

The target material in the samples to be analyzed may either be DNA or RNA, e.g.: genomic DNA, messenger RNA, viral RNA or amplified versions thereof. These molecules are also termed polynucleic acids.

It is possible to use genomic DNA or RNA molecules from HIV samples in the methods according to the present invention.

Well-known extraction and purification procedures are available for the isolation of RNA or DNA from a sample (fi. in Maniatis et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbour Laboratory Press (1989)).

The term “probe” refers to single stranded sequence-specific oligonucleotides, which have a sequence, which is complementary to the target sequence to be detected.

The term “target sequence” as referred to in the present invention describes the wild type nucleotide sequence, or the sequence comprising one or more polymorphic nucleotides of the protease gene to be specifically detected by a probe according to the present invention. This nucleotide sequence may encompass one or several nucleotide changes. Target sequences may refer to single nucleotide positions, codon positions, nucleotides encoding amino acids or to sequences spanning any of the foregoing nucleotide positions. In the present invention said target sequence often includes one or two variable nucleotide positions.

The term “polymorphic nucleotide” indicates a nucleotide in the protease gene of a particular HIV virus that is different from the nucleotide at the corresponding position in at least one other HIV virus. The polymorphic nucleotide may or may not give rise to resistance to an antiviral drug. It is to be understood that the complement of said target sequence is also a suitable target sequence in some cases. The target sequences as defined in the present invention provide sequences which should be complementary to the central part of the probe which is designed to hybridize specifically to said target region.

The term “complementary” as used herein means that the sequence of the single stranded probe is exactly the (inverse) complement of the sequence of the single-stranded target, with the target being defined as the sequence where the mutation to be detected is located.

“Specific hybridization” of a probe to a target sequence of the HIV polynucleic acids means that said probe forms a duplex with part of this region or with the entire region under the experimental conditions used, and that under those conditions said probe does not form a duplex with other regions of the polynucleic acids present in the sample to be analyzed.

Since the current application requires the detection of single basepair mismatches, very stringent conditions for hybridization are required, allowing in principle only hybridization of exactly complementary sequences. However, variations arm possible in the length of the probes (see below), and it should be noted that, since the central part of the probe is essential for its hybridization characteristics, possible deviations of the probe sequence versus the target sequence may be allowable towards head and tail of the probe, when longer probe sequences are used. These variations, which may be conceived from the common knowledge in the art, should however always be evaluated experimentally, in order to check if they result in equivalent hybridization characteristics than the exactly complementary probes.

Preferably, the probes of the invention are about 5 to 50 nucleotides long, more preferably from about 10 to 25 nucleotides. Particularly preferred lengths of probes include 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides. The nucleotides as used in the present invention may be ribonucleotides, deoxyribonucleotides and modified nucleotides such as inosine or nucleotides containing modified groups, which do not essentially alter their hybridization characteristics.

Probe sequences are represented throughout the specification as single stranded DNA oligonucleotides from the 5′ to the 3′ end. It is obvious to the man skilled in the art that any of the below-specified probes can be used as such, or in their complementary form, or in their RNA form (wherein U replaces T).

The probes according to the invention can be prepared by cloning of recombinant plasmids containing inserts including the corresponding nucleotide sequences, if need be by cleaving the latter out from the cloned plasmids upon using the adequate nucleases and recovering them, e.g. by fractionation according to molecular weight. The probes according to the present invention can also be synthesized chemically, for instance by the conventional phospho-triester method.

The term “solid support” can refer to any substrate to which an oligonucleotide probe can be coupled, provided that it retains its hybridization characteristics and provided that the background level of hybridization remains low. Usually the solid substrate will be a microtiter plate, a membrane (e.g. nylon or nitrocellulose) or a microsphere (bead) or a chip. Prior to application to the membrane or fixation it may be convenient to modify the nucleic acid probe in order to facilitate fixation or improve the hybridization efficiency. Such modifications may encompass homopolymer tailing, coupling with different reactive groups such as aliphatic groups, NH

2

groups, SH groups, carboxylic groups, or coupling with biotin, haptens or proteins.

The term “labeled” refers to the use of labeled nucleic acids. Labeling may be carried out by the use of labeled nucleotides incorporated during the polymerase step of the amplification such as illustrated by Saiki et al. (1988) or Bej et al. (1990) or labeled primers, or by any other method known to the person skilled in the art. The nature of the label may be isotopic (

32

P,

35

S, etc.) or non-isotopic (biotin, digoxigenin, etc.).

The term “primer” refers to a single stranded oligonucleotide sequence capable of acting as a point of initiation for synthesis of a primer extension product, which is complementary to the nucleic acid strand to be copied. The length and the sequence of the primer must be such that they allow to prime the synthesis of the extension products. Preferably the primer is about 5-50 nucleotides long. Specific length and sequence will depend on the complexity of the required DNA or RNA targets, as well as on the conditions of primer use such as temperature and ionic strength.

The term “primer pair” refers to a set of primers comprising at least one 5′ primer and one 3′ primer. The primer pair may consist of more than two primers, the complexity of the number of primers will depend on the hybridization conditions, variability of the sequences in the regions to be amplified and the target sequences to be detected.

The fact that amplification primers do not have to match exactly with the corresponding template sequence to warrant proper amplification is amply documented in the literature (Kwok et al., 1990).

The amplification method used can be either polymerase chain reaction (PCR; Saiki et al., 1988), ligase chain reaction (LCR; Landgren et al., 1988; Wu & Wallace, 1989; Barany, 1991), nucleic acid sequence-based amplification (NASBA; Guatelli et al., 1990; Compton, 1991), transcription-based amplification system (TAS; Kwoh et al., 1989), strand displacement amplification (SDA; Duck, 1990) or amplification by means of QB replicase (Lomeli et al., 1989) or any other suitable method to amplify nucleic acid molecules known in the art.

The oligonucleotides used as primers or probes may also comprise nucleotide analogues such as phosphorothiates (Matsukura et al., 1987), alkylphosphorothiates (Miller et al., 1979) or peptide nucleic acids (Nielsen et al., 1991; Nielsen et al., 1993) or may contain intercalating agents (Asseline et al., 1984).

As most other variations or modifications introduced into the original DNA sequences of the invention these variations will necessitate adaptations with respect to the conditions under which the oligonucleotide should be used to obtain the required specificity and sensitivity. However the eventual results of hybridization will be essentially the same as those obtained with the unmodified oligonucleotides.

The introduction of these modifications may be advantageous in order to positively influence characteristics such as hybridization kinetics, reversibility of the hybrid-formation, biological stability of the oligonucleotide molecules, etc.

The “sample” may be any biological material taken either directly from the infected human being (or animal), or after culturing (enrichment). Biological material may be e.g. expectorations of any kind, broncheolavages, blood, skin tissue, biopsies, sperm, lymphocyte blood culture material, colonies, liquid cultures, fecal samples, urine etc.

The sets of probes of the present invention will include at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more probes. Said probes may be applied in two or more distinct and known positions an a solid substrate. Often it is preferable to apply two or more probes together in one and the same position of said solid support.

For designing probes with desired characteristics, the following useful guidelines known to the person skilled in the art can be applied.

Because the extent and specificity of hybridization reactions such as those described herein are affected by a number of factors, manipulation of one or more of those factors will determine the exact sensitivity and specificity of a particular probe, whether perfectly complementary to its target or not. The importance and effect of various assay conditions, explained further herein, are known to those skilled in the art.

The stability of the [probe:target] nucleic acid hybrid should be chosen to be compatible with the assay conditions. This may be accomplished by avoiding long AT-rich sequences, by terminating the hybrids with G:C base pairs, and by designing the probe with an appropriate Tm. The beginning and end points of the probe should be chosen so that the length and % GC result in a Tm about 2-10° C. higher than the temperature at which the final assay will be performed. The base composition of the probe is significant because G-C base pairs exhibit greater thermal stability as compared to A-T base pairs due to additional hydrogen bonding. Thus, hybridization involving complementary nucleic acids of higher G-C content will be stable at higher temperatures.

Conditions such as ionic strength and incubation temperature under which a probe will be used should also be taken into account when designing a probe. It is known that hybridization will increase as the ionic strength of the reaction mixture increases, and that the thermal stability of the hybrids will increase with increasing ionic strength. On the other hand, chemical reagents, such as formamide, urea, DMSO and alcohols, which disrupt hydrogen bonds, will increase the stringency of hybridization. Destabilization of the hydrogen bonds by such reagents can greatly reduce the Tm. In general, optimal hybridization for synthetic oligonucleotide probes of about 10-50 bases in length occurs approximately 5° C. below the melting temperature for a given duplex. Incubation at tempts below the optimum may allow mismatched base sequences to hybridize and can therefore result in reduced specificity.

It is desirable to have probes, which hybridize only under conditions of high stringency. Under high stringency conditions only highly complementary nucleic acid hybrids will form; hybrids without a sufficient degree of complementarity will not form. Accordingly, the stringency of the assay conditions determines the amount of complementarity needed between two nucleic acid strands forming a hybrid. The degree of stringency is chosen such as to maximize the difference in stability between the hybrid formed with the target and the nontarget nucleic acid. In the present case, single base pair changes need to be detected, which requires conditions of very high stringency.

The length of the target nucleic acid sequence and, accordingly, the length of the probe sequence can also be important. In some cases, there may be several sequences from a particular region, varying in location and length, which will yield probes with the desired hybridization characteristics. In other cases, one sequence may be significantly better than another that differs merely by a single base. While it is possible for nucleic acids that are not perfectly complementary to hybridize, the longest stretch of perfectly complementary base sequence will normally primarily determine hybrid stability. While oligonucleotide probes of different lengths and base composition may be used, preferred oligonucleotide probes of this invention arm between about 5 to 50 (more particularly 10-25) bases in length and have a sufficient stretch in the sequence which is perfectly complementary to the target nucleic acid sequence.

Regions in the target DNA or RNA, which are known to form strong internal structures inhibitory to hybridization, arc less preferred. Likewise, probes with extensive self-complementarity should be avoided. As explained above, hybridization is the association of two single strands of complementary nucleic acids to form a hydrogen bonded double strand. It is implicit that if one of the two strands is wholly or partially involved in a hybrid that it will be less able to participate in formation of a new hybrid. There can be intramolecular and intermolecular hybrids formed within the molecules of one type of probe if there is sufficient self complementarity. Such structures can be avoided through careful probe design. By designing a probe so that a substantial portion of the sequence of interest is single stranded, the rate and extent of hybridization may be greatly increased. Computer programs are available to search for this type of interaction. However, in certain instances, it may not be possible to avoid this type of interaction.

Standard hybridization and wash conditions are disclosed in the Materials & Methods section of the Examples. Other conditions are for instance 3×SSC (Sodium Saline Citrate), 20% deionized FA (Formamide) at 50° C.

Other solutions (SSPE (Sodium saline phosphate EDTA), TMACl (tetramethyl ammonium Chloride), etc.) and temperatures can also be used provided that the specificity and sensitivity of the probes is maintained. If need be, slight modifications of the probes in length or in sequence have to be carried out to maintain the specificity and sensitivity required under the given circumstances.

Primers may be labeled with a label of choice (e.g. biotin). Different primer-based target amplification systems may be used, and preferably PCR-amplification, as set out in the examples. Single-round or nested PCR may be used.

The term “hybridization buffer” means a buffer enabling a hybridization reaction to occur between the probes and the polynucleic acids present in the sample, or the amplified product, under the appropriate stringency conditions.

The term “wash solution” means a solution enabling washing of the hybrids formed under the appropriate stringency conditions.

The following examples only serve to illustrate the present invention. These examples are in no way intended to limit the scope of the present invention.

FIGURE AND TABLE LEGENDS

FIG.

1

: Natural and drug selected variability in the vicinity of codons 30, 46, 48, 50, 54, 82, 84, and 90 of the HIV-1 protease gene. The most frequently observed wild-type sequence is shown in the top line (SEQ ID NO: 520 for codon 30, SEQ ID NO: 521 for codon 46/48, SEQ ID NO: 522 for codon 50, SEQ ID NO: 523 for codon for codon 54, SEQ ID NO: 524 for codon 82/84, and SEQ ID NO: 525 for codon 90). Naturally occurring variations are indicated below and occur independently from each other. Variants sequences for each of the indicated codons are as follows: SEQ ID NO: 7-46 for codon 30, SEQ ID NO: 47-120 for codon 46/48, SEQ ID NO: 121-175 for codon 50, SEQ ID NO: 176-227 for codon 54, SEQ ID NO: 228-357 for codon 82/84, and SEQ ID NO: 358-477 for codon 90. Drug-selected variants are indicated in bold.

FIG.

2

A: Reactivities of the selected probes for codon 30 immobilized on LiPA strips with reference material. The information in the boxed surface is not relevant for the discussion of probes for codon 30. The position of each selected probe on the membrane strip is shown at the left of each panel. The sequence of the relevant part of the selected probes is shown at the left and is given in Table 1. Each strip is incubated with a biotinylated PCR fragment from the reference panel. The reference panel accession numbers are indicated in Table 1 (SEQ ID NO: 31 corresponds to w25, SEQ ID NO: 35 corresponds to w29, SEQ ID NO: 32 corresponds to w32, SEQ ID NO: 42 corresponds to w36, and SEQ ID NO: 29 corresponds to m23). For several probes multiple reference panel possibilities are available, but only one relevant accession number given each time. *: False positive reactivities. At the bottom the strips, the amino acids at the relevant codon, as derived from the probe reactivity, is indicated.

FIG.

2

B: Reactivities of the selected probes for codons 46 and 48 immobilized on LiPA strips with reference material. The information in the boxed surface is not relevant for the discussion of probes for codons 46 and 48. The position of each selected probe on the membrane strip is shown at the left of each panel. The sequence of the relevant part of the selected probes is given in Table 1. Each strip is incubated with a biotinylated PCR fragment from the reference panel. The reference panel accession numbers are indicated in Table 1 (SEQ ID NO: 93 corresponds to w47, SEQ ID NO: 91 corresponds to w45, SEQ ID NO: 120 corresponds to w72, and SEQ ID NO: 87 corresponds to m41). For several probes multiple reference panel possibilities are available, but only one relevant accession number given each time. *: False positive reactivities. On top of the strips, the amino acids at the relevant codon, as derived from the probe reactivity, is indicated.

FIG.

2

C: Reactivities of the selected probes for codon 50 immobilized on LiPA strips With reference material. The information in the boxed surface is not relevant for the discussion of probes for codon 50. The position of each selected probe on the membrane strip is shown at the left of each panel. The sequence of the relevant part of the selected probes is given in Table 1. Each strip is incubated with a biotinylated PCR fragment from the reference panel. The reference panel accession numbers are indicated in Table 1 (SEQ ID NO: 151 corresponds to w31, SEQ ID NO: 164 corresponds to w44, SEQ ID NO: 172 corresponds to w51, and SEQ ID NO: 157 corresponds to m37). For several probes multiple reference panel possibilities are available, but only one relevant accession number given each time. *: False positive reactivities. At the bottom of the strips, the amino acids at the relevant codon, as derived from the probe reactivity, is indicated.

FIG.

2

D: Reactivities of the selected probes for codon 54 immobilized on LiPA strips with reference material. The information in the boxed surface is not relevant for the discussion of probes for codon 54. The position of each selected probe on the membrane strip is shown at the left of each panel. The sequence of the relevant part of the selected probes is given in Table 1. Each strip is incubated with a biotinylated PCR fragment from the reference panel. The reference panel accession numbers are indicated in Table 1 (SEQ ID NO: 178 corresponds to w3, SEQ ID NO: 212 corresponds to w34, SEQ ID NO: 189 corresponds to w14, SEQ ID NO: 194 corresponds to w19, SEQ ID NO: 197 corresponds to w22, SEQ ID NO: 202 corresponds to w26, SEQ ID NO: 204 corresponds to w27, SEQ ID NO: 213 corresponds to m35, and SEQ ID NO: 215 corresponds to m37). For several probes multiple reference panel possibilities are available, but only one relevant accession number given each time. *: False positive reactivities. At the bottom of the strips, the amino acids at the relevant codon, as derived from the probe reactivity, is indicated.

FIG.

2

E: Reactivities of the selected probes for codons 82 and 84 immobilized on LiPA strips with reference material. The information in the boxed surface is not relevant for the discussion of probes for codons 82 and 84. The position of each selected probe on the membrane strip is shown at the left of each panel. The sequence of the relevant part of the selected probes is given in Table 1. Each strip is incubated with a biotinylated PCR fragment from the reference panel. The reference panel accession numbers are indicated in Table 1 (SEQ ID NO: 318 corresponds to w91, SEQ ID NO: 287 corresponds to w60, SEQ ID NO: 338 corresponds to w111, SEQ ID NO: 316 corresponds to w89, SEQ ID NO: 269 corresponds to w42 SEQ ID NO: 263 corresponds to m36, SEQ ID NO: 294 corresponds to m67. SEQ ID NO: 265 corresponds to m38, SEQ ID NO: 332 corresponds to m105, SEQ ID NO: 354 corresponds to m127, SEQ ID NO: 267 corresponds to m40, SEQ ID NO: 290 corresponds to m63, and SEQ ID NO: 328 corresponds to m101). For several probes multiple reference panel possibilities are available, but only one relevant accession number given each time. *: False positive reactivities. At the bottom of the strips, the amino acids at the relevant codon, as derived from the probe reactivity, is indicated.

FIG.

2

F: Reactivities of the selected probes for codon 90 immobilized on LiPA strips with reference material. The information in the boxed surface is not relevant for the discussion of probes for codon 90. The position of each selected probe on the membrane strip is shown at the left of each panel. The sequence of the relevant part of the selected probes is given in Table 1. Each strip is incubated with a biotinylated PCR fragment from the reference panel. The reference panel accession numbers are indicated in Table 1 (SEQ ID NO: 384 corresponds to w27, SEQ ID NO: 394 corresponds to w37, SEQ ID NO: 396 corresponds to w39, SEQ ID NO: 407 corresponds to w50, SEQ ID NO: 409 corresponds to w52, SEQ ID NO: 426 corresponds to w69, SEQ ID NO: 430 corresponds to w73, SEQ ID NO: 436 corresponds to w79, SEQ ID NO: 400 corresponds to m43, and SEQ ID NO: 413 corresponds to m56). For several probes multiple reference panel possibilities are available, but only one relevant accession number given each time. *: False positive reactivities. At the bottom of the strips, the amino acids at the relevant codon, as derived from the probe reactivity, is indicated.

FIG.

3

: Sequence and position of the HIV-1 protease amplification primers. To obtain the reactivity with probes selected to determine the susceptibility to antiviral drugs involving codons 30, 46, 48, 50, 54, 82, and 84, nested amplification primers prot2bio (5′ primer, SEQ ID NO: 526) and Prot31bio (3′ primer, SEQ ID NO: 527) were designed. To obtain the reactivity with probes selected to determine the susceptibility to antiviral drugs involving codon 90, nested amplification primers Prot41bio (5′ primer, SEQ ID NO: 528) and Prot6bio (3′ primer, SEQ ID NO: 529) were designed.

FIG.

4

A: Phylogenetic analysis on 312 protease sequences allowed to separate genotype B strains from non-B strains. Reactivities of the selected probes for codon 30 immobilized on LiPA strips with a biotinylated PCR fragment of genotype B strains and non-B strains is shown, the exact percentages are indicated in table 5. The probes are indicated at the bottom. The sequence of the relevant part of the probes is given in Table 1.

FIG.

4

B: Phylogenetic analysis on 312 protease sequences allowed to separate genotype B strains from non-B strains. Reactivities of the selected probes for codons 46/48 immobilized on LiPA strips with a biotinylated PCR fragment of genotype B strains and non-B strains is shown, the exact percentages are indicated in table 5. The probes are indicated at the bottom. The sequence of the relevant part of the probes is given in Table 1.

FIG.

4

C: Phylogenetic analysis on 312 protease sequences allowed to separate genotype B strains from non-B strains. Reactivities of the selected probes for codon 50 immobilized on LiPA strips with a biotinylated PCR fragment of genotype B strains and non-B sons is shown, the exact percentages are indicated in table 5. The probes are indicated at the bottom. The sequence of the relevant part of the probes is given in Table 1.

FIG.

4

D: Phylogenetic analysis on 312 protease sequences allowed to separate genotype B strains from non-B strains. Reactivities of the selected probes for codon 54 immobilized on LiPA strips with a biotinylated PCR fragment of genotype B strains and non-B stains is shown, the exact percentages are indicated in table 5. The probes are indicated at the bottom. The sequence of the relevant part of the probes is given in Table 1.

FIG.

4

E: Phylogenetic analysis on 312 protease sequences allowed to separate genotype B strains from non-B strains. Reactivities of the selected probes for codons 82/84 immobilized an LiPA strips with a biotinylated PCR fragment of genotype B strains and non-B stains is shown, the exact percentages are indicated in table 5. The probes are indicated at the bottom. The sequence of the relevant part of the probes is given in Table 1.

FIG.

4

F: Phylogenetic analysis on 312 protease sequences allowed to separate genotype B strains from non-B strains. Reactivities of the selected probes for codon 90 immobilized on LiPA strips with a biotinylated PCR fragment of genotype B strains and non-B strains is shown, the exact percentages are indicated in table 5. The probes are indicated at the bottom. The sequence of the relevant part of the probes is given in Table 1.

FIG.

5

A: Geographical origin of 856 samples and reactivities with the different probes at codon position 30. The exact percentages are indicated in table 6. The probes are indicated at the bottom.

FIG.

5

B: Geographical origin of 856 samples and reactivities with the different probes at codon positions 46/48. The exact percentages are indicated in table 6. The probes are indicated at the bottom.

FIG.

5

C: Geographical origin of 856 samples and reactivities with the different probes at codon position 50. The exact percentages arm indicated in table 6. The probes are indicated at the bottom.

FIG.

5

D: Geographical origin of 856 samples and reactivities with the different probes at codon position 54. The exact percentages arm indicated in table 6. The probes are indicated at the bottom.

FIG.

5

E: Geographical origin of 856 samples and reactivities with the different probes at codon positions 82/84. The exact percentages are indicated in table 6. The probes are indicated at the bottom.

FIG.

5

F: Geographical origin of 856 samples and reactivities with the different probes at codon position 90. The exact percentages are indicated in table 6. The probes are indicated at the bottom.

Table 1: HIV-1 protease wild-type and drug-selected mutation probes with their corresponding sequences as applied on the HIV-1 protease LiPA strip. The most frequently observed wild-type sequence is shown at the top line. Probe names corresponding to the selected motifs are indicated in the left column, the relevant part of each probe applied on the strip is shown under the consensus sequence.

Table 2: Protease Inhibitors.

Table 3: HIV-1 protease wild-type and drug-selected mutation probes with their corresponding sequences as synthesized, immobilized and tested on LiPA strips. The most frequently observed wild-type sequence is shown at the top line. Probe names corresponding to the selected motifs arc indicated in the left column, the relevant part of each probe applied on the strip is shown under the consensus sequence. The probes retained are indicated in table 1.

Table 4: Polymorphic nucleotide sequences.

Table 5: % Reactivities of the HIV-1 protease wild-type and drug-selected mutation probes applied on the HIV-1 protease LiPA strip with genotype B strains and non-B strains.

Table 6: % Reactivities of the HIV-1 protease wild-type and drug-selected mutation probes applied on the HIV-1 protease LiPA strip with samples of different geographical origin.

Table 7: HIV-1 protease wild-type and drug-selected mutation probes with their corresponding sequences as applied on the HIV-1 protease LiPA strip. The most frequently observed wild-type sequence is shown at the top line. Probe names corresponding to the selected motifs are indicated in the left column, the relevant part of each probe applied on the strip is shown under the consensus sequence.

EXAMPLES

Example 1

Selection of the Plasma Samples, PCR Amplification and Cloning of the PCR Products

Plasma samples (n=557) were taken from HIV type-1 infected patients and stored at −20° C. until use. Plasma samples were obtained from naive and drug-treated patients. The drugs involved ritonavr, indinavir and saquinavir. The serum samples were collected from patients residing in Europe (Belgium, Luxembourg, France, Spain and UK), USA and Brazil.

HIV RNA was prepared from these samples using the guanidinium-phenol procedure. Fifty μl plasma was mixed with 150 μl Trizol®LS Reagent (Life Technologies, Gent, Belgium) at room temperature (volume ratio: 1 unit sample/3 units Trizol). Lysis and denaturation occurred by carefully pipetting up and down several times, followed by an incubation step at room temperature for at least 5 minutes. Fourthy μl CHCl

3

was added and the mixture was shaken vigorously by hand for at least 15 seconds, and incubated for 15 minutes at room temperature. The samples were centrifuged at maximum 12,000 g for 15 minutes at 4° C., and the colorless aqueous phase was collected and mixed with 100 μl isopropanol. To visualize the minute amounts of viral RNA, 20 μl of 1 μg/μl Dextran T500 (Pharmacia) was added, mixed and left at room temperature for 10 minutes. Following centrifugation at max. 12,000 g for 10 minutes at 4° C. and aspiration of the supernatant, the RNA pellet was washed with 200 μl ethanol, mixed by vortexing and collected by centrifugation at 7,500 g for 5 minutes at 4° C. Finally the RNA pellet was briefly air-dried and stored at −20° C. Alternatively, the High Pure Viral Nucleic Acid Kit (Boehringer Mannheim ) was used to extract RNA from the samples.

For cDNA synthesis and PCR amplification, the RNA pellet was dissolved in 15 μl random primes (20 ng/μl, pdN

6

, Pharmacia), prepared in DEPC-treated or HPLC grade water. After denaturation at 70° C. for 10 minutes, 5 μl cDNA mix was added, composed of 4 μl 5× AMV-RT buffer (250 mM Tris.HCl pH 8.5, 100 mM KCl, 30 mM MgCl

2

, 25 mM DTT)), 0.4 μL 25 mM dXTPs, 0.2 μl or 25 U Ribonuclease Inhibitor (HPRI, Amersham), and 0.3 μl or 8 U AMV-RT (Stratagene). cDNA synthesis occurred during the 90 minutes incubation at 42° C. The HIV-1 protease gene was than amplified using the following reaction mixture: 5 μl cDNA, 4.5 μl 10× Taq buffer, 0.3 μl 25 mM dXTPs, 1 μl (10 pmol) of each PCR primer, 38 μl H

2

O, and 0.2 μl (1 U) Taq. . Alternatively, the Titon One Tube RT-PCR system (Boehringer Mannheim) was used to perform RT-PCR. Codon positions involving resistance to saquinavir, ritonavir, indinavir, nelfinavir and VX-478 have been described (Shinazi et al) and PCR amplification primers were chosen outside these regions. The primer design was based on HIV-1 published sequences (mainly genotype B clade) (Myers et al.) and located in regions that showed a high degree of nucleotide conservation between the different HIV-1 clades. The final amplified region covered the HIV-1 protease gene from codon 9 to codon 99. The primers for amplification had the following sequence: outer sense primer Pr16: 5′ bio-CAGAGCCAACAGCCCCACCAG 3′ (SEQ ID NO 1); nested sense primer Prot2bio: 5′ CCT CAR ATC ACT CTT TGG CAA CG 3′ (SEQ ED NO 3); nested antisense primer Prot6bio: 3′ TAA TCR GGA TAA CTY TGA CAT GGT C 5′ (SEQ ID NO 4); and outer antisense primer RT12: 5′ bioATCAGGATGGAGTTCATAACCCATCCA 3′ (SEQ ID NO 2). Annealing occurred at 57° C., extension at 72° C. and denaturation at 94° C. Each step of the cycle took 1 minute, the outer PCR contained 40 cycles, the nested round 35. Nested round PCR products were analyzed on agarose gel and only clearly visible amplification products were used in the LiPA procedure. Quantification of viral RNA was obtained with the HIV Monitor™test (Roche, Brussels, Belgium). Later on, new sets of primers for amplification were selected. For the amplification of HIV protease codon 30-84: outer sense primer prot16: 5′-CAGAGCCAACAGCCCCACCAG-3′ (SEQ ID NO 501), outer antisense primer prot5: 5′-TTTTCTTCTGTCAATGGCCATTGTTT-3′ (SEQ ID NO 502) were used. Annealing occurred at 50° C., extension at 68° C. and denaturation at 94° C. for 35 cycles for the outer PCR. For the nested PCR annealing occurred at 45° C., denaturation at 94° C. and extension at 92° C. with primers: nested sense primers prot2a-bio: 5′-bio-CCTCAAATCACTCTTTGGCAACG-3′ (SEQ ID NO 503)and prot2b-bio: 5′-bio-CCTCSGSTCSCTCTTTGGCSSCG-3′ (SEQ ED NO 504), and nested antisense primer prot31-bio: 5′-bio-AGTCAACAGATTTCTTTCCAAT-3′ (SEQ ID NO 6). For the amplification of HIV protease codon 90, the outer PCR was as specified for HIV protease codon 30-84. For the nested PCR, nested sense primer prot41-bio: 5′-bio-CCTGTCAACATAATTGCAAG-3′ (SEQ ID NO 505) and nested antisense primers prot6a: 5′-bio-CTGGTACAGTTTCAATAGGGCTAAT-3′ (SEQ ID NO 506), prot6b: 5′-bio-CTGGTACAGTTTCAATAGGACTAAT-3′ (SEQ ID NO 507), prot6c: 5′-bio-CTGGTACAGTCTCAATAGGACTAAT-3′ (SEQ ID NO 508), prot6d: 5′-bio-CTGGTACAGTCTCAATAGGGCTAAT-3′ (SEQ ID NO 509) were used. For the nested PCR the annealing temperature occurred at 45° C. Primers were tested on a plasmid, which contained an HIV fragment of 1301 bp ligated in a pGEM-T vector. The fragment contains protease, reverse transcriptase and the primer sites of first and second round PCR. By restriction with SacI the plasmid is linearised.

Selected PCR products were cloned into the pretreated EcoRV site of the pGEMT vector (Promega). Recombinant clones were selected after α-complementation and restriction fragment length analysis, and sequenced using standard sequencing techniques with plasmid primers and internal HIV protease primers. Sometimes biotinylated fragments were directly sequenced with a dye-terminator protocol (Applied Biosystems) using the amplification primers. Alternatively, nested PCR was carried out with analogs of the nested primers, in which the biotin group was replaced with the T7- and SP6-primer sequence, respectively. These amplicons were than sequenced with an SP6- and T7-dye-primer procedure.

Example 2

Selection of a Reference Panel

Codon positions involving resistance to saquinavir, ritonavir, indinavir, nelfmiavir and VX-478 have been described (Shiazi et al. 1997). It was the aim to clone in plasmids those viral protease genes that are covering the different genetic motifs at those important codon positions conferring resistance against the described protease inhibitors.

After careful analysis of 312 protease gene sequences, obtained after direct sequencing of PCR fragments, a selection of 47 PCR fragments which covered the different target polymorphisms and mutations were retained and cloned in plasmids using described cloning techniques. The selection of samples originated from naive or drug-treated European, Brazilian or US patients. These 47 recombinant plasmids are used as a reference panel, a panel that was sequenced on both strands, and biotinylated PCR products from this panel were used to optimize probes for specificity and sensitivity.

Although this panel of 47 samples is a representative selection of clones at this moment, it is important to mention here that this selection is an fact only a temporally picture of the variability of the virus, and a continuous update of this panel will be mandatory. This includes on ongoing screening for the new variants of the virus, and recombinant cloning of these new motifs.

Probe Selection and LiPA Testing

To cover all the different genetic motifs in the reference panel, a total of 471 probes were designed (codon 30: 40 probes; codon 46/48: 72 probes; codon 50: 55 probes; codon 54: 54 probes, codon 82/84: 130 probes; codon 90: 120 probes). Table 3 shows the different probes that were selected for the different codon positions.

It was the aim to adapt all probes to react specifically under the same hybridization and wash conditions by carefully considering the % (G+C), the probe length, the final concentration of the buffer components, and hybridization temperature (Stuyver et al., 1997). Therefore, probes were provided enzymatically with a poly-T-tail using the TdT (Pharmacia) in a standard reaction condition, and purified via precipitation. For a limited number of probes with 3′ T-ending sequences, an additional G was incorporated between the probe sequence and the poly-T-tail in order to limit the hybridizing part to the specific probe sequence and to exclude hybridization with the tail sequence. Probe pellets were dissolved in standard saline citrate (SSC) buffer and applied as horizontal parallel lines on a membrane strip. Control lines for amplification (probe 5′ TAGGGGGAATTGGAGGTTTTAG 3 ′ (SEQ ID NO: 125), HIV protease aa 47 to aa 54) and conjugate incubation (biotinylated DNA) were applied alongside. Probes were immobilized onto membranes by baking, and the membranes were sliced into 4 mm strips also called LiPA strips.

Selection of the amplification primers and PCR amplification was as described in example 1. In order to select specific reacting probes out of the 471 candidate probes, LiPA tests were performed with biotinylated PCR fragments from the reference panel. To perform LiPA tests, equal amounts (10 μl) of biotinylated amplification products and denaturation mixture (0.4 N NaOH/0.1l% SDS) were mixed, followed by an incubation at room temperature for 5 minutes. Following this denaturation step, 2 ml hybridization buffer (2×SSC, 0.1% SDS, 50 mM Tris pH7.5) was added together with a membrane strip and hybridization was carried out at 39° C. for 30 min. Then, the hybridization mix was replaced by stringent washing buffer (same composition as hybridization buffer), and stringent washing occurred first at room temperature for 5 minutes and than at 39° C. for another 25 minutes. Buffers were than replaced to be suitable for the streptavidine alkaline phosphatase conjugate incubations. After 30 minutes incubation at room temperature, conjugate was rinsed away and replaced by the substrate components for alkaline phosphatase, Nitro-Blue-Tetrazolium and 5-Bromo-4-Chloro-3-Indolyl Phosphate. After 30 minutes incubation at room temperature, probes where hybridization occurred became visible because of the purple brown precipitate at these positions.

After careful analysis of the 471 probes, the most specific and sensitive probes (n=46) were finally selected, covering the natural and drug-selected variability in the vicinity of aa 30, 46, 48, 50, 54, 82, 84, and 90.

FIG. 2

shows the reactivity of the finally selected probes with the reference panel.

Example 3

LiPA Testing on Clinical Samples

A total of 856 samples were tested on this selection of 46 specific probes. The geographical origin of these samples is as follows: USA:359; France: 154; UK:36; Brazil 58; Spain 35; Belgium 199; Luxembourg: 15.

From this population, a total of 144 samples were sequenced which allowed to separate the genotype B samples (94) from the non-B samples (50). After analysis of these genotyped samples on LiPA, the genotypic reactivity on the selected probes was scored.

FIGS. 4A

to

4

F show these results for the different codon positions and for the genotype B versus non-B group. From these tables, it is clear that there is little difference in sequence usage for the different codon positions with resect to specific reactivities at the different probes.

The total collection of 856 samples was then tested on the available 46 probes. After dissection of those reactivities over the different probes and different geographical origin, the picture looks as is presented in

FIGS. 5A

to

5

F. Again here, the majority of the sequences used at the different codon positions are restricted to some very abundant wild type motifs. It is important to mention here that the majority of these samples are taken from patients never treated with protease inhibitors, en therefore, the majority of the reactivities are found in wild type motifs. Nevertheless, it is clear from some codon positions that the variability at some codon positions in the mutant motif might be considerable, and again, a continuous update on heavily treated patients is mandatory. Another issue is the amount of double blank reactivities, which is in this approach reaching up to 5% in global; with some peak values for some countries for some codon positions: for example 13.8% for codon 82/85 in Brazil; and 18.1% for codon 90 in Belgium.

The continuous update resulted in a further selection of probes. This later configuration of the strip is indicated in table 7.

REFERENCES

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Barany F. Genetic disease detection and DNA amplification using cloned thermostable ligase. Proc Natl Acad Sci USA 1991; 88: 189-193.

Bej A, Mahbubani M, Miller R, Di Cesare J, Haff L, Atlas R. Mutiplex PCR amplification and immobilized capture probes for detection of bacterial pathogens and indicators in water. Mol Cell Probes 1990; 4:353-365.

Compton J. Nucleic acid sequence-based amplification. Nature 1991; 350: 91-92.

Condra et al. 1995. In vivo emergence of HIV-1 variants resistant to multiple protease inhibitors. Nature 374: 569-571.

Condra et al. 1995. In vivo emergence of HIV-1 variants resistant to multiple protease inhibitors. Nature 374: 569-571.

Duck P. Probe amplifier s n based on chimeric cycling oligonucleotides. Biotechniques 1990; 9: 142-147.

Eberle et al. 1995. Resistance of HIV type 1 to proteinase inhibitor Ro 31-8959. AIDS Research and Human Retroviruses 11: 671-676.

Emini et al. 1994. Phenotypic and genotypic characterization of HIV-1 variants selected during treatment with the protease inhibitor L-735, L-524. Third International Workshop on HIV Drug Resistance, Kauai, Hi., USA.

Guatelli J, Whitfield K, Kwoh D, Barringer K, Richman D, Gengeras T. Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication. Proc Natl Acad Sci USA 1990; 87: 1874-1878.

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TABLE 1

26

27

28

29

30

31

32

33

34

3

Tm

lengte

Seq ID

ACA

GGA

GCA

GAT

GAT

ACA

GTA

TTA

GAA

GAA

pc30w25

GCA

GAT

GAT

ACA

GT

40

14

31

pc30w29

A

GCG

GAT

GAT

ACA

36

13

35

pc30w32

GCA

GAT

GAC

ACA

GT

42

14

38

pc30w36

GCA

GAC

GAT

ACA

GG

40

14

42

pc30m23

A

GCA

GAT

AAT

ACA

GT

40

15

29

44

45

46

47

48

49

50

51

52

CCA

AAA

ATG

ATA

GGG

GGA

ATT

GGA

GGT

pc48w47

AAA

ATG

ATA

GGG

GGA

42

15

93

pc48w45

A

ATG

ATA

GGA

GGA

ATT

42

16

91

pc48w72

A

AAA

ATA

ATA

GGG

GGA

42

16

120

pc48m41

ATG

ATA

GTG

GGA

ATT

40

15

87

48

49

50

51

52

53

54

GGG

GGA

ATT

GGA

GGT

TTT

ATC

pc50w31

GGA

ATT

GGA

GGT

TTT

42

15

151

pc50w44

GGA

ATT

GGG

GGT

TTG

42

15

164

pc50w52

GA

ATT

GGA

GGC

TTG

14

172

pc50m37

GGG

GGA

GTT

GGA

40

12

157

51

52

53

54

55

56

57

58

GGA

GGT

TTT

ATC

AAA

GTA

AGA

CAG

pc54w3

GT

TTT

ATC

AAA

GTA

AGA

42

17

178

pc54w34

GA

GGT

TTT

ATC

AAA

GT

42

16

212

pc54w14

GGT

TTT

ATC

AAG

GTA

A

42

16

189

pc54w19

A

GGC

TTT

ATC

AAA

GTA

42

16

194

pc54w22

GA

GGT

TTT

ATT

AAA

GTA

42

17

197

pc54w26

A

GGT

TTC

ATT

AAG

GTA

42

16

202

pc54w27

GGT

TTT

ATT

AAG

GTA

A

40

16

204

pc54m55

A

GGT

TTT

GCC

AAA

GT

38

15

pc54m35

GGT

TTT

GTC

AAA

GTA

40

15

213

pc54m37

GGT

TTT

GTC

AGA

GTA

42

15

215

78

79

80

81

82

83

84

85

86

87

GGA

CCT

ACA

CCT

GTC

AAC

ATA

ATT

GGA

AGA

pc82w91

ACA

CCT

GTC

AAC

ATA

A

44

16

318

pc82w60

CA

CCT

GTC

AAT

ATA

ATG

42

17

287

pc82w111

A

CCG

GTC

AAC

ATA

ATT

44

16

338

pc82w89

ACA

CCT

GTT

AAC

ATA

AG

42

17

316

pc82w42

CA

CCT

GTC

AAC

GTA

42

14

269

pc82m36

ACA

CCT

ACC

AAC

ATA

42

15

263

pc82m67

ACA

CCT

ACC

AAC

GT

42

14

294

pc82m38

ACA

CCT

TTC

AAC

ATA

40

15

265

pc82m105

ACG

CCC

TTC

AAC

ATA

44

15

332

pc82m127

CA

CCT

TTC

AAC

GTA

ATG

44

17

354

pc82m40

ACA

CCT

GCC

AAC

ATA

44

15

267

pc82m63

CA

CCT

GCC

AAT

ATA

AG

42

16

290

pc82m101

ACA

CCT

ATC

AAC

ATA

ATG

44

18

328

86

87

88

89

90

91

92

93

94

GGA

AGA

AAT

CTG

TTG

ACT

CAG

ATT

GGT

pc90w27

AAT

CTG

TTG

ACT

CA

38

14

384

pc90w37

AAT

CTG

TTG

ACT

CAG

ATG

42

18

394

pc90w39

GA

ACT

CTG

TTG

ACT

C

44

15

396

pc90w50

AAT

ATG

TTG

ACT

CAG

40

15

407

pc90w52

AAT

TTG

TTG

ACT

CAG

40

15

409

pc90w69

GA

AAC

CTG

TTG

ACT

40

14

426

pc90w73

TG

TTG

ACA

CAG

CTT

G

44

15

430

pc90w79

TG

TTG

ACC

CAG

ATT

G

44

15

436

pc90m43

A

AAT

CTG

ATG

ACT

CA

40

15

400

pc90m56

AAT

ATG

ATG

ACC

CAG

42

15

413

TABLE 2

Protease Inhibitors

Compound

Amino

Protease

acid

Inhibitors

change

Codon change

A-77003

R8Q

CGA to CAA

R8K

CGA to AAA

V32I

GTA to ATA

M46I

ATG to ATA

M46L

ATG to TTC

M46F

ATG to TTC

M46V

ATG to GTG

G48V

GGG to GTG

A71V

GCT to GTT

V82I

GTC to ATC

V82A

GTC to GCC

L63P

CTC to CCC

A71T

GCT to ACT

A71V

GCR to GTT

G73S

GGT to GCT

V82A

GTC to GCC

V82F

GTC to TTC

V82T

GTC to ACC

I84V

ATA to GTA

L90M

TTG to ATG

P9941

V82A

GTC to GCC

Ro 31-8959

L10I

CTC to ATC

(saquinavir)

G48V

GGG to GTG

I54V

ATC to GTC

I54V

ATA to GTA

G73S

GGT to AGT

V82A

GTC to GCC

I84V

ATA to GTA

L90M

TTG to ATG

RPI-312

I84V

ATA to GTA

SC-52151

L24V

TTA to GTA

G48V

GGG to GTG

A71V

GCT to GTT

V75I

GTA to ATA

P81T

CCT to ACT

V82A

GTC to GCC

N88D

AAT to GAT

SC-55389A

L10F

CTC to CGC

N88S

AAT to AGT

SKF108842

V82T

GTC to ACC

I84V

ATA to GTA

SKF108922

V82A

GTC to GCC

V82T

GTC to ACC

VB 11,328

L10F

CTC to GGC

M46I

ATG to ATA

I47V

ATA to CTA

I50V

ATT to GTT

184V

ATA to GTA

VX-478

L10F

CTC to CGC

(141W94)

M46I

ATG to ATA

I47V

ATA to CTA

I50V

ATT to GTT

I84V

ATA to GTA

XM323

L10F

CTC to CGC

K45I

AAA to ATA

M46L

ATG to CTG

V82A

GTC to GCC

V82I

GTC to ATC

V82F

GTC to TTC

I84V

ATA to GTA

L97V

TTA to GTA

I82T

ATC to ACC

A-75925

V32I

GTA to ATA

ABT-538

K20R

AAG to AAA

(ritonavir)

L33F

TTA to TTC

M36I

ATG to ATA

M46I

ATG to ATA

I54L

ATC to ?

I54V

ATC to GTC

A71V

GTC to GTT

V82F

GTC to TTC

V82A

GTC to GCC

V82T

GTC to ACC

V82S

GTC to TCC

I84V

ATA to GTA

L90M

TTG to ATG

AG1343

D30N

GAT to AAT

(nelfinavir)

M36I

M46I

ATG to ATA

L63P

CTC to CCC

A71V

GCT to GTT

V771

184V

ATA to GTA

N88D

L90M

TTG to ATG

BILA 1906

V32I

GTA to ATA

BS

M46I

ATG to ATA

M46L

ATG to TTG

A71V

GCT to CTT

I84A

ATA to GCA

184V

ATA to GTA

BILA 2011

V32I

GTA to ATA

(palinavir)

A71V

GCT to GTT

I84A

ATG to ATA

L63P

CTC to CCC

BILA 2185 BS

L23I

CTA to ATA

BMS 186,318

A71T

GCT to ACT

V82A

GTC to GCC

DMP450

L10F

CTC to TTC

M46I

ATG to ATA

D60E

GAT to GAA

I84V

ATA to GTA

KNI-272

V32I

GTA to ATA

MK-639

L10I

CTC to ATC

(L-735, 524,

L10R

CTC to CGC

indinavir)

L10V

CTC to GTC

K20M

AAG to ATG

K20R

AAG to AAA

L24I

TTA to ATA

V32I

GTA to ATA

M46I

ATG to ATA

M46L

ATG to TTG

I54V

ATC to GTC

TABLE 3

26

27

28

29

30

31

32

33

34

35

length

Seq ID

ACA

GGA

GCA

GAT

GAT

ACA

GTA

TTA

GAA

GAA

P30w1

A

GCA

GAT

GAT

ACA

GTA

TT

18

7

P30w2

GA

GCA

GAT

GAT

ACA

GTA

TT

19

8

P30w3

A

GCA

GAT

GAT

ACA

GTA

TTA

19

9

P30w4

GGA

GCA

GAT

GAT

ACA

GTA

TT

20

10

P30w5

GGA

GCA

GAT

GAT

ACA

GTA

TTA

21

11

P30w6

ACA

GGA

GCA

GAT

GAT

ACA

18

12

P30w7

CA

GGA

GCA

GAT

GAT

ACA

GT

19

13

P30w8

A

GGA

GCA

GAT

GAT

ACA

GTA

TG

20

14

P30w9

GGA

GCA

GAT

GAT

ACA

GTA

TG

19

15

P30w10

ACA

GGA

GCA

GAT

GAT

ACA

GG

19

16

P30m11

A

GCA

GAT

AAT

ACA

GTA

TT

18

17

P30m12

GA

GCA

GAT

AAT

ACA

GTA

TT

19

18

P30m13

A

GCA

GAT

AAT

ACA

GTA

TTA

19

19

P30m14

GGA

GCA

GAT

AAT

ACA

GTA

TT

20

20

P30m15

GGA

GCA

GAT

AAT

ACA

GTA

TTA

21

21

P30m15

ACA

GGA

GCA

GAT

AAT

ACA

18

22

P30m17

CA

GGA

GCA

GAT

AAT

ACA

GT

19

23

P30m18

A

GGA

GCA

GAT

AAT

ACA

GTA

TG

20

24

P30m19

GGA

GCA

GAT

AAT

ACA

GTA

TG

19

25

P30m20

ACA

GGA

GCA

GAT

AAT

ACA

GG

19

26

p30w21

A

GCA

GAT

GAT

ACA

GT

15

27

p30w22

A

GCA

GAT

GAT

ACA

GTA

G

16

28

p30m23

A

GCA

GAT

AAT

ACA

GTA

15

29

p30m24

A

GCA

GAT

AAT

ACA

GTA

G

16

30

p30w25

GCA

GAT

GAT

ACA

GT

14

31

p30w26

A

GCA

GAT

GAT

ACA

GG

14

32

p30w27

CA

GAT

GAT

ACA

GT

13

33

p30w28

GA

GCG

GAT

GAT

ACA

14

34

p30w29

A

GCG

GAT

GAT

ACA

13

35

p30m30

GCA

GAT

AAT

ACA

GTA

15

36

p30m31

GCA

GAT

AAT

ACA

GT

14

37

p30w32

GCA

GAT

GAC

ACA

GT

14

38

p30w33

CA

GAT

GAC

ACA

GTA

G

14

39

p30w34

CA

GAT

GAT

ACA

ATA

TT

16

40

p30w35

GCA

GAT

GAT

ACA

ATA

TG

16

41

p30w36

GCA

GAC

GAT

ACA

GG

13

42

p30w37

GCA

GAC

GAT

ACA

GT

14

43

p30w38

A

GAT

GAT

ACA

ATA

TT

15

44

p30w39

A

GAT

GAT

ACA

ATA

TTA

16

45

p30w40

GCA

GAT

GAT

ACA

ATA

15

46

44

45

46

47

48

49

50

51

52

53

54

length

Seq ID

CCA

AAA

ATG

ATA

GGG

GGA

ATT

GGA

GGT

TTT

ATC

P48w1

GTA

GGG

GGA

ATT

GGA

GGT

GG

18

47

P48w2

GTA

GGG

GGA

ATT

GGA

GGT

TG

19

48

P48w3

GTA

GGG

GGA

ATT

GGA

GGT

TTG

20

49

P48w4

GTA

GGG

GGA

ATT

GGA

GGT

TTT

21

50

P48w5

G

GTA

GGG

GGA

ATT

GGA

GGT

TTG

21

51

P48w6

ATG

GTA

GGG

GGA

ATT

GGA

18

52

P48w7

ATG

GTA

GGG

GGA

ATT

GGA

G

19

53

P48w8

A

ATG

GTA

GGG

GGA

ATT

GGA

19

54

P48w9

A

ATG

GTA

GGG

GGA

ATT

GGA

G

20

55

P48w10

A

ATG

GTA

GGG

GGA

ATT

GGA

GGG

GG

22

56

P48w21

ATA

ATA

GGG

GGA

ATT

GGA

18

57

P48w22

ATG

ATA

GGG

GGA

ATT

GGA

18

58

P48w23

A

ATA

ATA

GGG

GGA

ATT

GGA

19

59

P48w24

A

ATG

ATA

GGG

GGA

ATT

GGA

19

60

P48w25

ATA

GGG

GGA

ATT

GGA

GGT

GG

18

61

P48w26

ATA

GGG

GGA

ATT

GGA

GGT

TG

19

62

P48w28

ATA

GGG

GGA

ATT

GGA

GGT

TTG

20

63

P48w29

ATA

GGG

GGA

ATT

GGA

GGT

TTT

21

64

P48m11

GTA

GTG

GGA

ATT

GGA

GGT

GG

18

65

P48m12

GTA

GTG

GGA

ATT

GGA

GGT

TG

19

66

P48m13

GTA

GTG

GGA

ATT

GGA

GGT

TTG

20

67

P48m14

GTA

GTG

GGA

ATT

GGA

GGT

TTT

21

68

P48m15

G

GTA

GTG

GGA

ATT

GGA

GGT

TTG

21

69

P48m16

ATG

GTA

GTG

GGA

ATT

GGA

18

70

P48m17

ATG

GTA

GTG

GGA

ATT

GGA

G

19

71

P48m18

A

ATG

GTA

GTG

GGA

ATT

GGA

19

72

P48m19

A

ATG

GTA

GTG

GGA

ATT

GGA

G

20

73

P48m20

A

ATG

GTA

GTG

GGA

ATT

GGA

GGG

GG

22

74

P48m29

ATA

GTG

GGA

ATT

GGA

GGT

GG

18

75

P48m30

ATA

GTG

GGA

ATT

GGA

GGT

TG

19

76

P48m31

ATG

ATA

GTG

GGA

ATT

GGA

18

77

P48m32

ATG

ATA

GTG

GGA

ATT

GGA

G

19

78

P48m33

A

ATG

ATA

GTG

GGA

ATT

GGA

19

79

p48w34

G

ATA

GGG

GGA

ATT

G

14

80

p48w35

TG

ATA

GGG

GGA

ATT

G

15

81

p48w36

TG

ATA

GGG

GGA

ATT

GG

16

82

p48w37

ATG

ATA

GGG

GGA

ATT

15

83

p48m38

G

ATA

GTG

GGA

ATT

G

14

84

p48m39

TG

ATA

GTG

GGA

ATT

G

15

85

p48m40

TG

ATA

GTG

GGA

ATT

GG

16

86

p48m41

ATG

ATA

GTG

GGA

ATT

15

87

p48w42

ATA

ATA

GGG

GGA

ATT

15

88

p48w43

TG

ATA

GGG

GGA

GTT

14

89

p48w44

G

ATA

GGG

GGA

GTT

G

14

90

p48w45

A

ATG

ATA

GGA

GGA

ATT

16

91

p48w46

ATG

ATA

GGG

GGA

ATT

15

92

p48w47

AAA

ATG

ATA

GGG

GGA

15

93

p48w48

A

AAA

ATG

ATA

GGG

GG

15

94

p48w49

AA

ATG

ATA

GGG

GGA

AG

15

95

p48w50

AAA

ATA

ATA

GGG

GGA

AG

16

96

p48w51

AAA

ATA

AAA

AT

15

97

p48m52

AAA

ATG

ATA

GTG

GGA

AG

16

98

p48w52b

AAA

TTG

ATA

GGG

GG

14

99

p48m53

AAA

ATG

ATA

GTG

GGA

15

100

p48w53b

AAA

TTG

ATA

GGG

GGA

15

101

p48w54

CA

AAA

TTG

ATA

G

15

102

p48w55

ATG

GTA

GGG

GGA

ATT

15

103

p48w56

AA

ATG

GTA

GGG

GGA

14

104

p48w57

A

AAA

ATG

GTA

GGG

G

14

105

p48w58

ATG

ATA

GGG

GAA

ATT

15

106

p48w59

ATA

GGG

GAA

ATT

GGA

15

107

p48w60

ATA

GGG

GAA

ATT

GGA

G

16

108

p48w61

ATG

ATA

GGG

GGG

ATT

15

109

p48w62

ATA

GGG

GGG

ATT

GG

14

110

p48w63

A

GGG

GGG

ATT

GGA

13

111

p48m64

AAA

ATA

ATA

GTG

GGA

15

112

p48m65

A

AAA

ATA

ATA

GTG

GGA

16

113

p48m66

CA

AAA

ATA

ATA

GTG

GG

16

114

p48m67

AAA

TTG

ATA

GTG

GGA

15

115

p48m68

A

AAA

TTG

ATA

GTG

GGA

16

116

p48m69

CA

AAA

TTG

ATA

GTG

G

15

117

p48w70

AAA

ATG

ATA

GGG

GG

14

118

p48w71

A

AAA

ATG

ATA

GGG

G

14

119

pc48w72

A

AAA

ATA

ATA

GGG

GGA

16

120

45

46

47

48

49

50

51

52

53

54

length

Seq ID

AAA

ATG

GTA

GGG

GGA

ATT

GGA

GGT

TTT

ATC

P50w1

GGG

GGA

ATT

GGA

GGT

TTT

18

121

P50w2

A

GGG

GGA

ATT

GGA

GGT

TTT

19

122

P50w3

TA

GGG

GGA

ATT

GGA

GGT

TTT

20

123

P50w4

A

GGG

GGA

ATT

GGA

GGT

TTT

AG

20

124

P50w5

TA

GGG

GGA

ATT

GGA

GGT

TTT

AG

21

125

P50w6

GTA

GGG

GGA

ATT

GGA

GGT

TGG

19

126

P50w7

G

GTA

GGG

GGA

ATT

GGA

GGT

TGG

20

127

P50w8

GTA

GGG

GGA

ATT

GGA

GGT

TTG

20

128

P50w9

GTA

GGG

GGA

ATT

GGA

GGT

TTT

20

129

P50w10

TG

GTA

GGG

GGA

ATT

GGA

GGT

GG

20

130

p50w21

GG

GGA

ATT

GGA

GGT

TTT

17

131

P50w22

GG

GGA

ATT

GGA

GGT

TTG

16

132

P50w23

GG

GGA

ATT

GGA

GGT

TTT

AG

18

133

P50w24

GG

GGA

ATT

GGA

GGT

TG

15

134

P50w25

G

GGA

ATT

GGA

GGT

TTT

AT

18

135

P50w26

GG

GGA

ATT

GGA

GGT

TTT

17

136

P50m11

GGG

GGA

GTT

GGA

GGT

TTT

18

137

P50m12

A

GGG

GGA

GTT

GGA

GGT

TTT

19

138

P50m13

TA

GGG

GGA

GTT

GGA

GGT

TTT

20

139

P50m14

A

GGG

GGA

GTT

GGA

GGT

TTT

AG

20

140

P50m15

TA

GGG

GGA

GTT

GGA

GGT

TTT

AG

21

141

P50m16

GTA

GGG

GGA

GTT

GGA

GGT

TGG

19

142

P50m17

G

GTA

GGG

GGA

GTT

GGA

GGT

TGG

20

143

P50m18

GTA

GGG

GGA

GTT

GGA

GGT

TTG

20

144

P50m19

GTA

GGG

GGA

GTT

GGA

GGT

TTT

ATC

21

145

P50m20

TG

GTA

GGG

GGA

GTT

GGA

GGT

GG

20

146

P50m27

GG

GGA

GTT

GGA

GGT

TTG

19

147

P50m28

GG

GGA

GTT

GGA

GGT

TTT

AG

18

148

P50m29

GG

GGA

GTT

GGA

GGT

TG

15

149

P50m30

G

GGA

GTT

GGA

GGT

TTT

AT

18

150

p50w31

GGA

ATT

GGA

GGT

TTT

15

151

p50w32

G

GGA

ATT

GGA

GGT

TGG

15

152

p50m33

GGA

GTT

GGA

GGT

TTT

15

153

p50m34

G

GGA

GTT

GGA

GGT

TGG

14

154

p50m35

GGG

GGA

GTT

GGA

G

13

155

p50m36

GG

GGA

GTT

GGA

G

12

156

p50m37

GGG

GGA

GTT

GGA

12

157

p50w38

GGA

ATT

GGG

GGT

TTG

14

158

p50w39

GA

ATT

GGG

GGT

TTT

14

159

p50w40

GA

ATT

GGG

GGT

TTT

AG

15

160

p50w41

GGA

ATT

GGG

GGT

TG

13

161

p50w42

GGA

ATT

GGG

GGT

G

12

162

p50w43

GA

ATT

GGG

GGT

TG

12

163

p50w44

GA

ATT

GGG

GGT

TTG

13

164

p50w45

GGG

GGA

ATT

GCA

G

13

165

p50w46

GGA

ATT

GCA

GGT

TG

14

166

p50w47

GGA

ATT

GCA

GGT

G

13

167

p50w48

GGA

ATT

GGA

GGG

TTG

14

168

p50w49

GA

ATT

GGA

GGG

TTG

13

169

p50w50

GA

ATT

GGA

GGG

TTT

14

170

p50w51

GGA

ATT

GGA

GGC

TTG

14

171

p50w52

GA

ATT

GGA

GGC

TTG

13

172

p50w53

GA

ATT

GGA

GGC

TTT

14

173

p50m54

GGA

GTT

GGA

GGT

TTG

15

174

p50m55

GA

GTT

GGA

GGT

TTT

14

175

51

52

53

54

55

56

57

58

length

Seq ID

GGA

GGT

TTT

ATC

AAA

GTA

AGA

CAG

p54w1

GGT

TTT

ATC

AAA

GTA

A

16

176

p54w2

GT

TTT

ATC

AAA

GTA

AG

16

177

p54w3

GT

TTT

ATC

AAA

GTA

AGA

17

178

p54w4

T

TTT

ATC

AAA

GTA

AGA

16

179

p54w5

GGT

TTT

ATC

AAA

GTA

15

180

p54w6

GT

TTT

ATC

AAA

GTA

15

181

p54m7

GGT

TTT

GCC

AAA

GTA

15

182

p54m8

GT

TTT

GCC

AAA

GTA

A

15

183

p54m9

GT

TTT

GCC

AAA

GTA

AG

16

184

p54m10

T

TTT

GCC

AAA

GTA

AGA

16

185

p54m11

GGT

TTT

GCC

AAA

GT

14

186

p54m12

GT

TTT

GCC

AAA

GTA

14

187

p54w13

GT

TTT

ATC

AAG

GTA

AA

16

188

p54w14

GGT

TTT

ATC

AAG

GTA

A

16

189

p54w15

A

GGT

TTT

ATC

AAG

GTA

16

190

p54w16

GT

TTT

ATC

AAA

GTC

AGA

17

191

p54w17

TTT

ATC

AAA

GTC

AGA

C

16

192

p54w18

A

GGC

TTT

ATC

AAA

GTA

A

17

193

p54w19

A

GGC

TTT

ATC

AAA

GTA

16

194

p54m20

A

GGT

TTT

ATT

AAA

GTA

A

17

195

p54m21

GGT

TTT

ATT

AAA

GTA

AG

17

196

p54w22

GA

GGT

TTT

ATT

AAA

GTA

17

197

p54m22

GA

GGT

TTT

ATT

AAA

GTA

17

198

p54m23

GGT

TTT

ATT

GGT

TTT

AT

16

199

p54m24

GGT

TTC

ATT

AAG

GTA

15

200

p54m25

GGT

TTC

ATT

AAG

GTA

A

16

201

p54w26

A

GGT

TTC

ATT

AAG

GTA

16

202

p54m26

A

GGT

TTC

ATT

AAG

GTA

16

203

p54w27

GGT

TTT

ATT

AAG

GTA

A

16

204

p54m27

GGT

TTT

ATT

AAG

GTA

A

16

205

p54m28

A

GGT

TTT

ATT

AAG

GTA

16

206

p54m29

GA

GGT

TTT

ATT

AAG

GT

16

207

p54m30

GGT

TTT

ATT

AAG

GTA

AG

17

208

p54w31

GGT

TTT

ATC

AAA

GTA

A

16

209

p54w32

A

GGT

TTT

ATC

AAA

GTA

A

17

210

p54w33

A

GGT

TTT

ATC

AAA

GTA

16

211

p54w34

GA

GGT

TTT

ATC

AAA

GT

16

212

p54m35

GGT

TTT

GTC

AAA

GTA

15

213

p54m36

GGT

TTT

GTC

AAA

GTA

A

16

214

p54m37

GGT

TTT

GTC

AGA

GTA

15

215

p54m38

GGT

TTT

GTC

AGA

GTA

A

16

216

p54w39

GGG

TTT

ATC

AAA

GTA

15

217

p54w40

GGG

TTT

ATC

AAA

GTA

A

16

218

p54w41

GGC

TTC

ATC

AAA

GT

14

219

p54w42

GA

GGC

TTC

ATC

AAA

14

220

p54m48

GGT

TTT

GTC

AAA

GT

14

221

p54m49

GT

TTT

GTC

AGA

GTA

14

222

p54m50

GGT

TTT

GTC

AGA

GT

14

223

p54w51

A

GGT

TTA

ATC

AAA

GTA

16

224

p54w52

GA

GGT

TTA

ATC

AAA

GT

16

225

p54m53

GGT

TTT

ACC

AAA

GTA

15

226

p54m54

GGT

TTT

ACC

AAA

GT

14

227

78

79

80

81

82

83

84

85

86

87

length

Seq ID

GGA

CCT

ACA

CCT

GTC

AAC

ATA

ATT

GGA

AGA

P82w1

CCT

ACA

CCT

GTC

AAC

ATA

AG

19

228

P82w2

CCT

ACA

CCT

GTC

AAC

ATA

ATG

20

229

P82w3

CCT

ACA

CCT

GTC

AAC

ATA

ATT

21

230

P82w4

A

CCT

ACA

CCT

GTC

AAC

ATA

AG

20

231

P82w5

A

CCT

ACA

CCT

GTC

AAC

ATA

ATG

21

232

P82w6

A

CCT

ACA

CCT

GTC

AAC

ATA

19

233

P82w7

GA

CCT

ACA

CCT

GTC

AAC

ATA

20

234

P82w8

CA

CCT

GTC

AAC

ATA

ATT

GGA

20

235

P82w9

A

CCT

GTC

AAC

ATA

ATT

GGA

A

20

236

P82w10

ACA

CCT

GTC

AAC

ATA

ATT

GG

20

237

P82W21

A

CCT

GTC

AAC

ATA

ATT

GGA

19

238

P82m11

CCT

ACA

CCT

ACC

AAC

ATA

AG

19

239

P82m12

CCT

ACA

CCT

ACC

AAC

ATA

ATG

20

240

P82m13

CCT

ACA

CCT

ACC

AAC

ATA

ATT

21

241

P82m14

A

CCT

ACA

CCT

ACC

AAC

ATA

AG

20

242

P82m15

A

CCT

ACA

CCT

ACC

AAC

ATA

ATG

21

243

P82m16

A

CCT

ACA

CCT

ACC

AAC

ATA

19

244

P82m17

GA

CCT

ACA

CCT

ACC

AAC

ATA

20

245

P82m18

CA

CCT

ACC

AAC

ATA

ATT

GGA

20

246

P82m19

A

CCT

ACC

AAC

ATA

ATT

GGA

A

20

247

P82m20

ACA

CCT

ACC

AAC

ATA

ATT

G

19

248

P82m22

CCT

ACA

CCT

TTC

AAC

ATA

ATT

21

249

P82m23

CCT

ACA

CCT

GCC

AAC

ATA

ATT

21

250

P82m24

CCT

ACA

CCT

TCC

AAC

ATA

ATT

21

251

P82m25

A

CCT

TTC

AAC

ATA

ATT

GGA

A

20

252

P82m26

A

CCT

GCC

AAC

ATA

ATT

GGA

A

20

253

P82m27

A

CCT

TTC

AAC

ATA

ATT

GGA

A

20

254

P82m28

A

CCT

ACC

AAC

ATA

ATT

16

255

P82m29

A

CCT

TTC

AAC

ATA

ATT

GGA

19

256

P82m30

A

CCT

GCC

AAC

ATA

ATT

GGA

19

257

P82m31

A

CCT

TCC

AAC

ATA

ATT

GGA

19

258

P82w32

T

ACA

CCT

GTC

AAC

AT

15

259

P82w33

T

ACA

CCT

GTC

AAC

ATA

16

260

P82w34

ACA

CCT

GTC

AAC

ATA

15

261

P82w35

CA

CCT

GTC

AAC

ATA

14

262

P82m36

ACA

CCT

ACC

AAC

ATA

15

263

P82m37

CA

CCT

ACC

AAC

ATA

14

264

P82m38

ACA

CCT

TTC

AAC

ATA

15

265

P82m39

CA

CCT

TTC

AAC

ATA

14

266

P82m40

ACA

CCT

GCC

AAC

ATA

15

267

P82m41

CA

CCT

GCC

AAC

ATA

14

268

P82w42

CA

CCT

GTC

AAC

GTA

14

269

P82w43

CA

CCT

GTC

AAC

GT

13

270

P82w44

CCT

ACA

CCT

GTC

AAC

15

271

P82w45

T

ACG

CCT

GTC

AAC

AT

15

272

P82w46

CT

ACG

CCT

GTC

AAC

AG

15

273

P82m47

ACA

CCT

TCC

AAC

ATA

15

274

P82m48

CA

CCT

TCC

AAC

ATA

14

275

P82m49

ACA

CCT

TCC

AAC

AT

14

276

P82m50

ACA

CCT

ATC

AAC

ATA

15

277

P82m51

CA

CCT

ATC

AAC

ATA

AG

15

278

P82m52

CA

CCT

ATC

AAC

ATA

ATG

16

279

P82m53

A

CCT

ATC

AAC

ATA

ATG

15

280

P82w54

CCT

GTC

AAC

ATA

ATT

15

281

P82w55

CCT

GTT

AAC

ATA

ATT

G

16

282

P82w56

A

CCT

GTT

AAC

ATA

ATG

15

283

P82w57

CCG

GTC

AAC

ATA

ATT

15

284

P82w58

ACG

CCT

GTC

AAC

AT

14

285

P82w59

CCT

GTC

AAT

ATA

ATT

15

286

P82w60

CA

CCT

GTC

AAT

ATA

ATG

16

287

P82w61

ACA

CCT

GTC

AAT

ATA

AG

16

288

P82m62

CCT

GCC

AAT

ATA

ATT

15

289

P82m63

CA

CCT

GCC

AAT

ATA

AG

15

290

P82m64

CCT

ACC

AAC

GTA

ATT

15

291

P82m65

CCT

ACC

AAC

GTA

ATG

14

292

P82m66

CA

CCT

ACC

AAC

GTA

14

293

P82m67

ACA

CCT

ACC

AAC

GT

14

294

P82m68

CCT

TTC

AAC

GTA

ATT

15

295

P82m69

CA

CCT

TTC

AAC

GTA

AG

15

296

P82m70

ACA

CCT

TTC

AAC

GTA

15

297

P82m71

A

CCT

TTC

AAC

GTA

ATG

15

298

p82w72

CT

GTC

AAT

ATA

ATT

G

15

299

p82w73

CCT

GTC

AAT

ATA

ATT

G

16

300

p82w74

A

CCT

GTC

AAT

ATA

ATT

16

301

p82w75

CT

GTC

AAT

ATA

ATT

GG

16

302

p82w76

CCT

ACG

CCT

GTC

AA

14

303

p82w77

CT

ACG

CCT

GTC

AAC

14

304

p82w78

A

CCT

ACG

CCT

GTC

AA

15

305

p82w79

A

CCT

ACG

CCT

GTC

A

14

306

p82w80

T

ACA

CCG

GTC

AAC

A

14

307

p82w81

CT

ACA

CCG

GTC

AA

13

308

p82w82

CCT

ACA

CCG

GTC

A

13

309

p82w83

CA

CCT

GTC

AAC

ATA

A

15

310

p82w84

A

CCT

GTC

AAC

ATA

AT

15

311

p82w85

CT

ACA

CCT

GTC

AAC

A

15

312

p82w86

ACA

CCT

GTC

AAC

AT

14

313

p82w87

A

CCT

GTT

AAC

ATA

ATT

G

17

314

p82w88

CA

CCT

GTT

AAC

ATA

AG

15

315

p82w89

ACA

CCT

GTT

AAC

ATA

AG

16

316

p82w90

TCA

CCT

GTC

AAC

ATA

14

317

p82w91

ACA

CCT

GTC

AAC

ATA

A

16

318

p82w92

CA

CCT

GTC

AAC

ATA

AT

16

319

p82w93

CCT

GTC

AAC

ATA

ATT

15

320

p82w94

A

CCT

GTC

AAC

ATA

ATT

16

321

p82w95

CCT

GTC

AAC

ATA

ATT

G

16

322

p82w96

CCT

ACA

CCT

GTC

AA

14

323

p82w97

T

GTC

AAC

ATA

ATT

GG

15

324

p82w98

T

GTC

AAC

ATA

ATT

GGA

16

325

p82m99

ACA

CCT

TTC

AAC

ATA

A

16

326

p82m100

T

ACA

CCT

TTC

AAC

ATA

16

327

p82m101

ACA

CCT

ATC

AAC

ATA

ATG

17

328

p82m102

ACA

CCT

ATC

AAC

ATA

AG

16

329

p82m103

CA

CCT

GCC

AAT

ATA

ATG

16

330

p82m104

ACA

CCT

GCC

AAT

ATA

AG

16

331

p82m105

ACG

CCC

TTC

AAC

ATA

15

332

p82m106

CG

CCC

TTC

AAC

ATA

AG

15

333

p82m107

T

ACG

CCC

TTC

AAC

AT

15

334

p82w108

CT

ACA

CCG

GTC

AAC

14

335

p82w109

CCT

ACA

CCG

GTC

AA

14

336

p82w110

A

CCG

GTC

AAC

ATA

ATG

15

337

p82w111

A

CCG

GTC

AAC

ATA

ATT

16

338

p82w112

CT

ACA

CCA

GTC

AAC

14

339

p82w113

CT

ACA

CCA

GTC

AAC

A

15

340

p82w114

ACA

CCA

GTC

AAC

ATA

15

341

p82w115

ACA

CCA

GTC

AAC

ATA

AG

16

342

p82w116

T

ACG

CCT

GTC

AAC

AT

15

343

p82w117

ACG

CCT

GTC

AAC

ATA

15

344

p82w118

T

ACG

CCT

GTC

AAC

A

14

345

p82m119

CCT

ACA

CCT

TTC

AAC

15

346

p82m120

CT

ACA

CCT

TTC

AAC

14

347

p82m121

A

CCT

ACA

CCT

TTC

AA

15

348

p82w122

ACG

CCT

GTC

AAC

ATA

AGG

16

349

p82w123

T

ACG

CCT

GTC

AAC

ATA

16

350

p82w124

CG

CCT

GTC

AAC

ATA

AGG

15

351

p82m125

T

ACA

CCT

TTC

AAC

GTA

16

352

p82m126

ACA

CCT

TTC

AAC

GTA

AGG

16

353

p82m127

CA

CCT

TTC

AAC

GTA

ATG

16

354

p82m128

A

CCT

TTC

AAC

GTA

ATT

16

355

p82o129

C

AAC

GTA

ATT

GGA

AGA

16

356

p82o130

C

AAC

GTA

ATT

GGA

AG

15

357

86

87

88

89

90

91

92

93

94

length

Seq ID

GGA

AGA

AAT

CTG

TTG

ACT

CAG

ATT

GGT

P90w1

A

AAT

CTG

TTG

ACT

CAG

16

358

P90w2

GA

AAT

CTG

TTG

ACT

CAG

17

359

P90w3

GA

AAT

CTG

TTG

ACT

CAG

AGG

18

360

P90w4

A

AAT

CTG

TTG

ACT

CAG

AGG

17

361

P90w5

AGA

AAT

CTG

TTG

ACT

CAG

AGG

19

362

P90w6

AGA

AAT

CTG

TTG

ACT

CAG

ATG

20

363

P90w7

AGA

AAT

CTG

TTG

ACT

CAG

ATT

21

364

P90w8

AGA

AAT

CTG

TTG

ACT

CAG

ATT

GG

20

365

P90w9

GA

AGA

AAT

CTG

TTG

ACT

CAG

AGG

21

366

P90w10

A

AGA

AAT

CTG

TTG

ACT

CAG

ATG

21

367

P90m11

AGA

AAT

CTG

ATG

ACT

CAG

ATG

20

368

P90m12

AGA

AAT

CTG

ATG

ACT

CAG

ATT

21

369

P90m13

A

AGA

AAT

CTG

ATG

ACT

CAG

AGG

20

370

P90m14

GA

AGA

AAT

CTG

ATG

ACT

CAG

AGG

21

371

P90m15

A

AGA

AAT

CTG

ATG

ACT

CAG

ATG

21

372

P90m16

GA

AGA

AAT

CTG

ATG

ACT

CAG

ATT

20

373

P90m17

GGA

AGA

AAT

CTG

ATG

ACT

CAG

21

374

P90m18

A

AGA

AAT

CTG

ATG

ACT

CAG

19

375

P90m19

A

AAT

CTG

ATG

ACT

CAG

ATT

GG

21

376

P90m20

A

AAT

CTG

ATG

ACT

CAG

ATT

G

20

377

P90m21

A

AAT

CTG

ATG

ACT

CAG

CTT

G

20

378

P90m22

A

AAT

CTG

ATG

ACT

CAG

CTT

19

379

P90m23

AAT

CTG

ATG

ACT

CAG

CTT

G

18

380

P90w24

A

AAT

CTG

TTG

ACT

CAG

CTT

G

20

381

P90w25

A

AAT

CTG

TTG

ACT

CAG

CTT

19

382

P90w26

AAT

CTG

TTG

ACT

CAG

CTT

G

19

383

P90w27

AAT

CTG

TTG

ACT

CA

14

384

P90w28

AAT

CTG

TTG

ACT

CAG

15

385

P90w29

A

AAT

CTG

TTG

ACT

CA

15

386

P90w30

A

AAT

CTG

TTG

ACT

CAG

16

387

P90m31

AAT

CTG

ATG

ACT

CA

14

388

P90m32

AAT

CTG

ATG

ACT

CAG

15

389

P90m33

A

AAT

CTG

ATG

ACT

CA

15

390

P90m34

A

AAT

CTG

ATG

ACT

CAG

16

391

P90w35

GA

AAT

CTG

TTG

ACT

C

15

392

P90w36

GA

ACT

CTG

TTG

ACT

C

15

393

P90w37

T

CTG

TTG

ACT

CAG

ATG

15

394

P90w38

GA

AAT

CTG

TTG

ACT

C

15

395

P90w39

GA

ACT

CTG

TTG

ACT

C

15

396

P90w40

A

AAT

CTG

TTG

ACT

CA

15

397

P90w41

AAT

CTG

TTG

ACT

CAG

15

398

P90m42

AAT

CTG

ATG

ACT

CAG

15

399

P90m43

A

AAT

CTG

ATG

ACT

CA

15

400

P90w44

AT

CTG

TTG

ACT

CAG

AG

15

401

P90w45

CTG

TTG

ACT

CAG

ATT

15

402

P90w46

AGA

AAT

CTG

TTG

ACT

15

403

P90m47

AT

CTG

ATG

ACT

CAG

AG

15

404

P90m48

CTG

ATG

ACT

CAG

ATT

15

405

P90m49

AGA

AAT

CTG

ATG

ACT

CA

17

406

P90w50

AAT

ATG

TTG

ACT

CAG

15

407

P90w51

GA

AAT

ATG

TTG

ACT

CA

16

408

P90w52

AAT

TTG

TTG

ACT

CAG

15

409

P90w53

GA

AAT

TTG

TTG

ACT

CA

16

410

P90w54

AAT

ATG

TTG

ACC

CAG

15

411

P90w55

A

AAT

ATG

TTG

ACC

CA

15

412

P90m56

AAT

ATG

ATG

ACC

CAG

15

413

P90m57

A

CAG

ATG

ATG

ACC

CA

15

414

P90w58

AAC

ATG

TTG

ACT

CAG

15

415

P90w59

A

AAC

ATG

TTG

ACT

CAG

15

416

P90w60

TG

TTG

ACT

CAG

CTT

14

417

P90w61

CTG

TTG

ACT

CAG

CTG

14

418

P90m62

CT

ATG

ACT

CAG

CTT

14

419

P90m63

CTG

ATG

ACT

CAG

C-G

14

420

P90w64

TG

ACT

ACA

CAG

CTT

14

421

P90w65

CTG

TTG

ACA

CAG

C-G

14

422

P90w66

AAT

CTG

TTG

ACA

CAG

15

423

P90w67

AAC

CTG

TTG

ACT

CA

13

424

P90w68

A

AAC

CTG

TTG

ACT

C

13

425

P90w69

GA

AAC

CTG

TTG

ACT

13

426

p90w70

TG

TTG

ACT

CAG

ATT

G

15

427

p90w71

TG

TTG

ACT

CAG

ATT

GGG

16

428

p90w72

G

TTG

ACT

CAG

ATT

GGG

15

429

p90w73

TG

TTG

ACA

CAG

CTT

G

15

430

p90w74

CTG

TTG

ACA

CAG

CTT

15

431

p90w75

G

TTG

ACA

CAG

CTT

GGG

15

432

p90w76

TG

TTG

ACT

CAG

CTT

G

15

433

p90w77

G

TTG

ACT

CAG

ATG

15

434

p90w78

G

TTG

ACT

CAG

CTT

G

14

435

p90w79

TG

TTG

ACC

CAG

ATT

G

15

436

p90w80

G

TTG

ACC

CAG

ATT

G

14

437

p90w81

G

TTG

ACC

CAG

ATT

GGG

15

438

p90m82

TG

ATG

ACT

CAG

ATT

G

15

439

p90m83

TG

ATG

ACT

CAG

ATT

GGG

16

440

p90m84

G

ATG

ACT

CAG

ATT

GGG

15

441

p90m85

G

ATG

ACT

CAG

ATT

GGT

16

442

p90m86

CTG

ATG

ACT

CAG

CTT

15

443

p90m87

TG

ATG

ACT

CAG

CTT

G

15

444

P90w88

A

AAT

CTG

TTG

ACT

CA

15

445

P90w89

A

AAT

CTG

TTG

ACT

CA

15

446

p90w90

A

AAT

CTG

TTG

ACT

CA

15

447

p90w100

AAT

CTG

ATG

ACT

CAG

15

448

p90m92

A

AAT

CTG

ATG

ACT

CA

16

449

p90m93

GA

AAT

CTG

ATG

ACT

C

15

450

p90m94

CTG

ATG

ACT

CAG

ATG

15

451

p90m95

AGA

AAT

ATG

ATG

15

452

p90m96

A

AGA

AAT

ATG

ATG

ACT

16

453

p90m97

A

AGA

AAT

CTG

ATG

ACT

16

454

p90m98

A

AGA

AAT

ATA

ATG

ACT

16

455

p90m99

A

AAT

ATA

ATG

ACT

CAG

16

456

p90m100

AAT

ATG

ATG

ACC

CAG

15

457

p90m101

AAC

CTG

ATG

ACT

CAG

15

458

p90m102

AGA

AAT

TTG

ATG

ACT

C

16

459

p90m103

A

AAT

TTG

ATG

ACT

ATG

ACT

16

460

p90m104

AC

CTG

ATG

ACT

CAG

14

461

p90m105

AAT

CTG

ATG

ACT

CAG

A

16

462

p90m106

AT

CTG

ATG

ACT

CAG

ATG

16

463

p90m107

AT

CTG

ATG

ACT

CAG

14

464

p90m108

CTG

ATG

ACT

CAG

ATT

G

16

465

p90m109

AGA

AAT

CTG

ATG

ACT

C

16

466

p90m110

AGA

AAT

CTG

ATG

ACT

15

467

p90m111

GA

AGA

AAT

CTG

ATG

A

15

468

p90m112

GGA

AGA

AAT

CTG

ATG

A

16

469

p90m113

GA

AGA

AAT

CTG

ATG

AC

16

470

p90m114

AGA

AAT

CTG

ATG

AC

14

471

p90w115

AAT

CTG

TTA

ACT

CAG

15

472

p90w116

T

CTG

TTA

ACT

CAG

ATT

16

473

p90w117

AT

CTG

TTA

ACT

CAG

AG

15

474

p90w118

AGA

AAT

TTG

TTG

ACT

16

475

p90w119

GA

AAT

TTG

TTG

ACT

C

15

476

p90w120

AAT

TTG

TTG

ACT

CAG

15

477

TABLE 4

Polymorphic nucleotide sequences.

51

52

53

54

55

56

57

58

codon position

gga

ggt

ttt

atc

aaa

gta

aga

cag

consensus sequence

GGA

GGT

TTT

ATC

AAA

GTC

AGA

CAA

SEQ ID NO 478

GGA

GGT

TTC

ATT

AAG

GTA

AAA

CAG

SEQ ID NO 479

GGA

GGT

TTT

ATT

AAG

GTA

AGA

CAG

SEQ ID NO 480

GGA

GGT

TTT

ATT

AAA

GTA

AGA

CAA

SEQ ID NO 481

GGA

GGC

TTT

ATC

AAA

GTA

AGA

CAA

SEQ ID NO 482

GGA

GGT

TTT

ATC

AAA

GTC

AGA

CAA

SEQ ID NO 483

78

79

80

81

82

83

84

85

codon position

gga

cct

aca

cct

gtc

aac

ata

att

gg

consensus sequence

GGA

CCT

ACA

CCG

GTC

AAC

ATA

ATT

GG

SEQ ID NO 484

GGA

CCT

ACA

CCT

GCC

AAT

ATA

ATT

GG

SEQ ID NO 485

GGA

CCT

ACG

CCC

TTC

AAC

ATA

ATT

GG

SEQ ID NO 486

GGA

CCG

ACA

CCT

GTC

ACC

ATA

ATT

GG

SEQ ID NO 487

GGA

CCT

ATA

CCT

GTC

AAC

ATA

ATT

GG

SEQ ID NO 488

87

88

89

90

91

92

93

94

codon position

a

aga

aat

ctg

ttg

act

cag

att

ggc

consensus sequence

A

AAA

AAT

CTG

ATG

ACT

CAG

ATT

GGC

SEQ ID NO 489

A

AGA

ACT

CTG

TTG

ACT

CAG

CTT

GGA

SEQ ID NO 490

A

AGA

AAT

ATG

ATG

ACC

CAG

CTT

GGC

SEQ ID NO 491

A

AGA

AAT

ATA

ATG

ACT

CAG

CTT

GGA

SEQ ID NO 492

A

AGA

AAT

CTG

CTG

ACT

CAG

ATT

GGG

SEQ ID NO 493

A

AGA

AAT

CTG

TTG

ACA

CAG

CTT

GGC

SEQ ID NO 494

A

AGA

AAT

ATG

TTG

ACT

CAG

CTT

GGT

SEQ ID NO 495

A

AGA

AAT

TTG

TTG

ACT

CAG

ATT

GGG

SEQ ID NO 496

A

AGA

AAT

ATG

TTG

ACT

CAG

CTT

GGT

SEQ ID NO 497

A

AGA

AAT

ATG

TTG

ACT

CAG

CTT

GGA

SEQ ID NO 498

A

AGA

AAT

CTG

TTG

ACT

CAG

CTT

GGA

SEQ ID NO 499

A

AGA

AAC

CTG

TTG

ACT

CAA

CTT

GGT

SEQ ID NO 500

TABLE 5

probes for

probes for

probes for

codon p30

Type B

non-B

codon p48

Type B

non-B

codon p50

Type B

non-B

w25

95.7

98

w47

71.3

70

w31

95.7

98

w29

1.1

0

w45

11.7

22

w44

1.1

2

w32

1.1

1

w72

16

4

w52

8.5

4

w36

1.1

0

m41

3.2

0

m37

1.1

6

m23

1.1

0

neg.

0

8

neg.

1.1

0

neg.

0

1

probes for

probes for

probes for

codon p54

Type B

non-B

codon p82/84

Type B

non-B

codon p90

Type B

non-B

w3

71.3

48

w91

81.9

70

w27

50

2.5

w34

81.9

62

w60

2.1

12

w37

66.1

17.5

w14

3.2

18

w111

1.1

0

w39

7.1

0

w19

6.4

0

w89

1.1

10

w50

12.5

65

w22

4.3

8

w42

4.3

2

w52

7.1

2.5

w26

0

4

m36

2.1

0

w69

5.4

2.5

w27

0

4

m67

1.1

0

w73

5.4

22.5

m55

3.2

0

m38

2.1

2

w79

0

10

m35

14.9

4

m105

1.1

0

m43

19.6

5

m37

1.1

4

m127

1.1

0

m56

0

2.5

neg.

0

4

m40

14.9

2

neg.

3.6

12.5

m63

3.2

2

m101

2.1

12

neg.

3.2

8

TABLE 6

p30

USA

France

U.K.

Brazil

Spain

Luxemb.

Belgium

w25

98.9

99.4

88.9

98.3

94.3

100.0

97.0

w29

2.5

0.6

0.0

1.7

0.0

0.0

0.0

w32

3.3

0.6

5.6

5.2

5.7

6.7

1.5

w36

2.5

0.0

0.0

3.4

0.0

0.0

1.0

m23

3.1

0.0

0.0

0.0

0.0

0.0

1.0

neg.

0.6

0.6

5.6

0.0

0.0

0.0

1.0

p46/48

USA

France

U.K.

Brazil

Spain

Luxemb.

Belgium

w47

94.2

80.5

83.3

89.7

97.1

73.3

82.9

w45

8.6

15.6

0.0

1.7

5.7

6.7

11.1

w72

4.2

0.0

16.7

0.0

2.9

13.3

5.0

m41

0.0

0.0

0.0

10.3

0.0

13.3

1.0

neg.

2.8

4.5

0.0

0.0

0.0

0.0

2.5

p50

USA

France

U.K.

Brazil

Spain

Luxemb.

Belgium

w31

96.4

97.4

100.0

96.6

100.0

100.0

96.5

w44

1.7

0.6

0.0

1.7

0.0

0.0

1.0

w52

10.0

4.5

0.0

1.7

2.9

6.7

9.0

m37

2.5

0.6

0.0

1.7

0.0

6.7

0.5

neg.

3.1

2.6

0.0

3.4

0.0

0.0

1.5

p54

USA

France

U.K.

Brazil

Spain

Luxemb.

Belgium

w34

96.9

82.5

97.2

87.9

100.0

53.3

89.4

w3

84.7

77.9

94.4

69.0

82.9

46.7

76.9

w14

3.3

5.8

0.0

3.4

11.4

0.0

6.5

w19

9.2

2.6

0.0

1.7

2.9

6.7

5.5

w22

2.8

10.4

0.0

0.0

5.7

0.0

2.5

w26

0.0

1.3

0.0

0.0

0.0

0.0

0.0

w27

0.0

1.9

0.0

0.0

0.0

0.0

0.5

m55

0.0

0.0

0.0

0.0

0.0

13.3

0.5

m35

1.1

0.0

2.8

6.9

0.0

46.7

3.0

m37

0.0

0.0

0.0

0.0

0.0

13.3

0.0

neg.

0.6

1.3

0.0

1.7

0.0

0.0

2.0

p82/84

USA

France

U.K.

Brazil

Spain

Luxemb.

Belgium

w91

91.6

93.5

94.4

77.6

100.0

73.3

85.9

w60

6.4

2.6

0.0

1.7

2.9

13.3

5.5

w111

3.6

0.6

0.0

1.7

0.0

0.0

0.5

w89

7.0

1.9

0.0

3.4

0.0

0.0

3.0

w42

0.6

0.0

2.8

1.7

0.0

0.0

2.0

m36

0.3

0.0

0.0

0.0

0.0

0.0

0.0

m67

0.0

0.0

0.0

0.0

0.0

0.0

0.5

m38

0.0

0.0

0.0

0.0

0.0

6.7

0.0

m105

0.0

0.0

0.0

0.0

0.0

0.0

0.0

m127

0.0

0.0

0.0

0.0

0.0

0.0

0.0

m40

2.8

0.0

8.3

3.4

5.7

46.7

0.0

m63

0.3

0.0

0.0

1.7

2.9

13.3

0.5

m101

1.9

4.5

0.0

3.4

0.0

6.7

4.0

neg.

2.5

3.9

0.0

13.8

0.0

6.7

5.0

p90

USA

France

U.K.

Brazil

Spain

Belgium

w27

51.1

45.5

34.3

47.7

52.8

25.7

w37

91.9

73.4

80.0

81.8

88.9

55.2

w39

0.0

0.0

0.0

0.0

0.0

2.9

w50

2.6

23.8

2.9

13.6

11.1

21.9

w52

8.4

11.2

5.7

6.8

13.9

4.8

w69

5.2

1.4

5.7

2.3

0.0

3.8

w73

6.1

9.1

0.0

0.0

8.3

6.7

w79

7.1

11.2

8.6

9.1

5.6

5.7

m43

1.9

0.0

11.4

0.0

0.0

8.6

m56

0.3

1.4

0.0

0.0

0.0

0.0

neg.

1.0

0.0

0.0

0.0

0.0

18.1

TABLE 7

Tm

lengte

Seq ID

pc50w5

AGG

GGG

AAT

TGG

AGG

TTT

TA

20

511

26

27

28

29

30

31

32

33

34

35

ACA

GGA

GCA

GAT

GAT

ACA

GTA

TTA

GAA

GAA

pc30w25

GCA

GAT

GAT

ACA

GT

40

14

31

pc30w29

A

GCG

GAT

GAT

ACA

36

13

35

pc30w32

GCA

GAT

GAC

ACA

GT

42

14

38

pc30w36

GCA

GAC

GAT

ACA

GG

40

14

42

pc30m23

A

GCA

GAT

AAT

ACA

GT

40

15

29

44

45

46

47

48

49

50

51

52

CCA

AAA

ATG

ATA

GGG

GGA

ATT

GGA

GGT

pc48w37

ATG

ATA

GGG

GGA

ATT

15

512

pc48w47

AAA

ATG

ATA

GGG

GGA

42

15

93

pc48w73

A

AGA

ATG

ATA

GGG

G

14

513

pc48w45

AAA

ATG

ATA

GGA

GGA

ATT

42

18

91

pc48w72

A

AAA

ATA

ATA

GGG

GGA

42

16

120

pc48m41

ATG

ATA

GTG

GGA

ATT

40

15

87

48

49

50

51

52

53

54

GGG

GGA

ATT

GGA

GGT

TTT

ATC

pc50w31

GGA

ATT

GGA

GGT

TTT

42

15

151

pc50w44

GGA

ATT

GGG

GGT

TT

42

14

164

pc50w52

GA

ATT

GGA

GGC

TTG

14

172

pc50m37

GGG

GGA

GTT

GGA

40

12

157

51

52

53

54

55

56

57

58

GGA

GGT

TTT

ATC

AAA

GTA

AGA

CAG

pc54w34

GA

GGT

TTT

ATC

AAA

GT

42

16

212

pc54w14

GGT

TTT

ATC

AAG

GTA

A

42

16

189

pc54w19

A

GGC

TTT

ATC

AAA

GTA

42

16

194

pc54w22

GA

GGT

TTT

ATT

AAA

GTA

42

17

197

pc54w26

A

GGT

TTC

ATT

AAG

GTA

42

16

202

pc54w27

GGT

TTT

ATT

AAG

GTA

A

40

16

204

pc54m35

GGT

TTT

GTC

AAA

GTA

40

15

213

pc54m37

GGT

TTT

GTC

AGA

GTA

42

15

215

pc54m55

A

GGT

TTT

GCC

AAA

GT

15

516

78

79

80

81

82

83

84

85

86

87

GGA

CCT

ACA

CCT

GTC

AAC

ATA

ATT

GGA

AGA

pc82w91

ACA

CCT

GTC

AAC

ATA

A

44

16

318

pc82w60

CA

CCT

GTC

AAT

ATA

ATG

42

17

287

pc82w111

A

CCG

GTC

AAC

ATA

ATT

44

16

338

pc82w89

ACA

CCT

GTT

AAC

ATA

AG

42

17

316

pc82m101

ACA

CCT

ATC

AAC

ATA

AT

17

517

pc82w42

CA

CCT

GTC

AAC

GTA

42

14

269

pc82m38

ACA

CCT

TTC

AAC

ATA

40

15

265

pc82m105

ACG

CCC

TTC

AAC

ATA

44

15

332

pc82m127

CA

CCT

TTC

AAC

GTA

ATG

44

17

354

pc82m40

ACA

CCT

GCC

AAC

ATA

44

15

267

pc82m63

CA

CCT

GCC

AAT

ATA

AG

42

16

290

pc82m36

ACA

CCT

ACC

AAC

ATA

15

518

pc82m67

ACA

CCT

ACC

AAC

GT

14

519

86

87

88

89

90

91

92

93

94

GGA

AGA

AAT

CTG

TTG

ACT

CAG

ATT

GGT

pc90w27

ATT

CTG

TTG

ACT

CA

38

14

384

pc90w37

T

CTG

TTG

ACT

CAG

AT

15

514

pc90w39

GA

GTC

AAC

AGA

GTT

C

15

515

pc90w50

AAT

ATG

TTG

ACT

CAG

40

15

407

pc90w52

AAT

TTG

TTG

ACT

CAG

40

15

409

pc90w69

GA

AAC

CTG

TTG

ACT

40

14

426

pc90w73

TG

TTG

ACA

CAG

CTT

G

44

15

430

pc90w79

TG

TTG

ACC

CAG

ATT

G

44

15

436

pc90m138

GTC

ATC

AGA

TTT

CT

14

510

pc90m56

AAT

ATG

ATG

ACC

CAG

42

15

413

529

1

21

DNA

Aids-associated retrovirus

1
cagagccaac agccccacca g 21

2

27

DNA

Aids-associated retrovirus

2
atcaggatgg agttcataac ccatcca 27

3

23

DNA

Aids-associated retrovirus

3
cctcaratca ctctttggca acg 23

4

25

DNA

Aids-associated retrovirus

4
taatcrggat aactytgaca tggtc 25

5

20

DNA

Aids-associated retrovirus

5
cctgtcaaca taattggaag 20

6

21

DNA

Aids-associated retrovirus

6
agtcaacaga tttcttccaa t 21

7

18

DNA

Aids-associated retrovirus

7
agcagatgat acagtatt 18

8

19

DNA

Aids-associated retrovirus

8
gagcagatga tacagtatt 19

9

19

DNA

Aids-associated retrovirus

9
agcagatgat acagtatta 19

10

20

DNA

Aids-associated retrovirus

10
ggagcagatg atacagtatt 20

11

21

DNA

Aids-associated retrovirus

11
ggagcagatg atacagtatt a 21

12

18

DNA

Aids-associated retrovirus

12
acaggagcag atgataca 18

13

19

DNA

Aids-associated retrovirus

13
caggagcaga tgatacagt 19

14

21

DNA

Aids-associated retrovirus

14
aggagcagat gatacagtat g 21

15

20

DNA

Aids-associated retrovirus

15
ggagcagatg atacagtatg 20

16

20

DNA

Aids-associated retrovirus

16
acaggagcag atgatacagg 20

17

18

DNA

Aids-associated retrovirus

17
agcagataat acagtatt 18

18

19

DNA

Aids-associated retrovirus

18
gagcagataa tacagtatt 19

19

19

DNA

Aids-associated retrovirus

19
agcagataat acagtatta 19

20

20

DNA

Aids-associated retrovirus

20
ggagcagata atacagtatt 20

21

21

DNA

Aids-associated retrovirus

21
ggagcagata atacagtatt a 21

22

18

DNA

Aids-associated retrovirus

22
acaggagcag ataataca 18

23

19

DNA

Aids-associated retrovirus

23
caggagcaga taatacagt 19

24

21

DNA

Aids-associated retrovirus

24
aggagcagat aatacagtat g 21

25

20

DNA

Aids-associated retrovirus

25
ggagcagata atacagtatg 20

26

20

DNA

Aids-associated retrovirus

26
acaggagcag ataatacagg 20

27

15

DNA

Aids-associated retrovirus

27
agcagatgat acagt 15

28

17

DNA

Aids-associated retrovirus

28
agcagatgat acagtag 17

29

15

DNA

Aids-associated retrovirus

29
agcagataat acagt 15

30

17

DNA

Aids-associated retrovirus

30
agcagataat acagtag 17

31

14

DNA

Aids-associated retrovirus

31
gcagatgata cagt 14

32

15

DNA

Aids-associated retrovirus

32
agcagatgat acagg 15

33

13

DNA

Aids-associated retrovirus

33
cagatgatac agt 13

34

14

DNA

Aids-associated retrovirus

34
gagcggatga taca 14

35

13

DNA

Aids-associated retrovirus

35
agcggatgat aca 13

36

15

DNA

Aids-associated retrovirus

36
gcagataata cagta 15

37

14

DNA

Aids-associated retrovirus

37
gcagataata cagt 14

38

14

DNA

Aids-associated retrovirus

38
gcagatgaca cagt 14

39

15

DNA

Aids-associated retrovirus

39
cagatgacac agtag 15

40

16

DNA

Aids-associated retrovirus

40
cagatgatac aatatt 16

41

17

DNA

Aids-associated retrovirus

41
gcagatgata caatatg 17

42

14

DNA

Aids-associated retrovirus

42
gcagacgata cagg 14

43

14

DNA

Aids-associated retrovirus

43
gcagacgata cagt 14

44

15

DNA

Aids-associated retrovirus

44
agatgataca atatt 15

45

16

DNA

Aids-associated retrovirus

45
agatgataca atatta 16

46

15

DNA

Aids-associated retrovirus

46
gcagatgata caata 15

47

20

DNA

Aids-associated retrovirus

47
gtagggggaa ttggaggtgg 20

48

20

DNA

Aids-associated retrovirus

48
gtagggggaa ttggaggttg 20

49

21

DNA

Aids-associated retrovirus

49
gtagggggaa ttggaggttt g 21

50

21

DNA

Aids-associated retrovirus

50
gtagggggaa ttggaggttt t 21

51

22

DNA

Aids-associated retrovirus

51
ggtaggggga attggaggtt tg 22

52

18

DNA

Aids-associated retrovirus

52
atggtagggg gaattgga 18

53

19

DNA

Aids-associated retrovirus

53
atggtagggg gaattggag 19

54

19

DNA

Aids-associated retrovirus

54
aatggtaggg ggaattgga 19

55

20

DNA

Aids-associated retrovirus

55
aatggtaggg ggaattggag 20

56

24

DNA

Aids-associated retrovirus

56
aatggtaggg ggaattggag gggg 24

57

18

DNA

Aids-associated retrovirus

57
ataatagggg gaattgga 18

58

18

DNA

Aids-associated retrovirus

58
atgatagggg gaattgga 18

59

19

DNA

Aids-associated retrovirus

59
aataataggg ggaattgga 19

60

19

DNA

Aids-associated retrovirus

60
aatgataggg ggaattgga 19

61

20

DNA

Aids-associated retrovirus

61
atagggggaa ttggaggtgg 20

62

20

DNA

Aids-associated retrovirus

62
atagggggaa ttggaggttg 20

63

21

DNA

Aids-associated retrovirus

63
atagggggaa ttggaggttt g 21

64

21

DNA

Aids-associated retrovirus

64
atagggggaa ttggaggttt t 21

65

20

DNA

Aids-associated retrovirus

65
gtagtgggaa ttggaggtgg 20

66

20

DNA

Aids-associated retrovirus

66
gtagtgggaa ttggaggttg 20

67

21

DNA

Aids-associated retrovirus

67
gtagtgggaa ttggaggttt g 21

68

21

DNA

Aids-associated retrovirus

68
gtagtgggaa ttggaggttt t 21

69

22

DNA

Aids-associated retrovirus

69
ggtagtggga attggaggtt tg 22

70

18

DNA

Aids-associated retrovirus

70
atggtagtgg gaattgga 18

71

19

DNA

Aids-associated retrovirus

71
atggtagtgg gaattggag 19

72

19

DNA

Aids-associated retrovirus

72
aatggtagtg ggaattgga 19

73

20

DNA

Aids-associated retrovirus

73
aatggtagtg ggaattggag 20

74

24

DNA

Aids-associated retrovirus

74
aatggtagtg ggaattggag gggg 24

75

20

DNA

Aids-associated retrovirus

75
atagtgggaa ttggaggtgg 20

76

20

DNA

Aids-associated retrovirus

76
atagtgggaa ttggaggttg 20

77

18

DNA

Aids-associated retrovirus

77
atgatagtgg gaattgga 18

78

19

DNA

Aids-associated retrovirus

78
atgatagtgg gaattggag 19

79

19

DNA

Aids-associated retrovirus

79
aatgatagtg ggaattgga 19

80

14

DNA

Aids-associated retrovirus

80
gataggggga attg 14

81

15

DNA

Aids-associated retrovirus

81
tgataggggg aattg 15

82

16

DNA

Aids-associated retrovirus

82
tgataggggg aattgg 16

83

15

DNA

Aids-associated retrovirus

83
atgatagggg gaatt 15

84

14

DNA

Aids-associated retrovirus

84
gatagtggga attg 14

85

15

DNA

Aids-associated retrovirus

85
tgatagtggg aattg 15

86

16

DNA

Aids-associated retrovirus

86
tgatagtggg aattgg 16

87

15

DNA

Aids-associated retrovirus

87
atgatagtgg gaatt 15

88

15

DNA

Aids-associated retrovirus

88
ataatagggg gaatt 15

89

14

DNA

Aids-associated retrovirus

89
tgataggggg agtt 14

90

14

DNA

Aids-associated retrovirus

90
gataggggga gttg 14

91

18

DNA

Aids-associated retrovirus

91
aaaatgatag gaggaatt 18

92

15

DNA

Aids-associated retrovirus

92
atgatagggg gaatt 15

93

15

DNA

Aids-associated retrovirus

93
aaaatgatag gggga 15

94

15

DNA

Aids-associated retrovirus

94
aaaaatgata ggggg 15

95

16

DNA

Aids-associated retrovirus

95
aaatgatagg gggaag 16

96

17

DNA

Aids-associated retrovirus

96
aaaataatag ggggaag 17

97

11

DNA

Aids-associated retrovirus

97
aaaataaaaa t 11

98

17

DNA

Aids-associated retrovirus

98
aaaatgatag tgggaag 17

99

14

DNA

Aids-associated retrovirus

99
aaattgatag gggg 14

100

15

DNA

Aids-associated retrovirus

100
aaaatgatag tggga 15

101

15

DNA

Aids-associated retrovirus

101
aaattgatag gggga 15

102

12

DNA

Aids-associated retrovirus

102
caaaattgat ag 12

103

15

DNA

Aids-associated retrovirus

103
atggtagggg gaatt 15

104

14

DNA

Aids-associated retrovirus

104
aaatggtagg ggga 14

105

14

DNA

Aids-associated retrovirus

105
aaaaatggta gggg 14

106

15

DNA

Aids-associated retrovirus

106
atgatagggg aaatt 15

107

15

DNA

Aids-associated retrovirus

107
ataggggaaa ttgga 15

108

16

DNA

Aids-associated retrovirus

108
ataggggaaa ttggag 16

109

15

DNA

Aids-associated retrovirus

109
atgatagggg ggatt 15

110

14

DNA

Aids-associated retrovirus

110
atagggggga ttgg 14

111

13

DNA

Aids-associated retrovirus

111
aggggggatt gga 13

112

15

DNA

Aids-associated retrovirus

112
aaaataatag tggga 15

113

16

DNA

Aids-associated retrovirus

113
aaaaataata gtggga 16

114

16

DNA

Aids-associated retrovirus

114
caaaaataat agtggg 16

115

15

DNA

Aids-associated retrovirus

115
aaattgatag tggga 15

116

16

DNA

Aids-associated retrovirus

116
aaaattgata gtggga 16

117

15

DNA

Aids-associated retrovirus

117
caaaattgat agtgg 15

118

14

DNA

Aids-associated retrovirus

118
aaaatgatag gggg 14

119

14

DNA

Aids-associated retrovirus

119
aaaaatgata gggg 14

120

16

DNA

Aids-associated retrovirus

120
aaaaataata ggggga 16

121

18

DNA

Aids-associated retrovirus

121
gggggaattg gaggtttt 18

122

19

DNA

Aids-associated retrovirus

122
agggggaatt ggaggtttt 19

123

20

DNA

Aids-associated retrovirus

123
tagggggaat tggaggtttt 20

124

21

DNA

Aids-associated retrovirus

124
agggggaatt ggaggtttta g 21

125

22

DNA

Aids-associated retrovirus

125
tagggggaat tggaggtttt ag 22

126

21

DNA

Aids-associated retrovirus

126
gtagggggaa ttggaggttg g 21

127

22

DNA

Aids-associated retrovirus

127
ggtaggggga attggaggtt gg 22

128

21

DNA

Aids-associated retrovirus

128
gtagggggaa ttggaggttt g 21

129

21

DNA

Aids-associated retrovirus

129
gtagggggaa ttggaggttt t 21

130

22

DNA

Aids-associated retrovirus

130
tggtaggggg aattggaggt gg 22

131

17

DNA

Aids-associated retrovirus

131
ggggaattgg aggtttt 17

132

17

DNA

Aids-associated retrovirus

132
ggggaattgg aggtttg 17

133

19

DNA

Aids-associated retrovirus

133
ggggaattgg aggttttag 19

134

16

DNA

Aids-associated retrovirus

134
ggggaattgg aggttg 16

135

18

DNA

Aids-associated retrovirus

135
gggaattgga ggttttat 18

136

17

DNA

Aids-associated retrovirus

136
ggggaattgg aggtttt 17

137

18

DNA

Aids-associated retrovirus

137
gggggagttg gaggtttt 18

138

19

DNA

Aids-associated retrovirus

138
agggggagtt ggaggtttt 19

139

20

DNA

Aids-associated retrovirus

139
tagggggagt tggaggtttt 20

140

21

DNA

Aids-associated retrovirus

140
agggggagtt ggaggtttta g 21

141

22

DNA

Aids-associated retrovirus

141
tagggggagt tggaggtttt ag 22

142

21

DNA

Aids-associated retrovirus

142
gtagggggag ttggaggttg g 21

143

22

DNA

Aids-associated retrovirus

143
ggtaggggga gttggaggtt gg 22

144

21

DNA

Aids-associated retrovirus

144
gtagggggag ttggaggttt g 21

145

24

DNA

Aids-associated retrovirus

145
gtagggggag ttggaggttt tatc 24

146

22

DNA

Aids-associated retrovirus

146
tggtaggggg agttggaggt gg 22

147

17

DNA

Aids-associated retrovirus

147
ggggagttgg aggtttg 17

148

19

DNA

Aids-associated retrovirus

148
ggggagttgg aggttttag 19

149

16

DNA

Aids-associated retrovirus

149
ggggagttgg aggttg 16

150

18

DNA

Aids-associated retrovirus

150
gggagttgga ggttttat 18

151

15

DNA

Aids-associated retrovirus

151
ggaattggag gtttt 15

152

16

DNA

Aids-associated retrovirus

152
gggaattgga ggttgg 16

153

15

DNA

Aids-associated retrovirus

153
ggagttggag gtttt 15

154

16

DNA

Aids-associated retrovirus

154
gggagttgga ggttgg 16

155

13

DNA

Aids-associated retrovirus

155
gggggagttg gag 13

156

12

DNA

Aids-associated retrovirus

156
ggggagttgg ag 12

157

12

DNA

Aids-associated retrovirus

157
gggggagttg ga 12

158

15

DNA

Aids-associated retrovirus

158
ggaattgggg gtttg 15

159

14

DNA

Aids-associated retrovirus

159
gaattggggg tttt 14

160

16

DNA

Aids-associated retrovirus

160
gaattggggg ttttag 16

161

14

DNA

Aids-associated retrovirus

161
ggaattgggg gttg 14

162

13

DNA

Aids-associated retrovirus

162
ggaattgggg gtg 13

163

13

DNA

Aids-associated retrovirus

163
gaattggggg ttg 13

164

14

DNA

Aids-associated retrovirus

164
ggaattgggg gttt 14

165

13

DNA

Aids-associated retrovirus

165
gggggaattg cag 13

166

14

DNA

Aids-associated retrovirus

166
ggaattgcag gttg 14

167

13

DNA

Aids-associated retrovirus

167
ggaattgcag gtg 13

168

15

DNA

Aids-associated retrovirus

168
ggaattggag ggttg 15

169

14

DNA

Aids-associated retrovirus

169
gaattggagg gttg 14

170

14

DNA

Aids-associated retrovirus

170
gaattggagg gttt 14

171

15

DNA

Aids-associated retrovirus

171
ggaattggag gcttg 15

172

14

DNA

Aids-associated retrovirus

172
gaattggagg cttg 14

173

14

DNA

Aids-associated retrovirus

173
gaattggagg cttt 14

174

15

DNA

Aids-associated retrovirus

174
ggagttggag gtttg 15

175

14

DNA

Aids-associated retrovirus

175
gagttggagg tttt 14

176

16

DNA

Aids-associated retrovirus

176
ggttttatca aagtaa 16

177

16

DNA

Aids-associated retrovirus

177
gttttatcaa agtaag 16

178

17

DNA

Aids-associated retrovirus

178
gttttatcaa agtaaga 17

179

16

DNA

Aids-associated retrovirus

179
ttttatcaaa gtaaga 16

180

15

DNA

Aids-associated retrovirus

180
ggttttatca aagta 15

181

14

DNA

Aids-associated retrovirus

181
gttttatcaa agta 14

182

15

DNA

Aids-associated retrovirus

182
ggttttgcca aagta 15

183

15

DNA

Aids-associated retrovirus

183
gttttgccaa agtaa 15

184

16

DNA

Aids-associated retrovirus

184
gttttgccaa agtaag 16

185

16

DNA

Aids-associated retrovirus

185
ttttgccaaa gtaaga 16

186

14

DNA

Aids-associated retrovirus

186
ggttttgcca aagt 14

187

14

DNA

Aids-associated retrovirus

187
gttttgccaa agta 14

188

16

DNA

Aids-associated retrovirus

188
gttttatcaa ggtaaa 16

189

16

DNA

Aids-associated retrovirus

189
ggttttatca aggtaa 16

190

16

DNA

Aids-associated retrovirus

190
aggttttatc aaggta 16

191

17

DNA

Aids-associated retrovirus

191
gttttatcaa agtcaga 17

192

16

DNA

Aids-associated retrovirus

192
tttatcaaag tcagac 16

193

17

DNA

Aids-associated retrovirus

193
aggctttatc aaagtaa 17

194

16

DNA

Aids-associated retrovirus

194
aggctttatc aaagta 16

195

17

DNA

Aids-associated retrovirus

195
aggttttatt aaagtaa 17

196

17

DNA

Aids-associated retrovirus

196
ggttttatta aagtaag 17

197

17

DNA

Aids-associated retrovirus

197
gaggttttat taaagta 17

198

17

DNA

Aids-associated retrovirus

198
gaggttttat taaagta 17

199

17

DNA

Aids-associated retrovirus

199
ggttttattg gttttat 17

200

15

DNA

Aids-associated retrovirus

200
ggtttcatta aggta 15

201

16

DNA

Aids-associated retrovirus

201
ggtttcatta aggtaa 16

202

16

DNA

Aids-associated retrovirus

202
aggtttcatt aaggta 16

203

16

DNA

Aids-associated retrovirus

203
aggtttcatt aaggta 16

204

16

DNA

Aids-associated retrovirus

204
ggttttatta aggtaa 16

205

16

DNA

Aids-associated retrovirus

205
ggttttatta aggtaa 16

206

16

DNA

Aids-associated retrovirus

206
aggttttatt aaggta 16

207

16

DNA

Aids-associated retrovirus

207
gaggttttat taaggt 16

208

17

DNA

Aids-associated retrovirus

208
ggttttatta aggtaag 17

209

16

DNA

Aids-associated retrovirus

209
ggttttatca aagtaa 16

210

17

DNA

Aids-associated retrovirus

210
aggttttatc aaagtaa 17

211

16

DNA

Aids-associated retrovirus

211
aggttttatc aaagta 16

212

16

DNA

Aids-associated retrovirus

212
gaggttttat caaagt 16

213

15

DNA

Aids-associated retrovirus

213
ggttttgtca aagta 15

214

16

DNA

Aids-associated retrovirus

214
ggttttgtca aagtaa 16

215

15

DNA

Aids-associated retrovirus

215
ggttttgtca gagta 15

216

16

DNA

Aids-associated retrovirus

216
ggttttgtca gagtaa 16

217

15

DNA

Aids-associated retrovirus

217
gggtttatca aagta 15

218

16

DNA

Aids-associated retrovirus

218
gggtttatca aagtaa 16

219

14

DNA

Aids-associated retrovirus

219
ggcttcatca aagt 14

220

14

DNA

Aids-associated retrovirus

220
gaggcttcat caaa 14

221

14

DNA

Aids-associated retrovirus

221
ggttttgtca aagt 14

222

14

DNA

Aids-associated retrovirus

222
gttttgtcag agta 14

223

14

DNA

Aids-associated retrovirus

223
ggttttgtca gagt 14

224

16

DNA

Aids-associated retrovirus

224
aggtttaatc aaagta 16

225

16

DNA

Aids-associated retrovirus

225
gaggtttaat caaagt 16

226

15

DNA

Aids-associated retrovirus

226
ggttttacca aagta 15

227

14

DNA

Aids-associated retrovirus

227
ggttttacca aagt 14

228

20

DNA

Aids-associated retrovirus

228
cctacacctg tcaacataag 20

229

21

DNA

Aids-associated retrovirus

229
cctacacctg tcaacataat g 21

230

21

DNA

Aids-associated retrovirus

230
cctacacctg tcaacataat t 21

231

21

DNA

Aids-associated retrovirus

231
acctacacct gtcaacataa g 21

232

22

DNA

Aids-associated retrovirus

232
acctacacct gtcaacataa tg 22

233

19

DNA

Aids-associated retrovirus

233
acctacacct gtcaacata 19

234

20

DNA

Aids-associated retrovirus

234
gacctacacc tgtcaacata 20

235

20

DNA

Aids-associated retrovirus

235
cacctgtcaa cataattgga 20

236

20

DNA

Aids-associated retrovirus

236
acctgtcaac ataattggaa 20

237

20

DNA

Aids-associated retrovirus

237
acacctgtca acataattgg 20

238

19

DNA

Aids-associated retrovirus

238
acctgtcaac ataattgga 19

239

20

DNA

Aids-associated retrovirus

239
cctacaccta ccaacataag 20

240

21

DNA

Aids-associated retrovirus

240
cctacaccta ccaacataat g 21

241

21

DNA

Aids-associated retrovirus

241
cctacaccta ccaacataat t 21

242

21

DNA

Aids-associated retrovirus

242
acctacacct accaacataa g 21

243

22

DNA

Aids-associated retrovirus

243
acctacacct accaacataa tg 22

244

19

DNA

Aids-associated retrovirus

244
acctacacct accaacata 19

245

20

DNA

Aids-associated retrovirus

245
gacctacacc taccaacata 20

246

20

DNA

Aids-associated retrovirus

246
cacctaccaa cataattgga 20

247

20

DNA

Aids-associated retrovirus

247
acctaccaac ataattggaa 20

248

19

DNA

Aids-associated retrovirus

248
acacctacca acataattg 19

249

21

DNA

Aids-associated retrovirus

249
cctacacctt tcaacataat t 21

250

21

DNA

Aids-associated retrovirus

250
cctacacctg ccaacataat t 21

251

21

DNA

Aids-associated retrovirus

251
cctacacctt ccaacataat t 21

252

20

DNA

Aids-associated retrovirus

252
acctttcaac ataattggaa 20

253

20

DNA

Aids-associated retrovirus

253
acctgccaac ataattggaa 20

254

20

DNA

Aids-associated retrovirus

254
acctttcaac ataattggaa 20

255

16

DNA

Aids-associated retrovirus

255
acctaccaac ataatt 16

256

19

DNA

Aids-associated retrovirus

256
acctttcaac ataattgga 19

257

19

DNA

Aids-associated retrovirus

257
acctgccaac ataattgga 19

258

19

DNA

Aids-associated retrovirus

258
accttccaac ataattgga 19

259

15

DNA

Aids-associated retrovirus

259
tacacctgtc aacat 15

260

16

DNA

Aids-associated retrovirus

260
tacacctgtc aacata 16

261

15

DNA

Aids-associated retrovirus

261
acacctgtca acata 15

262

14

DNA

Aids-associated retrovirus

262
cacctgtcaa cata 14

263

15

DNA

Aids-associated retrovirus

263
acacctacca acata 15

264

14

DNA

Aids-associated retrovirus

264
cacctaccaa cata 14

265

15

DNA

Aids-associated retrovirus

265
acacctttca acata 15

266

14

DNA

Aids-associated retrovirus

266
cacctttcaa cata 14

267

15

DNA

Aids-associated retrovirus

267
acacctgcca acata 15

268

14

DNA

Aids-associated retrovirus

268
cacctgccaa cata 14

269

14

DNA

Aids-associated retrovirus

269
cacctgtcaa cgta 14

270

13

DNA

Aids-associated retrovirus

270
cacctgtcaa cgt 13

271

15

DNA

Aids-associated retrovirus

271
cctacacctg tcaac 15

272

15

DNA

Aids-associated retrovirus

272
tacgcctgtc aacat 15

273

16

DNA

Aids-associated retrovirus

273
ctacgcctgt caacag 16

274

15

DNA

Aids-associated retrovirus

274
acaccttcca acata 15

275

14

DNA

Aids-associated retrovirus

275
caccttccaa cata 14

276

14

DNA

Aids-associated retrovirus

276
acaccttcca acat 14

277

15

DNA

Aids-associated retrovirus

277
acacctatca acata 15

278

16

DNA

Aids-associated retrovirus

278
cacctatcaa cataag 16

279

17

DNA

Aids-associated retrovirus

279
cacctatcaa cataatg 17

280

16

DNA

Aids-associated retrovirus

280
acctatcaac ataatg 16

281

15

DNA

Aids-associated retrovirus

281
cctgtcaaca taatt 15

282

16

DNA

Aids-associated retrovirus

282
cctgttaaca taattg 16

283

16

DNA

Aids-associated retrovirus

283
acctgttaac ataatg 16

284

15

DNA

Aids-associated retrovirus

284
ccggtcaaca taatt 15

285

14

DNA

Aids-associated retrovirus

285
acgcctgtca acat 14

286

15

DNA

Aids-associated retrovirus

286
cctgtcaata taatt 15

287

17

DNA

Aids-associated retrovirus

287
cacctgtcaa tataatg 17

288

17

DNA

Aids-associated retrovirus

288
acacctgtca atataag 17

289

15

DNA

Aids-associated retrovirus

289
cctgccaata taatt 15

290

16

DNA

Aids-associated retrovirus

290
cacctgccaa tataag 16

291

15

DNA

Aids-associated retrovirus

291
cctaccaacg taatt 15

292

15

DNA

Aids-associated retrovirus

292
cctaccaacg taatg 15

293

14

DNA

Aids-associated retrovirus

293
cacctaccaa cgta 14

294

14

DNA

Aids-associated retrovirus

294
acacctacca acgt 14

295

15

DNA

Aids-associated retrovirus

295
cctttcaacg taatt 15

296

16

DNA

Aids-associated retrovirus

296
cacctttcaa cgtaag 16

297

15

DNA

Aids-associated retrovirus

297
acacctttca acgta 15

298

16

DNA

Aids-associated retrovirus

298
acctttcaac gtaatg 16

299

15

DNA

Aids-associated retrovirus

299
ctgtcaatat aattg 15

300

16

DNA

Aids-associated

300
cctgtcaata taattg 16

301

16

DNA

Aids-associated retrovirus

301
acctgtcaat ataatt 16

302

16

DNA

Aids-associated retrovirus

302
ctgtcaatat aattgg 16

303

14

DNA

Aids-associated retrovirus

303
cctacgcctg tcaa 14

304

14

DNA

Aids-associated retrovirus

304
ctacgcctgt caac 14

305

15

DNA

Aids-associated retrovirus

305
acctacgcct gtcaa 15

306

14

DNA

Aids-associated retrovirus

306
acctacgcct gtca 14

307

14

DNA

Aids-associated retrovirus

307
tacaccggtc aaca 14

308

13

DNA

Aids-associated retrovirus

308
ctacaccggt caa 13

309

13

DNA

Aids-associated retrovirus

309
cctacaccgg tca 13

310

15

DNA

Aids-associated retrovirus

310
cacctgtcaa cataa 15

311

15

DNA

Aids-associated retrovirus

311
acctgtcaac ataat 15

312

15

DNA

Aids-associated retrovirus

312
ctacacctgt caaca 15

313

14

DNA

Aids-associated retrovirus

313
acacctgtca acat 14

314

17

DNA

Aids-associated retrovirus

314
acctgttaac ataattg 17

315

16

DNA

Aids-associated retrovirus

315
cacctgttaa cataag 16

316

17

DNA

Aids-associated retrovirus

316
acacctgtta acataag 17

317

15

DNA

Aids-associated retrovirus

317
tcacctgtca acata 15

318

16

DNA

Aids-associated retrovirus

318
acacctgtca acataa 16

319

16

DNA

Aids-associated retrovirus

319
cacctgtcaa cataat 16

320

15

DNA

Aids-associated retrovirus

320
cctgtcaaca taatt 15

321

16

DNA

Aids-associated retrovirus

321
acctgtcaac ataatt 16

322

16

DNA

Aids-associated retrovirus

322
cctgtcaaca taattg 16

323

14

DNA

Aids-associated retrovirus

323
cctacacctg tcaa 14

324

15

DNA

Aids-associated retrovirus

324
tgtcaacata attgg 15

325

16

DNA

Aids-associated retrovirus

325
tgtcaacata attgga 16

326

16

DNA

Aids-associated retrovirus

326
acacctttca acataa 16

327

16

DNA

Aids-associated retrovirus

327
tacacctttc aacata 16

328

18

DNA

Aids-associated retrovirus

328
acacctatca acataatg 18

329

17

DNA

Aids-associated retrovirus

329
acacctatca acataag 17

330

17

DNA

Aids-associated retrovirus

330
cacctgccaa tataatg 17

331

17

DNA

Aids-associated retrovirus

331
acacctgcca atataag 17

332

15

DNA

Aids-associated retrovirus

332
acgcccttca acata 15

333

16

DNA

Aids-associated retrovirus

333
cgcccttcaa cataag 16

334

15

DNA

Aids-associated retrovirus

334
tacgcccttc aacat 15

335

14

DNA

Aids-associated retrovirus

335
ctacaccggt caac 14

336

14

DNA

Aids-associated retrovirus

336
cctacaccgg tcaa 14

337

16

DNA

Aids-associated retrovirus

337
accggtcaac ataatg 16

338

16

DNA

Aids-associated retrovirus

338
accggtcaac ataatt 16

339

14

DNA

Aids-associated retrovirus

339
ctacaccagt caac 14

340

15

DNA

Aids-associated retrovirus

340
ctacaccagt caaca 15

341

15

DNA

Aids-associated retrovirus

341
acaccagtca acata 15

342

17

DNA

Aids-associated retrovirus

342
acaccagtca acataag 17

343

15

DNA

Aids-associated retrovirus

343
tacgcctgtc aacat 15

344

15

DNA

Aids-associated retrovirus

344
acgcctgtca acata 15

345

14

DNA

Aids-associated retrovirus

345
tacgcctgtc aaca 14

346

15

DNA

Aids-associated retrovirus

346
cctacacctt tcaac 15

347

14

DNA

Aids-associated retrovirus

347
ctacaccttt caac 14

348

15

DNA

Aids-associated retrovirus

348
acctacacct ttcaa 15

349

18

DNA

Aids-associated retrovirus

349
acgcctgtca acataagg 18

350

16

DNA

Aids-associated retrovirus

350
tacgcctgtc aacata 16

351

17

DNA

Aids-associated retrovirus

351
cgcctgtcaa cataagg 17

352

16

DNA

Aids-associated retrovirus

352
tacacctttc aacgta 16

353

18

DNA

Aids-associated retrovirus

353
acacctttca acgtaagg 18

354

17

DNA

Aids-associated retrovirus

354
cacctttcaa cgtaatg 17

355

16

DNA

Aids-associated retrovirus

355
acctttcaac gtaatt 16

356

16

DNA

Aids-associated retrovirus

356
caacgtaatt ggaaga 16

357

15

DNA

Aids-associated retrovirus

357
caacgtaatt ggaag 15

358

16

DNA

Aids-associated retrovirus

358
aaatctgttg actcag 16

359

17

DNA

Aids-associated retrovirus

359
gaaatctgtt gactcag 17

360

20

DNA

Aids-associated retrovirus

360
gaaatctgtt gactcagagg 20

361

19

DNA

Aids-associated retrovirus

361
aaatctgttg actcagagg 19

362

21

DNA

Aids-associated retrovirus

362
agaaatctgt tgactcagag g 21

363

21

DNA

Aids-associated retrovirus

363
agaaatctgt tgactcagat g 21

364

21

DNA

Aids-associated retrovirus

364
agaaatctgt tgactcagat t 21

365

23

DNA

Aids-associated retrovirus

365
agaaatctgt tgactcagat tgg 23

366

23

DNA

Aids-associated retrovirus

366
gaagaaatct gttgactcag agg 23

367

22

DNA

Aids-associated retrovirus

367
aagaaatctg ttgactcaga tg 22

368

21

DNA

Aids-associated retrovirus

368
agaaatctga tgactcagat g 21

369

21

DNA

Aids-associated retrovirus

369
agaaatctga tgactcagat t 21

370

22

DNA

Aids-associated retrovirus

370
aagaaatctg atgactcaga gg 22

371

23

DNA

Aids-associated retrovirus

371
gaagaaatct gatgactcag agg 23

372

22

DNA

Aids-associated retrovirus

372
aagaaatctg atgactcaga tg 22

373

23

DNA

Aids-associated retrovirus

373
gaagaaatct gatgactcag att 23

374

21

DNA

Aids-associated retrovirus

374
ggaagaaatc tgatgactca g 21

375

19

DNA

Aids-associated retrovirus

375
aagaaatctg atgactcag 19

376

21

DNA

Aids-associated retrovirus

376
aaatctgatg actcagattg g 21

377

20

DNA

Aids-associated retrovirus

377
aaatctgatg actcagattg 20

378

20

DNA

Aids-associated retrovirus

378
aaatctgatg actcagcttg 20

379

19

DNA

Aids-associated retrovirus

379
aaatctgatg actcagctt 19

380

19

DNA

Aids-associated retrovirus

380
aatctgatga ctcagcttg 19

381

20

DNA

Aids-associated retrovirus

381
aaatctgttg actcagcttg 20

382

19

DNA

Aids-associated retrovirus

382
aaatctgttg actcagctt 19

383

19

DNA

Aids-associated retrovirus

383
aatctgttga ctcagcttg 19

384

14

DNA

Aids-associated retrovirus

384
aatctgttga ctca 14

385

15

DNA

Aids-associated retrovirus

385
aatctgttga ctcag 15

386

15

DNA

Aids-associated retrovirus

386
aaatctgttg actca 15

387

16

DNA

Aids-associated retrovirus

387
aaatctgttg actcag 16

388

14

DNA

Aids-associated retrovirus

388
aatctgatga ctca 14

389

15

DNA

Aids-associated retrovirus

389
aatctgatga ctcag 15

390

15

DNA

Aids-associated retrovirus

390
aaatctgatg actca 15

391

16

DNA

Aids-associated retrovirus

391
aaatctgatg actcag 16

392

15

DNA

Aids-associated retrovirus

392
gaaatctgtt gactc 15

393

15

DNA

Aids-associated retrovirus

393
gaactctgtt gactc 15

394

16

DNA

Aids-associated retrovirus

394
tctgttgact cagatg 16

395

15

DNA

Aids-associated retrovirus

395
gaaatctgtt gactc 15

396

15

DNA

Aids-associated retrovirus

396
gaactctgtt gactc 15

397

15

DNA

Aids-associated retrovirus

397
aaatctgttg actca 15

398

15

DNA

Aids-associated retrovirus

398
aatctgttga ctcag 15

399

15

DNA

Aids-associated retrovirus

399
aatctgatga ctcag 15

400

15

DNA

Aids-associated retrovirus

400
aaatctgatg actca 15

401

16

DNA

Aids-associated retrovirus

401
atctgttgac tcagag 16

402

15

DNA

Aids-associated retrovirus

402
ctgttgactc agatt 15

403

15

DNA

Aids-associated retrovirus

403
agaaatctgt tgact 15

404

16

DNA

Aids-associated retrovirus

404
atctgatgac tcagag 16

405

15

DNA

Aids-associated retrovirus

405
ctgatgactc agatt 15

406

17

DNA

Aids-associated retrovirus

406
agaaatctga tgactca 17

407

15

DNA

Aids-associated retrovirus

407
aatatgttga ctcag 15

408

16

DNA

Aids-associated retrovirus

408
gaaatatgtt gactca 16

409

15

DNA

Aids-associated retrovirus

409
aatttgttga ctcag 15

410

16

DNA

Aids-associated retrovirus

410
gaaatttgtt gactca 16

411

15

DNA

Aids-associated retrovirus

411
aatatgttga cccag 15

412

15

DNA

Aids-associated retrovirus

412
aaatatgttg accca 15

413

15

DNA

Aids-associated retrovirus

413
aatatgatga cccag 15

414

15

DNA

Aids-associated retrovirus

414
acagatgatg accca 15

415

15

DNA

Aids-associated retrovirus

415
aacatgttga ctcag 15

416

16

DNA

Aids-associated retrovirus

416
aaacatgttg actcag 16

417

14

DNA

Aids-associated retrovirus

417
tgttgactca gctt 14

418

15

DNA

Aids-associated retrovirus

418
ctgttgactc agctg 15

419

14

DNA

Aids-associated retrovirus

419
ctatgactca gctt 14

420

14

DNA

Aids-associated retrovirus

420
ctgatgactc agcg 14

421

14

DNA

Aids-associated retrovirus

421
tgactacaca gctt 14

422

14

DNA

Aids-associated retrovirus

422
ctgttgacac agcg 14

423

15

DNA

Aids-associated retrovirus

423
aatctgttga cacag 15

424

14

DNA

Aids-associated retrovirus

424
aacctgttga ctca 14

425

14

DNA

Aids-associated retrovirus

425
aaacctgttg actc 14

426

14

DNA

Aids-associated retrovirus

426
gaaacctgtt gact 14

427

15

DNA

Aids-associated retrovirus

427
tgttgactca gattg 15

428

17

DNA

Aids-associated retrovirus

428
tgttgactca gattggg 17

429

16

DNA

Aids-associated retrovirus

429
gttgactcag attggg 16

430

15

DNA

Aids-associated retrovirus

430
tgttgacaca gcttg 15

431

15

DNA

Aids-associated retrovirus

431
ctgttgacac agctt 15

432

16

DNA

Aids-associated retrovirus

432
gttgacacag cttggg 16

433

15

DNA

Aids-associated retrovirus

433
tgttgactca gcttg 15

434

13

DNA

Aids-associated retrovirus

434
gttgactcag atg 13

435

14

DNA

Aids-associated retrovirus

435
gttgactcag cttg 14

436

15

DNA

Aids-associated retrovirus

436
tgttgaccca gattg 15

437

14

DNA

Aids-associated retrovirus

437
gttgacccag attg 14

438

16

DNA

Aids-associated retrovirus

438
gttgacccag attggg 16

439

15

DNA

Aids-associated retrovirus

439
tgatgactca gattg 15

440

17

DNA

Aids-associated retrovirus

440
tgatgactca gattggg 17

441

16

DNA

Aids-associated retrovirus

441
gatgactcag attggg 16

442

16

DNA

Aids-associated retrovirus

442
gatgactcag attggt 16

443

15

DNA

Aids-associated retrovirus

443
ctgatgactc agctt 15

444

15

DNA

Aids-associated retrovirus

444
tgatgactca gcttg 15

445

15

DNA

Aids-associated retrovirus

445
aaatctgttg actca 15

446

15

DNA

Aids-associated retrovirus

446
aaatctgttg actca 15

447

15

DNA

Aids-associated retrovirus

447
aaatctgttg actca 15

448

15

DNA

Aids-associated retrovirus

448
aatctgatga ctcag 15

449

15

DNA

Aids-associated retrovirus

449
aaatctgatg actca 15

450

15

DNA

Aids-associated retrovirus

450
gaaatctgat gactc 15

451

15

DNA

Aids-associated retrovirus

451
ctgatgactc agatg 15

452

12

DNA

Aids-associated retrovirus

452
agaaatatga tg 12

453

16

DNA

Aids-associated retrovirus

453
aagaaatatg atgact 16

454

16

DNA

Aids-associated retrovirus

454
aagaaatctg atgact 16

455

16

DNA

Aids-associated retrovirus

455
aagaaatata atgact 16

456

16

DNA

Aids-associated retrovirus

456
aaatataatg actcag 16

457

15

DNA

Aids-associated retrovirus

457
aatatgatga cccag 15

458

15

DNA

Aids-associated retrovirus

458
aacctgatga ctcag 15

459

16

DNA

Aids-associated retrovirus

459
agaaatttga tgactc 16

460

19

DNA

Aids-associated retrovirus

460
aaatttgatg actatgact 19

461

14

DNA

Aids-associated retrovirus

461
acctgatgac tcag 14

462

16

DNA

Aids-associated retrovirus

462
aatctgatga ctcaga 16

463

17

DNA

Aids-associated retrovirus

463
atctgatgac tcagatg 17

464

14

DNA

Aids-associated retrovirus

464
atctgatgac tcag 14

465

16

DNA

Aids-associated retrovirus

465
ctgatgactc agattg 16

466

16

DNA

Aids-associated retrovirus

466
agaaatctga tgactc 16

467

15

DNA

Aids-associated retrovirus

467
agaaatctga tgact 15

468

15

DNA

Aids-associated retrovirus

468
gaagaaatct gatga 15

469

16

DNA

Aids-associated retrovirus

469
ggaagaaatc tgatga 16

470

16

DNA

Aids-associated retrovirus

470
gaagaaatct gatgac 16

471

14

DNA

Aids-associated retrovirus

471
agaaatctga tgac 14

472

15

DNA

Aids-associated retrovirus

472
aatctgttaa ctcag 15

473

16

DNA

Aids-associated retrovirus

473
tctgttaact cagatt 16

474

16

DNA

Aids-associated retrovirus

474
atctgttaac tcagag 16

475

15

DNA

Aids-associated retrovirus

475
agaaatttgt tgact 15

476

15

DNA

Aids-associated retrovirus

476
gaaatttgtt gactc 15

477

15

DNA

Aids-associated retrovirus

477
aatttgttga ctcag 15

478

24

DNA

Aids-associated retrovirus

478
ggaggtttta tcaaagtcag acaa 24

479

24

DNA

Aids-associated retrovirus

479
ggaggtttca ttaaggtaaa acag 24

480

24

DNA

Aids-associated retrovirus

480
ggaggtttta ttaaggtaag acag 24

481

24

DNA

Aids-associated retrovirus

481
ggaggtttta ttaaagtaag acaa 24

482

24

DNA

Aids-associated retrovirus

482
ggaggcttta tcaaagtaag acaa 24

483

24

DNA

Aids-associated retrovirus

483
ggaggtttta tcaaagtcag acaa 24

484

26

DNA

Aids-associated retrovirus

484
ggacctacac cggtcaacat aattgg 26

485

26

DNA

Aids-associated retrovirus

485
ggacctacac ctgccaatat aattgg 26

486

26

DNA

Aids-associated retrovirus

486
ggacctacgc ccttcaacat aattgg 26

487

26

DNA

Aids-associated retrovirus

487
ggaccgacac ctgtcaccat aattgg 26

488

26

DNA

Aids-associated retrovirus

488
ggacctatac ctgtcaacat aattgg 26

489

25

DNA

Aids-associated retrovirus

489
aaaaaatctg atgactcaga ttggc 25

490

25

DNA

Aids-associated retrovirus

490
aagaactctg ttgactcagc ttgga 25

491

25

DNA

Aids-associated retrovirus

491
aagaaatatg atgacccagc ttggc 25

492

25

DNA

Aids-associated retrovirus

492
aagaaatata atgactcagc ttgga 25

493

25

DNA

Aids-associated retrovirus

493
aagaaatctg ctgactcaga ttggg 25

494

25

DNA

Aids-associated retrovirus

494
aagaaatctg ttgacacagc ttggc 25

495

25

DNA

Aids-associated retrovirus

495
aagaaatatg ttgactcagc ttggt 25

496

25

DNA

Aids-associated retrovirus

496
aagaaatttg ttgactcaga ttggg 25

497

25

DNA

Aids-associated retrovirus

497
aagaaatatg ttgactcagc ttggt 25

498

25

DNA

Aids-associated retrovirus

498
aagaaatatg ttgactcagc ttgga 25

499

25

DNA

Aids-associated retrovirus

499
aagaaatctg ttgactcagc ttgga 25

500

25

DNA

Aids-associated retrovirus

500
aagaaacctg ttgactcaac ttggt 25

501

21

DNA

Aids-associated retrovirus

501
cagagccaac agccccacca g 21

502

26

DNA

Aids-associated retrovirus

502
ttttcttctg tcaatggcca ttgttt 26

503

23

DNA

Aids-associated retrovirus

503
cctcaaatca ctctttggca acg 23

504

23

DNA

Aids-associated retrovirus

504
cctcagatca ctctttggca acg 23

505

20

DNA

Aids-associated retrovirus

505
cctgtcaaca taattgcaag 20

506

25

DNA

Aids-associated retrovirus

506
ctggtacagt ttcaataggg ctaat 25

507

25

DNA

Aids-associated retrovirus

507
ctggtacagt ttcaatagga ctaat 25

508

25

DNA

Aids-associated retrovirus

508
ctggtacagt ctcaatagga ctaat 25

509

25

DNA

Aids-associated retrovirus

509
ctggtacagt ctcaataggg ctaat 25

510

14

DNA

Aids-associated retrovirus

510
gtcatcagat ttct 14

511

20

DNA

Aids-associated retrovirus

511
agggggaatt ggaggtttta 20

512

15

DNA

Aids-associated retrovirus

512
atgatagggg gaatt 15

513

14

DNA

Aids-associated retrovirus

513
aagaatgata gggg 14

514

15

DNA

Aids-associated retrovirus

514
tctgttgact cagat 15

515

15

DNA

Aids-associated retrovirus

515
gagtcaacag agttc 15

516

15

DNA

Aids-associated retrovirus

516
aggttttgcc aaagt 15

517

17

DNA

Aids-associated retrovirus

517
acacctatca acataat 17

518

15

DNA

Aids-associated retrovirus

518
acacctacca acata 15

519

14

DNA

Aids-associated retrovirus

519
acacctacca acgt 14

520

30

DNA

AIDS-associated retrovirus

520
acaggagcag atgatacagt attagaagaa 30

521

33

DNA

AIDS-associated retrovirus

521
ccaaaaatga tagggggaat tggaggtttt atc 33

522

30

DNA

AIDS-associated retrovirus

522
aaaatgatag ggggaattgg aggttttatc 30

523

24

DNA

AIDS-associated retrovirus

523
ggaggtttta tcaaagtaag acag 24

524

30

DNA

AIDS-associated retrovirus

524
ggacctacac ctgtcaacat aaatggaaga 30

525

27

DNA

AIDS-associated retrovirus

525
ggaagaaatc tgttgactca gattggt 27

526

23

DNA

Artificial Sequence

Synthetic oligonucleotide

526
cctcaratca ctctttggga acg 23

527

21

DNA

Artificial Sequence

Synthetic oligonucleotide

527
taaccttctt tagacaactg a 21

528

20

DNA

Artificial Sequence

Synthetic oligonucleotide

528
cctgtcaaca taattggaag 20

529

25

DNA

Artificial Sequence

Synthetic oligonucleotide

529
taatcrggat aactytgaca tggtc 25

Method for detection of drug-selected mutations in the HIV protease gene

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