Patent Application
20120058515
The present invention relates generally to a method of regulating oligonucleotide functionality and, more particularly, to a method of regulating the functionality of a primer or probe. The method of the invention is designed to provide a means to selectively inactivate or activate the functionality of an oligonucleotide, such as a primer, thereby providing means to regulate the progress of any method using that oligonucleotide. The development of a means to regulate the functionality of an oligonucleotide, such as a primer, is useful in a range of applications including, but not limited to, amplification reactions such as PCR, isothermal amplification and nucleic acid strand extension. With respect to amplification reactions, these have wide utility including the diagnosis and/or monitoring of disease conditions which are characterised by specific gene sequences and the characterisation or analysis of specific gene regions of interest.
The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.
Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.
The polymerase chain reaction (PCR) is a technique which is utilised to amplify specific regions of a DNA strand. This may be a single gene, just a part of a gene or a non-coding sequence. Most PCR methods typically amplify DNA fragments of up to 10 kilo base pairs (kb), although some techniques allow for amplification of fragments up to 40 kb in size (Cheng et al., 1994, Proc Natl Acad Sci. 91:5695-5699).
PCR, as currently practiced, requires several basic components (Sambrook and Russel, 2001, Molecular Cloning: A Laboratory Manual, 3rd Ed.). These components are:
PCR is carried out in small reaction tubes (0.2-0.5 ml volumes), containing a reaction volume typically of 15-100 μl which are inserted into a thermal cycler. This machine heats and cools the reaction tubes within it to the precise temperature required for each step of the reaction. Most thermal cyclers comprise heated lids to prevent condensation on the inside of the reaction tube caps. Alternatively, a layer of oil may be placed on the reaction mixture to prevent evaporation.
Accordingly, PCR is a method that allows exponential amplification of DNA sequences within a longer DNA molecule. The reaction involves a number of cycles of amplification, and in each cycle the template for each molecular reaction is either a strand of the initial DNA in the sample or a strand of DNA synthesised in a preceding cycle. Each PCR cycle involves the following steps
Typically the PCR reagents and conditions are chosen so that denaturation, hybridisation and extension occur at close to maximum efficiency and as a result the amount of the desired sequence increases with each cycle by a factor of close to 2. Substantial amplification occurs by the end of the PCR eg a 30 cycle PCR will result in amplification of the original template by a factor of almost 230 (1,000,000,000). This degree of amplification facilitates detection and analysis of the amplified product.
After a number of cycles of amplification, the PCR may be terminated and the product analysed in various ways, most commonly by gel electrophoresis. When the PCR is carried out using a fixed number of cycles, the amount of amplified product is usually not closely related to the amount of input target DNA, and this type of PCR is rather a qualitative tool for detecting the presence or absence of a particular DNA and/or for providing sufficient target DNA for further analysis.
In order to measure messenger RNA (mRNA), the method uses reverse transcriptase to initially convert mRNA into complementary DNA (cDNA) which is then amplified by PCR and analyzed by agarose gel electrophoresis. Reverse transcription followed by end-point PCR is similarly essentially a qualitative technique.
In order to provide quantification capability, real-time PCR was developed. This procedure follows the general pattern of PCR, but the amplified DNA is quantified during each cycle. Two common methods of quantification are the use of fluorescent dyes that intercalate with double-stranded DNA and modified DNA oligonucleotide primers or probes the fluorescence of which changes during one of the steps of the PCR. Frequently, real-time polymerase chain reaction is combined with reverse transcriptase polymerase chain reaction to quantify low abundance messenger RNA (mRNA), enabling a researcher to quantify relative gene expression at a particular time or in a particular cell or tissue type.
(i) Real-Time PCR Using Dyes Binding to Double-Stranded DNA
A DNA-binding dye, such as Sybr Green, binds to all double-stranded (ds)DNA in a PCR reaction, causing increased fluorescence of the dye. An increase in DNA product during PCR therefore leads to an increase in fluorescence intensity which is measured at each cycle, thus allowing DNA concentrations to be quantified.
(ii) Fluorescent Reporter Sequence Methods
A number of different methods using fluorescent reporter primers or probes have been developed and they tend to be more accurate and reliable than use of DNA binding dyes. They use one or more DNA primers or probes to quantify only the DNA to which the primer or probe hybridises. Use of a reporter probe, such as a Taqman probe, significantly increases specificity and may allow quantification even in the presence of some non-specific DNA amplification. Use of sequence-specific primers or probes allows for multiplexing—assaying for several different amplified products in the same reaction by using specific sequences or probes with different-coloured labels, provided that all targets are amplified with similar efficiency.
In terms of quantification, relative concentrations of DNA present during the exponential phase of the reaction are determined by plotting fluorescence against cycle number on a logarithmic scale. A threshold for increase of fluorescence above background or decrease below background (depending on the precise method) is determined. The cycle at which the fluorescence from a sample crosses the threshold is called the cycle threshold, Ct.
The amount of target DNA is then determined by comparing the test results to the results produced by one or more standards. When the target DNA is genomic DNA, then a series of standards, usually 10 fold dilutions of a known amount of the target DNA, are commonly used. When the target DNA is cDNA, then one or more internal standards of the cDNA of another gene are commonly used.
A variation of traditional PCR, designed to increase the specificity of the PCR amplification, is the nested PCR reaction. In this amplification reaction, two sets of primers are used in two successive reactions. In the first, one pair of primers is used to generate DNA products, which may also contain products amplified from non-target areas. The products from the first PCR are then used to start a second, using one (‘hemi-nesting’) or two different primers whose binding sites are located (nested) within the first set. The specificity of all of the primers is combined, usually leading to a single product.
Nested PCR is conventionally performed by carrying out an initial PCR in one reaction tube, transferring an aliquot of the amplified products into a second reaction tube, and then carrying out a second PCR. This procedure has two disadvantages. It is more complex than a single PCR and, more importantly, it carries the risk of contaminating the environment with the amplified products of the first PCR, which may lead to contamination of subsequent experimental procedures. For this reason, several methods have been developed for carrying out the successive PCRs in the one reaction tube.
Carrying out two rounds of PCR in the one reaction tube involves adding the primers for the two rounds into the initial reaction mixture. The methods that have then been used for producing two sequential rounds of PCR, the first using the outer pair of primers and the second using the inner pair, include:
The principle underlying all of these methods is to produce, at some point, a rapid or gradual loss of the activity of the primers for the first round PCR so that ongoing amplification depends progressively on the activity of the primers for the second round PCR. However, all of these methods have disadvantages, the nature of the disadvantage depending upon the method. Their robustness varies and the reaction conditions may need to be adjusted depending on the sequence of interest which is to be amplified. Amplification may be inefficient, in some cases throughout the first round PCR and in other cases during the transition from the first round to the second round PCR. As a consequence, some of the approaches are not widely used in practice, whereas others are used only for detection and not for quantification.
Most amplification methods which are routinely performed rely on thermal cycling to achieve amplification. There have also been developed a range of isothermal amplification techniques, such as transcription mediated amplification, strand displacement amplification, loop mediated isothermal amplification, isothermal multiple displacement amplification and helicase dependent amplification. However, these isothermal amplification reactions tend to be more complex than the polymerase chain reaction and somewhat more difficult to optimise. They therefore tend to be used more for end-point detection rather than for quantification. They do nonetheless have the advantage that a thermal cycler is not required thereby making these methods significantly simpler to set up. This can be a significant issue in environments where access to sophisticated equipment, such as thermal cyclers, is not possible. It is also possible to perform nested isothermal amplification although this is not often done. These various isothermal reactions are either completely DNA-based or at some stage involve cyclical production of RNA, but they all involve the use of DNA primers which hybridise and extend under the action of a DNA polymerase.
In work leading up to the present invention, there has been developed a method of regulating oligonucleotide functionality, such as primer functionality, in terms of both its activation and inactivation, based on the use of antisense oligonucleotides. This development has enabled improvement in the efficiency of, for example, primer-based technologies such as nucleic acid amplification. For example, the method of the present invention has enabled the development of a single tube nested PCR in which specificity and efficiency are both improved based on the ability to control primer functionality. This enables efficient amplification using selected primers followed by efficient amplification using other primers. This amplification method is particularly useful due to its utility with both isothermal and thermal nucleic acid amplification reactions. However, the present invention is useful beyond merely its application in the context of amplification reactions and extends to utility with respect to any reaction in which the activity of an oligonucleotide is required to be modulated during the course of the reaction.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
As used herein, the term “derived from” shall be taken to indicate that a particular integer or group of integers has originated from the species specified, but has not necessarily been obtained directly from the specified source. Further, as used herein the singular forms of “a”, “and” and “the” include plural referents unless the context clearly dictates otherwise.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
One aspect of the present invention is directed to a method of modulating the functionality of an oligonucleotide of interest, said method comprising:
In another embodiment the present invention is directed to a method of modulating the capacity of a primer to undergo extension along a target nucleic acid, said method comprising:
A further aspect of the present invention is directed to a method of modulating the capacity of an oligonucleotide of interest to undergo extension along a target nucleic acid, said method comprising hybridising said oligonucleotide to the 3′ end of an antisense oligonucleotide and facilitating the 3′ extension of said oligonucleotide of interest along said antisense oligonucleotide wherein the antisense nucleotide sequence along which said oligonucleotide of interest extends generates an extension of said oligonucleotide which is either:
In another aspect there is provided a method of modulating the functionality of an oligonucleotide of interest, said method comprising:
In one embodiment, said nucleic acid is DNA.
In another embodiment said oligonucleotide of interest is directed to a target molecule which is either a protein or a nucleic acid molecule.
In another embodiment functionality of said oligonucleotide of interest is functionality as a primer, aptamer, DNAzyme, RNase or ribozyme.
In another embodiment there is provided a method of amplifying a target DNA, said method comprising:
In still another embodiment the present invention is directed to a method of amplifying a target DNA, said method comprising:
In yet another embodiment the present invention is directed to a method of amplifying a target DNA, said method comprising:
In yet still another embodiment there is provided a method of amplifying a target DNA, said method comprising:
In still yet another embodiment there is provided a method of amplifying a target DNA t, said method comprising:
The present invention is predicated, in part, on the design of antisense technology which enables the inducible inactivation or activation of one or more oligonucleotides which participate in a reaction. For example, the method of the present invention is very useful when the oligonucleotides are primers in the context of a nested amplification reaction where one can design a single tube nested PCR method which maintains a constant and optimal level of efficiency. This development has enabled a degree of control and robustness not previously available in the context of primer-based technologies. Still further, in the context of amplification methods this method is applicable to both thermal and isothermal nucleic acid amplification reactions.
Accordingly, one aspect of the present invention is directed to a method of modulating the functionality of an oligonucleotide of interest, said method comprising:
Reference to an “oligonucleotide of interest” should be understood as a reference to any DNA or RNA molecule in respect of which functionality is sought to be regulated or modified. To this end, reference to “functionality” should be understood as a reference to the ability of the oligonucleotide to perform an action on or in association with a target molecule e.g. by acting as a primer which undergoes extension, by hybridising to a nucleic acid target or other target molecule, or by acting as a nucleic acid enzyme. Reference to “modulate” should be understood as referring to either the acquisition of a new function of the oligonucleotide or the effecting of a change in the level of a pre-existing function, which may be achieved by effecting a change in the concentration of free (i.e. unhybridised) oligonucleotide or by a change in its sequence or structure. It should be appreciated that to the extent that the oligonucleotide of interest is a primer, in some scenarios the primer may hybridise to its target nucleic acid but if it cannot extend, then that primer is regarded as non-functional in the context of the present invention. The oligonucleotide of interest is one which, in the context of its functionality, is required, among other things, to hybridise to a complementary molecular region, most commonly a target nucleic acid region. This may occur in the context of a wide variety of different types of reactions including, but not limited to, probe based technologies (such as Southern blotting), primer based technologies (such as nucleic acid amplification), nucleic acid extension, and reactions which involve the oligonucleotide binding to and/or modifying a target (such as when the oligonucleotide of interest acts as an aptamer, DNAzyme or ribozyme). In one embodiment, said oligonucleotide of interest is a primer.
According to this embodiment the present invention is directed to a method of modulating the capacity of a primer to undergo extension along a target nucleic acid, said method comprising:
Reference to a “target molecule”, should be understood as a reference to a molecule to which said oligonucleotide of interest hybridises or binds. The target molecule may be any non-proteinaceous or proteinaceous molecule, such as a nucleic acid or protein. It should be understood, however, that a target nucleic acid molecule is not the antisense oligonucleotide or another primer which uses said antisense oligonucleotide of interest as a template for extension or ligation.
To the extent that the target molecule is a nucleic acid, such as a nucleic acid which is sought to be amplified, reference to a nucleic acid “region of interest” or “target nucleic acid” should be understood as a reference to any region of DNA or RNA which is sought to be amplified or probed. This may be a gene, part of a gene or an intergenic region. To this end, reference to “gene” should be understood as a reference to a DNA molecule which codes for a protein product, whether that be a full length protein or a protein fragment. In terms of chromosomal DNA, the gene will include both intron and exon regions. However, to the extent that the DNA of interest is cDNA, such as might occur if the DNA of interest is vector DNA or reverse transcribed mRNA, there may not exist intron regions. Such DNA may nevertheless include 5′ or 3′ untranslated regions. Accordingly, reference to “gene” herein should be understood to encompass any form of DNA which codes for a protein or protein fragment including, for example, genomic DNA and cDNA. The subject nucleic acid region of interest may also be a non-coding portion of genomic DNA which is not known to be associated with any specific gene (such as the commonly termed “junk” DNA regions). It may be any region of genomic DNA produced by recombination, either between 2 regions of genomic DNA or 1 region of genomic DNA and a region of foreign DNA such as a virus or an introduced sequence. It may be a region of a partly or wholly synthetically or recombinantly generated nucleic acid molecule. The subject nucleic acid region of interest may also be a region of DNA which has been previously amplified by any nucleic acid amplification method, including polymerase chain reaction (PCR) (i.e. it has been generated by an amplification method).
The subject “nucleic acid” region or “oligonucleotide” may be DNA or RNA or derivative or analogue thereof. Where the region of interest is a DNA sequence which encodes a proteinaceous molecule it may take the form of genomic DNA, cDNA which has been generated from a mRNA transcript, or DNA generated by nucleic acid amplification. However where the subject DNA does not encode a protein, either genomic DNA or synthetically or recombinantly generated DNA may be the subject of analysis. As would be appreciated by the skilled person, both synthetically and recombinantly generated DNA may also encode all or part of a protein. However, if the subject method is directed to an RNA region of interest, it would be appreciated that it will usually first be necessary to reverse transcribe the RNA to DNA, such as using RT-PCR. The subject RNA may be any form of RNA, such as mRNA, primary RNA transcript, ribosomal RNA, transfer RNA, micro RNA or the like. Preferably, said nucleic acid region of interest is a DNA region of interest. To this end, said DNA includes DNA generated by reverse transcription from RNA which is ultimately the subject of analysis, and DNA generated by a nucleic acid amplification method such as PCR.
Accordingly, another aspect of the present invention is directed to a method of modulating the functionality of a DNA oligonucleotide of interest, said method comprising:
In one embodiment, said oligonucleotide of interest is directed to a target molecule which is either a protein or a nucleic acid molecule.
In another embodiment, said oligonucleotide of interest is a primer, aptamer, DNAzyme, ribozyme or RNase.
The present invention more particularly provides a method of modulating the capacity of a primer to undergo extension along a target DNA, said method comprising:
Reference to “DNA” should be understood as a reference to deoxyribonucleic acid or derivative or analogue thereof. In this regard, it should be understood to encompass all forms of DNA, including cDNA and genomic DNA. The nucleic acid molecules of the present invention may be of any origin including naturally occurring (such as would be derived from a biological sample), recombinantly produced or synthetically produced.
Reference to “derivatives” should be understood to include reference to fragments, homologs or orthologs of said DNA from natural, synthetic or recombinant sources. “Functional derivatives” should be understood as derivatives which exhibit any one or more of the functional activities of DNA. The derivatives of said DNA sequences include fragments having particular regions of the DNA molecule fused to other proteinaceous or non-proteinaceous molecules. “Analogs” contemplated herein include, but are not limited to, modifications to the nucleotide or nucleic acid molecule such as modifications to its chemical makeup or overall conformation. This includes, for example, incorporation of novel or modified purine or pyrimidine bases or modification to the manner in which nucleotides or nucleic acid molecules interact with other nucleotides or nucleic acid molecules such as at the level of backbone formation or complementary base pair hybridisation. The biotinylation or other form of labelling of a nucleotide or nucleic acid molecules is an example of a “functional derivative” as herein defined.
Preferably, said DNA is a gene or gene fragment, a chromosomal gene translocation breakpoint or DNA produced by prior nucleic acid amplification, such as PCR. The DNA of interest may be chemically synthesised or may be derived from the DNA or RNA of any organism including, but not limited to, any animal, plant, bacterium or virus.
Without limiting the present invention to any one theory or mode of action, it has been determined that oligonucleotide functionality can be regulated via antisense technology. Specifically, a method for either inactivating and/or thereafter activating the functionality of an oligonucleotide, such as a primer, has been developed based on the use of antisense molecules together with effecting a sequence change in either the oligonucleotide or the antisense molecule such that the functionality of the oligonucleotide (e.g. the capacity of a primer to hybridise or undergo extension) is modulated. Reference to “extension” in this context should be understood as a reference to the ability of that primer to be induced by a polymerase to undergo 3′ nucleic acid extension along a template to which it is hybridised. Such extension reactions are predominantly utilised in the context of nucleic acid amplification reactions such as PCR. However, it would be appreciated by the person of skill in the art that there are also other applications for primer extension reactions, such as production of single stranded DNA. The sequence change can be effected by any suitable means including, for example, the use of antisense molecules which directly block the capacity of an oligonucleotide to extend subsequently to its hybridisation to a target (or conversely can be degraded to reveal a functional primer) or which themselves undergo extension or form the template for oligonucleotide extension in order to similarly regulate the functionality of the primer.
In classical PCR, the primers and reaction conditions are designed so that hybridisation and extension of the forward and reverse primers occur at or close to maximum efficiency so that the number of amplicons approximately doubles with each cycle, resulting in efficient exponential amplification. An adequate concentration of primers is important in achieving optimal efficiency. Greater specificity of DNA amplification can be obtained if two or more sets of primers are used in successive reactions. In this way, the impact of any non-specific products amplified from non-target areas can be minimised by conducting a further amplification using primers whose binding sites are located internal to those of the first set. The specificity of all the primers is combined, usually leading to a single product. A nested PCR can be performed as a sequential series of separate reactions or it can be performed in a single reaction container. Although the appeal of a single tube nested PCR reaction is obvious, the approach has suffered from problems such as the inconsistency of efficiency between different rounds of amplification, this leading to significant limitations where it has been desired to obtain a quantitative result. In order to effect amplification from different primer sets in a sequential order, many of the prior art methods have largely focussed on the method of lowering the concentration of the outer primers and having a high annealing temperature for the first phase and a lower temperature for the second phase. However, the efficiency of the amplification has differed significantly as between the different primer sets, thereby limiting the utility of this method.
Accordingly, in the context of a nested PCR embodiment of the present invention, the inner primers can be inactivated while the outer primers are undergoing extension. Thereafter, the outer primers can be rendered inactive using antisense oligonucleotides which hybridise to them while inner primer functionality is restored, either by a change in the conditions of the reaction or via a hybridisation between or dissociation of the antisense oligonucleotides and the inner primers. This interaction can occur in any one of a number of ways, which will be discussed in more detail hereafter Hybridisation of the antisense oligonucleotide to the outer and/or inner primers will result in a decrease in concentration of free primers which in turn will lead to inefficiency of hybridisation of that primer to the DNA target template, whereas dissociation will have the opposite effect. Hybridisation is subject to a number of variables. The strength and extent of hybridisation of the antisense oligonucleotide to the primer will depend on its sequence and length, the presence of any mismatches or modifications which either decrease or increase hybridisation, the concentration of the oligonucleotide and the annealing temperature or time. Except for an antisense oligonucleotide for which the most 3′ base of the antisense oligonucleotide hybridises to the most 5′ base of the primer, hybridisation of the antisense oligonucleotide will result in it extending in the 3′ direction. This will produce a longer antisense oligonucleotide which will hybridise more strongly during subsequent cycles.
Reference to an “oligonucleotide” should be understood as a reference to any molecule comprising a sequence of nucleotides, or functional derivatives or analogues thereof. Reference to a “primer” should be understood as a reference to an oligonucleotide which has the function of hybridising to a nucleic acid target and has the actual or potential function of undergoing an extension or ligation reaction. Reference to an “antisense oligonucleotide” should be understood as a reference to an oligonucleotide part or all of which has a complementary sequence to part or all of the oligonucleotide of interest. It should be understood that an oligonucleotide, primer or antisense oligonucleotide may comprise non-nucleic acid components. For example, the oligonucleotide, primer or antisense oligonucleotide may also comprise a non-nucleic acid tag such as a fluorescent or enzymatic tag or some other non-nucleic acid component which facilitates the use of the molecule as a probe or which otherwise facilitates its detection or immobilisation. The oligonucleotide, primer or antisense oligonucleotide may also comprise additional nucleic acid components. In another example, the oligonucleotide, primer or antisense oligonucleotide may be a protein nucleic acid which comprises a peptide backbone exhibiting nucleic acid side chains. Preferably, said primer is a DNA primer.
The antisense oligonucleotide of the present invention is designed to hybridise to an oligonucleotide of interest, such as a primer or probe. Accordingly, by “directed to” is meant that the antisense oligonucleotide hybridises to a region of the subject oligonucleotide of interest. The antisense oligonucleotide may, therefore, hybridise across only part of the oligonucleotide of interest or it may hybridise across the full length of the oligonucleotide of interest. It should be understood that although the antisense oligonucleotide is referred to as an “antisense” oligonucleotide, the use of this terminology is intended to indicate that the nucleotide sequence of this antisense is designed to enable the antisense to hybridise to the subject oligonucleotide of interest, such as a primer. It should also be understood that in terms of the nomenclature used in this specification to describe the antisense oligonucleotide, the end of the antisense oligonucleotide which hybridises to the 3′ end of the oligonucleotide of interest is referred to as the 5′ end of the antisense oligonucleotide while the other end is the 3′ end of the antisense oligonucleotide.
Without limiting the present invention to any one theory or mode of action, the reaction of the present invention may be designed to use either one, or more than one, type of antisense oligonucleotide, depending on the number of oligonucleotides of interest which are present in the reaction and the function of which it is designed to modulate. It should also be understood that in the context of nested amplification reactions, the antisense oligonucleotides which are utilised may be directed towards either one or more forward primers or one or more reverse primers. In another alternative, the reaction may be designed to use both antisense oligonucleotides directed to one or more forward primers and antisense oligonucleotides directed to one or more reverse primers.
In one aspect, the method of the present invention is based on using an antisense:oligonucleotide of interest hybridisation event as the basis to effect a sequence change in either one of these molecules or a further oligonucleotide molecule associated with this complex such that the functionality of the oligonucleotide of interest is ultimately modulated. Reference to “modulate” or “modulation” is intended to mean either that an oligonucleotide of interest which is capable of hybridisation or binding to a target molecule and/or undergoing nucleotide extension is rendered non-functional or that the converse occurs, that is that an oligonucleotide of interest which is inactive in that it cannot undergo hybridisation or binding to a target molecule and/or extension, is rendered functional. In the latter situation, the oligonucleotide of interest may have been rendered inactive due to application of the method of the present invention or it may have been initially produced in this form and thereafter at the appropriate time it can be rendered functional. To this end, reference to “nucleotide sequence change” should be understood as a reference to any change to the nucleotide sequence of an oligonucleotide relative to the sequence prior to the change event and includes, but is not limited to, nucleotide extension of the oligonucleotide of interest, antisense molecule or other oligonucleotide forming part of an oligonucleotide complex, mutation or addition or deletion of one or more nucleotides or degradation of the nucleic acid molecule.
Methods for achieving this nucleotide sequence change include but are not limited to the following:
The antisense oligonucleotides in one aspect of the present invention function on the basis of providing a template against which a hybridised functional oligonucleotide of interest, such as a primer, extends and is thereby rendered non-functional or the reverse occurs. This is more specifically described as follows:
(i) 5′ Tag on the Antisense Oligonucleotide.
This mechanism for inhibiting extension of a primer is based on designing the antisense oligonucleotide to hybridise to the 3′ end of a primer and locating an oligonucleotide tag at the 5′ end of the antisense oligonucleotide (also herein interchangeably referred to as an oligonucleotide extension). A schematic diagram of this mechanism is shown in
When the primer hybridises to the tagged antisense oligonucleotide, the primer extends in the 3′ direction. Following dissociation of this duplex the extended primer may hybridise to the complementary template strand. For efficient extension along the template, the sequence of the primer extension must be complementary to that sequence of the template strand which hybridises to the extension, and for this to be the case the sequence of the oligonucleotide tag must be the same as that sequence of the template strand which hybridises to the extension. However, if the sequence of the oligonucleotide tag differs from that sequence of the template strand which hybridises to the extension, further extension and amplification will not take place ie. the primer will have been inactivated. As a consequence, there occurs progressive inactivation of primer molecules with increasing cycle number and this phenomenon synergises with the direct inhibitory effect produced by hybridisation of the antisense oligonucleotide to the outer primer.
Those skilled in the art will appreciate that the essential property of the tag is to act as a template for 3′ extension of the primer such that the extended primer is unable to act during subsequent amplification. This can be achieved by comprising the 5′ antisense oligonucleotide tag of either normal nucleotides or modified nucleotides, such as either iso-deoxycytosine or iso-deoxyguanine (the complementary nucleotide must be present in the reaction). The greater the difference between the sequence of the oligonucleotide tag and the sequence of the template strand which would hybridise to the extension, the greater the degree of primer inactivation. Particularly efficient inactivation is produced by making the sequence of the oligonucleotide tag exactly complementary to that sequence of the template strand which would hybridise to the extension. Accordingly, as the amplification reaction progresses, either with repetitive annealing during thermal cycling or with annealing during the constant temperature of an isothermal reaction, some molecules of the antisense oligonucleotide will hybridize to the primer. The hybridized primer molecules will extend in the 3′ direction along the tag, the sequence of which has been chosen so that the 3′ end of the extended primer is now mismatched to the corresponding sequence of the template. Thus extended primer molecules, although they can still hybridize to the nucleic acid template cannot extend. Thus as the reaction proceeds the primer molecules are progressively inactivated and are unable to lead to amplification.
Reference to “oligonucleotide tag” should be understood as a reference to a nucleotide sequence which is linked to the antisense oligonucleotide of the present invention. In one embodiment, the tag is 1-10 bases in length, preferably 2-5 bases in length and more preferably 2-3 bases in length.
The subject tag is designed such that the nucleotide sequence which is complementary to the sequence of the tag is “mismatched” relative to the nucleotide sequence of the DNA region 5′ of the hybridisation site of the 3′ end of the primer. By “mismatched” is meant that the sequence of the tag is such that subsequently to hybridisation of the primer to the antisense oligonucleotide and the extension of the primer along the tag, only the section of the extended primer which corresponds to the original primer will be able to hybridise to the DNA region of interest and the extended section will be of a sequence which does not facilitate its hybridisation to the DNA region of interest. In this way, any further extension of this primer during amplification is inhibited since the 3′ end of this primer cannot hybridise to the DNA region of interest. Accordingly, when the primer hybridises to the antisense oligonucleotide, the primer extends in the 3′ direction and produces a terminal sequence which prevents efficient extension in the 3′ direction when the primer modified in this way subsequently hybridises to its amplicon template.
During the course of the reaction, either due to a change in annealing temperature or to the course of time during a constant annealing temperature, the antisense oligonucleotide and the primer hybridise, the primer extends to generate a mismatch to its template which prevents extension.
(ii) Inactivation of a Primer Owing to the Formation of a Stem-Loop
A schematic diagram of this mechanism is shown in
Modification of the primer in this way may be produced during the reaction by a change in annealing temperature or may occur during the course of time with a constant annealing temperature. Inactivation of the primer by production of a stem loop structure may be of particular value when it is desired to inactivate the primer to prevent it hybridising to its template or to other regions of the genome. This may be useful when the binding site for the inner primer partly overlaps the binding sites for the outer primer since in this circumstance any hybridisation of the outer primer, even if it is inactive by being unable to extend, may inhibit hybridisation of the inner primer. When the 5′ tag of the antisense oligonucleotide is designed to produce stem-loop formation of the extended primer, in nearly all cases the sequence of the extended primer will be such that, even if the stem-loop unfolds and the extended primer binds to its template, there will still be a mismatch and the extended primer will be inactive.
(iii) Re-Functionalisation of Tagged Primer
A schematic diagram of this mechanism is shown in
As would be appreciated, the primer described in point (i) above can be rendered non-functional by virtue of the extension of the primer to incorporate a nucleotide sequence which is mismatched relative to part of the primer hybridisation site. Such a primer, whether generated according to the method described in point (i) or otherwise synthetically or recombinantly produced, can be reactivated using an antisense oligonucleotide which hybridises to the 3′ end of the primer but comprises an additional oligonucleotide tag, the sequence of which corresponds to the sequence of the nucleic acid region of interest. By facilitating extension of the primer along this tag, an extended primer molecule is generated which comprises regions of sequence at its 5′ and 3′ ends which are complementary to the nucleic acid region of interest. The intervening mismatched sequence is sufficiently short, relative to the complementary regions, thereby enabling hybridisation of the primer to the DNA region of interest and subsequent extension, despite the intervening mismatched sequence.
Accordingly, the primer is initially inactive owing to the presence of two or more nucleotides at its 3′ end which are mismatched to the template. When the antisense oligonucleotide and primer hybridise, the primer extends along the oligonucleotide tag, the sequence of which is such that the sequence of the extension of the primer is now a match to the template.
(iv) Activation of Primer Via Tm Modulation
Another variation of the activation of a primer by extension along an antisense oligonucleotide can be achieved via modulation of the Tm. A schematic diagram of this mechanism is shown in
In relation to the second aspect of the invention, the regulation of functionality of an oligonucleotide of interest is based on extension or degradation of the antisense molecule itself. Essentially, this method can be used to either increase or decrease the concentration of free oligonucleotide of interest which can function as a probe or primer. Specifically:
(i) Use of an Antisense Oligonucleotide which Hybridizes to the 3′ End of the Oligonucleotide of Interest.
As the amplification reaction progresses, either with repetitive annealing during the thermal cycles or with annealing during the constant temperature of an isothermal reaction, some molecules of the antisense oligonucleotide will hybridize to the oligonucleotide of interest and extend in the 3′ direction along the oligonucleotide of interest. A schematic diagram of this mechanism is shown in
A variation to this method, a schematic diagram of which is shown in
(ii) Oligonucleotide Complex
In yet another embodiment, the antisense molecule may form part of an oligonucleotide complex. By “oligonucleotide complex” is meant a complex which comprises two or more associated oligonucleotides. It should be understood that in the context of the present invention, additional oligonucleotides may become associated with the complex or, alternatively, one or more oligonucleotides which form part of the complex may become dissociated or degraded. It should also be understood that the subject “complex” includes reference to separate oligonucleotides which are associated via hybridisation or oligonucleotides which are joined, via, for example, by phosphodiester or other chemical bonds, to form a single sequence which can then form a stem loop structure. This complex therefore comprises a single DNA strand which has functionally distinct regions. For example, the complex may take the form of a stem-loop structure wherein at one end of this structure is the oligonucleotide of interest while at the other end is the antisense sequence. Where the antisense end of this structure folds back on itself to hybridise with the oligonucleotide of interest end, antisense “hybridisation” has occurred. Accordingly, it should be understood that the “antisense oligonucleotide” may be a separate molecule to the oligonucleotide of interest or it may be attached to or otherwise continuous with the oligonucleotide of interest, as described above.
In one example, a schematic diagram of which is shown in
In another example, a schematic diagram of which is shown in
The features of a stem-loop oligonucleotide complex may include, but are not limited to:
However this is a dynamic situation, with the stem opening and closing and the oligonucleotide of interest binding and unbinding. Consequently, the amplification cycles can be designed to facilitate 3′ extension of the stem-loop molecule. With each cycle the antisense molecule will be extended and develop a large stem which will not open up in subsequent cycles. Oligonucleotide of interest molecules will therefore be unable to hybridise and will be able to bind to their DNA region of interest. Again it is desirable that the polymerase mediating 3′ extension of the stem should not have 5′-3′ nuclease activity.
Where there is utilised an antisense oligonucleotide which comprises a stem loop structure with a secondary antisense sequence, the length and Tm of the mini-stem are chosen so that as the reaction progresses, there occurs 3′ extension of the secondary antisense sequence, so that the overall antisense oligonucleotide forms a definitive stem-loop structure with an extended stem. Antisense activity is thus progressively lost and oligonucleotide of interest activity progressively increases.
The antisense sequence may comprise one or a number of ribonucleotides or modified ribonucleotides. If so, then the rate of hydrolysis of dinucleotide bonds will be increased. The ribonucleotide rate of hydrolysis can be further increased incorporating into the reaction a ribonuclease such as RNase A or RNase H together with a divalent cation. Progressive hydrolysis of the antisense sequence will result in dissociation of a linear antisense oligonucleotide from the oligonucleotide of interest or unfolding of the stem-loop and loss of antisense inhibition, so that oligonucleotide of interest activity will gradually increase.
Accordingly, one aspect of the present invention is directed to a method of modulating the capacity of an oligonucleotide of interest to undergo extension along a target nucleic acid, said method comprising hybridising said oligonucleotide to the 3′ end of an antisense oligonucleotide and facilitating the 3′ extension of said oligonucleotide of interest along said antisense oligonucleotide wherein the antisense nucleotide sequence along which said oligonucleotide of interest extends generates an extension of said oligonucleotide which is either:
In one embodiment, said oligonucleotide of interest is a primer.
In another aspect there is provided a method of modulating the functionality of an oligonucleotide of interest, said method comprising:
In one embodiment, said nucleic acid is DNA.
In another embodiment said oligonucleotide of interest is directed to a target molecule which is either a protein or a nucleic acid molecule.
In another embodiment functionality of said oligonucleotide of interest is functionality as a primer, aptamer, DNAzyme, RNase or ribozyme.
Accordingly, there is provided a method of modulating the capacity of a primer to undergo extension along a target DNA, said method comprising hybridising said primer to the 3′ end of an antisense oligonucleotide and facilitating the 3′ extension of said primer along said antisense oligonucleotide wherein the antisense nucleotide sequence along which said primer extends generates a primer extension which is either:
In another aspect there is provided a method of modulating the functionality of a DNA primer said method comprising:
It should be understood that the method of the present invention may be designed such that all the antisense oligonucleotides are of the same type of structure or it may be designed such that any two or more antisense oligonucleotide structure types are used in the one method.
As detailed hereinbefore, there is a wide range of applications for the methods of the present invention including, but not limited to:
Inactivation can be designed to involve the outer primers and the reaction would be performed under conditions which would ensure that the inner primers were active once the outer primers had become inactive. Those skilled in the art would know that such conditions would include (i) an annealing temperature sufficient to enable activity of the inner primers throughout the reaction, or (ii) a stepwise or gradual decrease in annealing temperature such that the inner primers were initially inactive but became active during the course of the reaction, or (iii) use of an antisense oligonucleotide, as previously described, to convert one or more inactive inner primers to activity during the course of the reaction. The use of antisense oligonucleotides to enable performance of a nested PCR in a closed single tube is described in Brisco et al. (2011).
Production of Single-Stranded DNA.
Production of single-stranded DNA towards the end of the nucleic acid amplification reaction can be useful for various purposes including sequencing, quantification, and the ability to use a wide range of probes to detect amplified product. The production of single-stranded DNA by amplification reactions involving one or more antisense oligonucleotides can be achieved by adjusting reaction conditions so that one primer is inactivated earlier in time than the other primer. Those skilled in the art will realise that one way of achieving this is to use only one antisense oligonucleotide and to adjust reaction conditions, such as annealing temperature, so that the target primer becomes inactivated after a defined number of cycles, thus allowing the other primer to continue annealing and extending to produce increasing amounts of single-stranded DNA. An example of this use is shown in
Again, conditions can be such that activation occurs gradually during the course of the reaction, at a constant annealing temperature, or the temperature conditions can be adjusted to control the point in the reaction at which activation occurs.
Accordingly, in one embodiment the present invention enables the inducible inactivation or activation of the primers which are sought to be used in a nested amplification reaction, thereby enabling the design of a single tube nested PCR method which maintains a constant and optimal level of efficiency. More specifically, this development has enabled a degree of control and robustness not previously available in the context of a single tube nested PCR. The method of the present invention is useful in the context of any application which requires the analysis of a specific DNA region of interest, such as a specific gene. Still further, this method is applicable to improve both thermal and isothermal nucleic acid amplification reactions.
According to this embodiment there is provided a method of amplifying a target DNA, said method comprising:
Appreciation of the various factors influencing hybridisation will help those skilled in the art to design a single-tube nested PCR involving one or more antisense oligonucleotide primers such that (a) hybridisation of an antisense oligonucleotide to a primer is minimal or absent during the phase where that primer is required to anneal and extend but occurs during the phase when other primers are required to preferentially anneal and extend and/or (b) hybridisation of an antisense oligonucleotide to another primer is minimal or absent during the phase where that primer is required not to be able to anneal and extend and other primers are required to preferentially anneal and extend. but occurs during the phase when that primer is required to preferentially anneal and extend.
Reference to “forward primer” should be understood as a reference to a primer which amplifies the target DNA in the DNA sample of interest and in the PCR by hybridising to the antisense strand of the target DNA.
Reference to “reverse primer” should be understood as a reference to a primer which amplifies the target DNA in the DNA sample of interest and in the PCR by hybridising to the sense strand of the target DNA.
Reference to “downstream” or “upstream” in relation to location should be understood as reference to location relative to the sense DNA strand, with “downstream” referring to 3′ and “upstream” referring to 5′.
As detailed hereinbefore, a nested nucleic acid amplification reaction, such as a PCR or isothermal reaction, is predicated on the use of two or more sets of forward and reverse primers which are directed to hybridising to progressively more internal sequences within the target DNA. In the context of the present invention, reference to the “first” forward and reverse primer should be understood as a reference to the primers which hybridise at the outermost positions of the target DNA. The “second” forward and reverse primers should be understood as a reference to internal primers. That is, these second primers are designed to hybridise to a sequence which is downstream of the first forward primer and upstream of the first reverse primer, respectively. It is within the skill of the person in the art to design at what intervals along the target DNA these primers will hybridise. It should be understood that one may also adopt the method to incorporate a third forward and reverse primer set which is designed to hybridise to a sequence which is downstream of the second forward primer and upstream of the second reverse primer, respectively.
Without limiting the present invention to any one theory or mode of action, the reaction of the present invention may be designed to use a semi-nested rather than a nested reaction. In this case, either a second forward or a second reverse primer is not used and antisense oligonucleotides directed to the single forward or single reverse primer that remains are not present.
In one embodiment the present invention is directed to a method of amplifying a target DNA, said method comprising:
In another embodiment the present invention is directed to a method of amplifying a target DNA, said method comprising:
In another embodiment there is provided a method of amplifying a target DNA, said method comprising:
In one embodiment, said antisense oligonucleotide hybridises to the 3′ end of said primer and extends along said primer.
In another embodiment, the 3′ end of said antisense oligonucleotide hybridises to the 3′ end of said primer and the 3′ end of said antisense oligonucleotide extends along said primer and the 3′ end of said primer extends along said antisense oligonucleotide.
In still another embodiment there is provided a method of amplifying a target DNA, said method comprising:
The method of the present invention therefore enables an initial amplification off the outermost primers to initially proceed efficiently as the dominant amplification reaction. However, since it is sought to conclude this amplification and to proceed with amplification off the internal second primer set, effecting the hybridisation of the antisense oligonucleotide to the first forward primer results in the generation of primers which are effectively blocked from undergoing any further extension in the context of the target DNA. Together with release of the internal primers from the antisense oligonucleotide to which they are bound, the ongoing unwanted amplification of the outer primers is minimised and the amplification of the inner primers can proceed under conditions which facilitate efficient amplification. It should be understood that one need not necessarily utilise antisense oligonucleotides directed to the inner primers, for example if the outer primers effectively themselves hinder hybridisation of the inner primer to their hybridisation site.
As detailed hereinbefore, the present invention is predicated on the fact that in some amplification situations it may be desirable to have one or a pair of primers active during initial cycles of amplification and to then suddenly or gradually become inactive, and to have one or another pair of primers inactive during the initial cycles and to then suddenly or gradually become active during the later cycles.
Facilitating the interaction of the primer with antisense oligonucleotide and the primer with the target DNA may be performed by any suitable method. Those methods will be known to those skilled in the art. To this end, it should be understood that the antisense oligonucleotide and/or the inner primers can be incorporated into the reaction tube at any suitable time point. While incorporation is generally prior to the commencement of the initial amplification cycles, that is together with the forward and reverse outer primers, incorporation of one or more may be performed subsequently to the initial amplification cycles with the outer primers. The mode of incorporation of the antisense oligonucleotide and/or the inner primers will depend on how the skilled person is seeking to perform the amplification reaction but, in general, for ease of use and avoidance of contamination, it is usually desirable to be able to perform the entire reaction in a single tube. Nevertheless, any other method of achieving the steps of the invention can be used.
Methods for achieving primer directed amplification are also very well known to those of skill in the art. In a preferred method, said amplification is polymerase chain reaction.
Reference to a “sample” should be understood as a reference to either a biological or a non-biological sample. Examples of non-biological samples includes, for example, the nucleic acid products of synthetically produced nucleic acid populations. Reference to a “biological sample” should be understood as a reference to any sample of biological material derived from any living creature such as, but not limited to an animal, plant or microorganism (including cultures of microorganisms) and such as, but not limited to, cellular material, blood, mucus, faeces, urine, tissue biopsy specimens, fluid which has been introduced into the body of an animal and subsequently removed (such as, for example, the saline solution extracted from the lung following lung lavage or the solution retrieved from an enema wash), plant material or plant propagation material such as seeds or flowers or a microorganism colony. The biological sample which is tested according to the method of the present invention may be tested directly or may require some form of treatment prior to testing. For example, a biopsy sample may require homogenisation prior to testing. Further, to the extent that the biological sample is not in liquid form, it may require the addition of a reagent, such as a buffer, to mobilise the sample.
To the extent that the target DNA or other molecule is present in a biological sample, the biological sample may be directly tested or else all or some of the nucleic acid material present in the biological sample may be isolated prior to testing. It is within the scope of the present invention for the target nucleic acid molecule to be pre-treated prior to testing, for example inactivation of live virus or being run on a gel. It should also be understood that the biological sample may be freshly harvested or it may have been stored (for example by freezing) prior to testing or otherwise treated prior to testing (such as by undergoing culturing).
Reference to “contacting” the sample with the primer or antisense oligonucleotide should be understood as a reference to facilitating the mixing of the primer with the sample such that interaction (for example, hybridisation) can occur. Means of achieving this objective would be well known to those of skill in the art. Without limiting the present invention to any one theory or mode of action, the extent of contact is determined by the reaction conditions as well as the respective Tm values of the primer and the antisense oligonucleotide. Contact might be manipulated by controlling annealing temperature, so that contact does not occur at a high temperature but occurs at a lower temperature. Or contact might be allowed to occur at a constant rate during a constant temperature. Or extent of contact might be changed progressively owing to progressive change in annealing temperature such as during a touchdown PCR.
The choice of what type of sample is most suitable for testing in accordance with the method disclosed herein will be dependent on the nature of the situation, such as the nature of the condition being monitored. For example, in a preferred embodiment a neoplastic condition is the subject of analysis. If the neoplastic condition is a leukaemia, a blood sample, lymph fluid sample or bone marrow aspirate would likely provide a suitable testing sample. Where the neoplastic condition is a lymphoma, a lymph node biopsy or a blood or marrow sample would likely provide a suitable source of tissue for testing. Consideration would also be required as to whether one is monitoring the original source of the neoplastic cells or whether the presence of metastases or other forms of spreading of the neoplasia from the point of origin is to be monitored. In this regard, it may be desirable to harvest and test a number of different samples from any one mammal. In another example, in another preferred embodiment an infectious disease condition is the subject of analysis. In order to determine the nature and site of infection, samples of blood, bodily fluid, excretion or tissue would be likely to provide a suitable sample for testing and the primers necessary for amplification of particular nucleic acid sequences would be chosen so that detection would be directed against the range of organisms likely to be responsible for the infection. Choosing an appropriate sample and amplification system for any given detection scenario would fall within the skills of the person of ordinary skill in the art.
The term “mammal” to the extent that it is used herein includes humans, primates, livestock animals (e.g. horses, cattle, sheep, pigs, donkeys), laboratory test animals (e.g. mice, rats, rabbits, guinea pigs), companion animals (eg. dogs, cats) and captive wild animals (eg. kangaroos, deer, foxes). Preferably, the mammal is a human or a laboratory test animal. Even more preferably the mammal is a human. The term “organism” to the extent that it is used herein refers to any living entity, including but not limited to animals, plants, bacteria, viruses and fungi.
The present invention is further described by reference to the following non-limiting examples.
Table 1 shows the results of 2 experiments illustrating conversion by antisense of the active outer primer to an inactive state and of the inactive inner primer to an active state.
Tables 2, 3, and 4 show various aspects of the protocol used.
The following method involves the use of antisense oligonucleotides to switch off outer primers and switch on inner primers during the course of a PCR. Conditions are adjusted so that the inner primers are switched on to full functionality before the point at which functionality of the outer primers has been lost.
PCR: Use a 25 μL reaction containing 100 ng of each outer primer, 100 ng of each inner primer, each antisense oligonucleotide at a molar concentration equal to that of the corresponding outer and inner primer, 2.5 mM magnesium, 200 nM dATP, dTTP, dGTP and dCTP, lu Platinum Taq (Invitrogen), a Taqman hydrolysis probe and various masses of DNA in a reaction containing 20 mM Tris-HCl pH 8.4, 50 mM KCl, 2.5 mM MgCl2. The cycling conditions are: 96° C. for 2 minutes then 40 cycles of 94° C. for 15 seconds, 58° C. for 60 seconds, 72° C. for 60 seconds.
Outer and inner Primers: To calculate Tm values use a base stacking model (based on Borer P. N. et al. (1974) J. Mol. Biol. 86:843 & SantaLucia, J. (1998) Proc. Nat. Acad. Sci. USA 95:1460, available at http://www.promega.com/biomath/calc11.htm.), and assuming 2.5 mM magnesium, 200 nM dATP, dTTP, dGTP and dCTP and 100 ng of primer. Use a gradient of annealing temperature to determine the 3ge of temperatures over which the primers produce efficient amplification. Outer and inner primers with a Tm of 60-65° C., are satisfactory.
The inner primers are designed so that the two bases at the 3′ end produce a mismatch when the primers hybridise to the template strand. This results in the primers being inactive during the initial cycles of the PCR.
Antisense Oligonucleotides:
Since the outer primer would extend along the 5′ tag after hybridisation, the 3′-5′ sequence of the tag is complementary to the 3′-5′ sequence of the template strand immediately downstream of the point at which the native primer would hybridise. This design results in a 3 base mismatch between the 3′ end of the extended primer and its template, which blocks the outer primer from extending further, and thus prevents amplification of the target. The sequence of the 3 bases at the 3′ end of the oligonucleotide is such that each potentially hybridising base of the oligonucleotide is the same as the potentially hybridising base of the primer. Again, this design results in a 3 base mismatch, which blocks extension of the oligonucleotide. This strategy prevents production of an unduly long antisense oligonucleotide which has a higher Tm and which might exert unwanted inhibition during the initial high-temperature phase of the PCR. Extension of the antisense oligonucleotide in the 3′ direction can also be prevented by an amine modification at the 3′ end.
The following protocol uses antisense oligonucleotides to sequentially switch off the activity of one set of primers and switch on the activity of another set of primers during isothermal nucleic acid amplification. The primers of the second set are targeted to sites internal to the sites targeted by the first set of primers, ie., the reaction is a nested amplification. Helicase dependent amplification (HDA) is the method used for isothermal amplification.
HDA involves use of the IsoAmp II Universal tHDA kit supplied by Biohelix Corporation. (http://www.biohelix.com/default.asp). The detailed amplification protocol and instructions for HDA are provided at http://www.biohelix.com/pdf/-H0110S_full_version_BH.pdf. and protocol C is the protocol generally used.
Use of antisense oligonucleotides involves the following modifications and/or additions to the reaction:
100 ng each of all 4 primers.
The results of the experiment are shown in
Single-stranded DNA has a number of uses. In this experiment, shown in
PCR
Unless otherwise stated, a 25 μl reaction contained 100 ng of each outer primer, 200 ng of each inner primer, each antisense oligonucleotide at a molar concentration equal to that of the corresponding outer primer, 2.5 mM magnesium, 200 nM each of dATP, dTTP, dGTP, and dCTP, 1 U of Platinum Taq (Invitrogen), a Taqman hydrolysis probe, and 30 pg to 100 ng of DNA. The reactions were set up manually, and PCR was performed in 20 mM Tris-HCl (pH 8.4), 50 mM KCl, and 2.5 mM MgCl2. Unless otherwise stated, the cycling conditions were as follows: hot start, 96° C. for 2 min; 15 cycles of a high-temperature “first phase”, 94° C. for 15 s and 72° C. for 60 s; incubation for 5 min at 58° C.; and then 40 cycles of a low-temperature “second phase”, 94° C. for 15 s, 58° C. for 90 s, and 72° C. for 60 s. This profile is termed the “two-phase protocol”. All results shown for cycle number refer to the total number of cycles from the beginning of the PCR (i.e., they include the initial 15 high-temperature cycles). PCRs were performed on a Bio-Rad IQ5 iCycler (software version 2.0.148.60623). DNA was from approximately 50 ml of blood of a healthy volunteer, extracted by the Qiagen QIAamp DNA Blood Maxi Kit according to the manufacturer's protocol (column purification, RNase not used), and quantified by the Invitrogen Qubit fluorometer with the Quant-iT dsDNA BR Assay Kit.
Outer and Inner Primers
The Tm values were calculated using a base stacking model (based on Borer et al. (J. Mol. Biol. 86 (1974) 843-853) and SantaLucia (J. SantaLucia Jr., Proc. Natl. Acad. Sci. USA 95 (1998) 1460-1465) and available at http://www.promega.com/biomath/calc11.htm) and assuming 2.5 mM magnesium and 600 nM primer. The range of temperatures over which the primers produced efficient amplification was then determined experimentally using a gradient of annealing temperature. Primers with a variety of predicted Tm values were investigated, and ultimately outer primers with Tm values of 77-78° C., which were found to produce efficient amplification at least up to 74° C. annealing, and inner primers with Tm values of 58-62° C., which were found to produce efficient amplification up to 61-64° C. annealing, were used.
Antisense Oligonucleotides
These molecules were based on the antisense sequence to the 3′ end of the outer primers. They were designed to have a Tm that would result in minimal hybridization to the outer primers during the initial high-temperature phase of the PCR but material hybridization during the later low-temperature phase. They carried 5′ and 3′ tags, each consisting of three nucleotides. Because the outer primer would extend along the 5′ tag after hybridization, the 3′-5′ sequence of the tag was the complement of the 3′-5′ sequence of the template strand immediately downstream of the point at which the native primer would hybridize. This design would result in a 3-base mismatch between the 3′ end of the extended primer and its template, with each mismatch being either a:a, g:g, c:c, or t:t. This would block the outer primer from extending further and, thus, prevent amplification of the target. The sequence of the 3 bases at the 3′ end of the oligonucleotide was such that each potentially hybridizing base of the oligonucleotide was the same as the potentially hybridizing base of the outer primer. Again, this design would result in a 3-base mismatch that would block extension of the oligonucleotide along the outer primer. This strategy prevented production, during the initial high-temperature phase of the PCR, of an unduly long antisense oligonucleotide that had a higher Tm and that might exert unwanted inhibition.
The Tm value of each antisense oligonucleotide was determined for the core sequence, excluding the 5′ and 3′ tags, and the members of each pair had approximately the same Tm. The inhibitory activity of each pair was tested by performing a PCR that contained the outer primers and the antisense oligonucleotides but not the inner primers, using as controls both a complete PCR that contained both sets of primers and the antisense oligonucleotides and a PCR that contained only the outer primers. It was found that pairs of oligonucleotides, with each member of the pair having a Tm between 48 and 55° C., resulted in satisfactory inhibition of the outer primers during the low-temperature second phase of the PCR without producing inhibition during the high-temperature first phase. The Tm values for the antisense oligonucleotides used in the final protocol were 49-54° C.
Data Reporting and Statistical Tests
Data are reported following the MIQE (minimum information for publication of quantitative real-time PCR experiments) guidelines (Bustin et al. Clin. Chem. 55 (2009) 611-622). Differences were tested with the Student's t test.
The antisense qPCR strategy was investigated for four genes: a 475-bp segment of NRAS (neuroblastoma RAS viral oncogene homologue, geneID 4893, MIM 164790), a 287-bp segment of BCR (breakpoint cluster region, geneID 613, MIM 151410), a 474-by segment of APC (adenomatous polyposis coli, geneID 324, MIM 611731), and a 184-bp segment of a rearranged immunoglobulin heavy chain (IGH) gene (IGH@, geneID 3492). A number of primers and antisense oligonucleotides were investigated during development of each system, and the primers and oligonucleotides finally used are shown in Table 5.
Preliminary investigations, correlating calculated Tm values with function at various annealing temperatures, showed that the individual primers amplified efficiently up to annealing temperatures equal to their calculated Tm value plus 5-7° C., pairs of primers amplified efficiently up to an annealing temperature equal to their mean Tm value plus 2-4° C., and pairs of antisense oligonucleotides produced measurable inhibition of a pair of outer primers up to an annealing temperature equal to the mean oligonucleotide Tm value plus approximately 10° C. A useful criterion for satisfactory inhibition of outer primers by a pair of antisense oligonucleotides was that when the only primers in the PCR were the outer primers, a two-phase PCR protocol was used and the mass of DNA added was such that the cycle threshold (Ct) was reached after 6 or 7 low-temperature cycles, and then adding the antisense oligonucleotides increased the Ct by 5-10 cycles. In addition to their Tm, the magnitude of the inhibitory effect of the antisense oligonucleotides was also found to be influenced by their molar concentration, the duration of annealing, and the annealing temperature. The inhibitory effect of the antisense oligonucleotides was decreased if only one oligonucleotide was used in the reaction, but the inhibitory effect could be satisfactorily increased by using an oligonucleotide with a Tm increased by 2-3° C. Omitting the 3′ tag of the antisense oligonucleotide resulted in the variable inhibition of amplification during the initial high-temperature phase of the PCR (data not shown).
Results using the final antisense qPCR protocol, as described in Materials and methods, are shown in
A direct comparison between amplification efficiency of antisense qPCR and that of standard qPCR was performed in 21 experiments-8 with N-RAS, 5 with BCR, 6 with APC, and 2 with IGH—with each experiment amplifying 0.1-100 ng of DNA. No statistically significant differences were detected. The mean slopes of the relationship between Ct and logarithm of the mass of DNA were −3.62 for antisense qPCR and −3.58 for standard PCR, with the standard error for the paired differences being 0.065. The mean difference between the Ct value for antisense qPCR and that for standard qPCR for any given mass of DNA was −0.467, with the standard error being 0.252 (t=1.85, P<0.1, two-tailed). The most obvious interpretation of the results is that there is no real difference in amplification efficiency between the two techniques. If there was in fact a small real difference, then the results would actually suggest that amplification by antisense qPCR is more efficient.
In one experiment studying the APC gene to analyze the results of amplification by the outer and inner primers during antisense PCR, the PCR was stopped after a variable number of cycles and an aliquot was assayed by a second qPCR to quantify the various amplicons produced. In this secondary assay, the outer primers quantified only the long amplicons produced by the outer primers in the primary PCR, whereas the inner primers quantified the total amplicons (i.e., both the short amplicons produced by the inner primers and the long amplicons produced by the outer primers). The results are shown in
The effect of antisense qPCR on nonspecific amplification was studied for each of the four gene targets. Masses of DNA ranging from 30 pg to 100 ng were amplified either by standard PCR, using the outer primers only, or by antisense qPCR and Ct values that were determined either by a Taqman probe, which is specific for the intended product, or by SYBR Green, which measures all PCR products—both the intended product and nonspecific products. In a first experiment with each gene, the two-phase temperature profile was used for both antisense qPCR and standard PCR (using outer primers only). Because the lower annealing temperature after cycle 15 might have been slightly suboptimal for the outer primers alone, a second comparison was performed with N-RAS, APC, and BCR in which antisense qPCR was performed with the usual temperature profile, whereas standard PCR was performed with an annealing temperature of 72° C. for all cycles. Study of amplification of the IGH gene using an annealing temperature of 72° C. for all cycles was not possible because annealing of the Taqman probe decreased above a temperature of 61° C. The results were essentially the same for all seven experiments, two of which are shown in
Quantification of rare targets requires a large number of PCR cycles, and in this situation it might be desired to perform more than 15 high-temperature cycles so as to decrease the number of low temperature cycles and, hence, minimize the possibility of nonspecific amplification during this phase. Therefore, we investigated whether progressive inactivation of the outer primers by the antisense oligonucleotides during the high-temperature phase might eventually reduce amplification efficiency. Three experiments were performed: one each studying N-RAS, BCR, and APC. Using an annealing temperature of 72° C., various masses of DNA were amplified by the outer primers so that Ct values of up to 35 cycles were observed. When antisense oligonucleotides were also present, there was no inhibition except in the experiment studying N-RAS, where two pairs of antisense oligonucleotides, one with a Tm value of 50° C. and one with Tm values of 55° C., were tested. Slight inhibition was observed after 32 cycles when the latter pair of primers was used. The data, therefore, suggested that up to 30 cycles could safely be used for the high-temperature first phase of the PCR without measurable inhibition by the antisense oligonucleotides.
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.
This application is a non-provisional of and claims the benefit of priority to U.S. provisional Patent Application Ser. No. 61/319,146, filed on Mar. 30, 2010, the disclosures of which are hereby expressly incorporated by reference in their entireties
| Number | Date | Country | |
|---|---|---|---|
| 61319146 | Mar 2010 | US |
