Patent Application
20140073530
Throughout this application, various publications are referenced. Disclosures of these publications in their entireties are hereby incorporated in their entireties by reference into this application in order to more fully describe the state of the art to which this invention pertains.
1. Field of Invention
The present invention relates to a method of making rapid, definitive identification of human leukocytic antigens (HLA) by DNA analysis and the device thereof. The present invention also relates to a method of making rapid, definitive identification of a human or any organism by DNA analysis and the device thereof.
2. Description of Related Arts
Accurate HLA typing is essential for matching donor and recipient in organ or marrow transplantation (Thomas, 1983) to prevent the development of acute graft-versus-host disease (GVHD). This is generally accomplished by standard serological typing (Mach et al., 1990). Recent studies have demonstrated that DNA genotyping can provide more accurate and definitive result (Kaneshige et. al., (1993); Chow and Tonai, 2003; Mach et al., 2004). Results of HLA-DQ, DR and DP genotyping provided data for accurate matching which is necessary in selecting potential organ donors (Tam, 1998; 2004; 2005; 2006).
HLA genotyping using sequence-specific primers polymerase chain reaction (SSP-PCR) amplifications has been previously reported. However, due to highly polymorphic nature of HLA-DQ, DR and DP loci, the number of SSP required will be in the hundreds, and, therefore, exceed the limit of multiplex-PCR for efficient PCR amplification. A kit that is available on the market today includes an amplification process of carrying out a 96 PCR separate reactions, then followed by analysis on gel electrophoresis size separation. This is not only time consuming and costly but also prone to error due to the complexity of the reaction setup and sizing uncertainty of gel electrophoresis.
Thus, DNA sequencing is still considered the method of choice for accurate genotyping of the HLA cluster. Unfortunately, because of the existence of highly homologous sequence of pseudogene(s) that may be co-amplified during the polymerase chain reaction (PCR) amplification process, accurate genotyping by DNA sequencing alone may prove more difficult and costly.
The present invention discloses a method of rapidly analyzing HLA loci of DP, DR and DQ beta sequences by Allelic-Specific-Oligonucleotide (ASO) probes using an improved flow-through format. Such flow-through format, which was described in U.S. Pat. Nos. 5,471,547 and 6,020,187, employs a fast annealing process that uses a very inexpensive device for accurate mutation detection, genotyping and fingerprinting analysis.
DNA fingerprinting by restriction fragment length polymorphism (RFLP) was first introduced in 1985 (Gill P, et. al.) for human identification, and was subsequently applied to identification of other organisms. In practical applications, DNA fingerprinting has been widely accepted as the best forensic tool for identification of suspects in criminal cases, for paternity disputes and for establishing or verifying the identity of a person. The time consuming RFLP method has been gradually replaced by high throughput automated processes. Using PCR amplification for analyzing the number of short tandem repeat (STR), first discovered in 1991 (Edwards et al., 1991), from 10, 16, 18 or more loci in the human genome, single cell identification is now possible. However, both STR and/or variable number of tandem repeats (VNTR) are relatively expensive because these methods require the use of sophisticated equipment, and labor intensive and time consuming process like Southern blotting hybridization. Sporadic mutations (Chakraborty and Stivers, 1996) may reduce the accuracy and the power for definitive identification. Furthermore, STR data suggested that the frequency of mutation, particularly in cancer patients, is not uncommon. Hence, new alternative method is needed.
Single nucleotide polymorphism (SNP) genotyping provides greater discriminating power by selecting an appropriate number of SNPs at unlink loci, and the mutation frequency in each locus (site) is lower than VNTR or STR systems for forensic or individual personal identification. The present invention provides a method of making rapid, definitive biometric identification of an individual, such as a human being, animal, plant or any organism, using SNP genotyping.
The present invention describes Allelic-Specific-Oligonucleotide Reversed-Dot-Blotting (ASO-RDB) direct flow-through hybridization is a better alternative for the detection of specific target HLA DNA sequences. The data obtained refer to the specific segments of HLA loci of DP, DR and DQ beta that are able to provide accurate determination of the genotypes. The present invention provides a cost effective procedure for HLA identification by using common primer pairs to perform a simple multiplex PCR followed by hybridization with the required numbers of ASO-probes in a Low-Density Array format. The amplified HLA fragments (including the pseudogenes) can be analyzed in a single membrane embedded with the ASO-probes for definitive HLA classification. Hence, this is a far superior method than other DNA or serological methods currently available. This invention provides a HLA typing technique which is faster and simpler, does not require expensive equipment, and is therefore less costly to manufacture and operate than direct DNA sequencing and multiplex PCR gel electrophoresis procedures.
The primers and oligo-probes shown in
Although HLA genotyping is exemplified in this application, the Single nucleotide polymorphism (SNP)-based genotyping technique can be applied to other genetic materials and/or sequences obtained from any organism following the teaching of this application, such as the procedures shown in
Other than for DNA fingerprinting, SNP genotyping can be utilized for identification of gene fragments, or polymorphism of genes that have altered or attenuated the function of the gene in question. For this reason, the present invention can be used for rapid, definitive identification of infectious agents, inherited disease caused by the specific DNA sequences, or the presence or absence of such infectious agents or DNA sequences that cause inherited diseases.
The present invention provides a method of performing rapid nucleic acid detection, comprising the steps of: (a) obtaining a sample comprising a target nucleic acid molecule; (b) mixing the target nucleic acid molecule with (i) a first probe that will bind to the nucleic acid molecule, and (ii) a second agent that will bind to the first probe, thereby forming a nucleic acid molecule complex in solution; (c) applying in a flow through manner the solution comprising the nucleic acid molecule complex to an array comprising a third probe that will bind to the complex, thereby capturing the nucleic acid molecule complex on the array; and (d) detecting the captured nucleic acid molecule on the array.
The present invention also provides a method of performing rapid nucleic acid detection, comprising the steps of: (a) obtaining a sample comprising a target nucleic acid molecule; (b) mixing the target nucleic acid molecule with (i) a first probe that will bind to the nucleic acid molecule, (ii) a first antibody, and (iii) a labeling agent, wherein the first probe will form a complex with the target nucleic acid molecule, and the first antibody will bind to the resulting complex; (c) applying in a flow through manner a solution comprising the nucleic acid molecule complex to an array comprising a second antibody that will bind to the first antibody, thereby capturing the nucleic acid molecule complex on the array; and (d) detecting the captured nucleic acid molecule on the array.
The present invention also provides a device capable of practicing the detection methods described above.
The present invention also provides a lateral flow hybridization device comprising: (a) a membrane for immobilizing capture molecules capable of capturing target analysts, wherein the membrane is positioned in an reaction chamber where it can be maintained in controlled conditions; (b) controlling elements that can be regulated to maintain the reaction chamber in the controlled conditions; (c) connecting elements for connection to a power supply and control unit that can regulate and maintain the controlled conditions; and (d) liquid delivery elements capable of accepting and removing solution to and from the reaction chamber, wherein the solution is maintained in a lateral flow direction that allows the solution flows through the membrane pores in cross-section so that all target analysts pass through the capture molecules immobilized on the membrane, thereby providing higher sensitivity of analysts detection.
The present invention also provides a lateral flow hybridization system comprising more than one of the hybridization device described herein, wherein each of the hybridization devices are connected to a power supply and control unit capable of supplying energy and providing regulatory control to the hybridization devices.
The present invention also provides a lateral flow hybridization device comprising: (a) more than one reaction chambers, each of which comprises a membrane for immobilizing capture molecules capable of capturing target analysts; (b) controlling elements that can be regulated to maintain the reaction chamber in controlled conditions; (c) connecting elements for connection to a power supply and control unit that can regulate and maintain the controlled conditions; and (d) liquid delivery elements capable of accepting and removing solution to and from the reaction chambers, wherein the solution is maintained in a lateral flow direction that allows the solution flows through the membrane pores in cross-section so that all target analysts pass through the capture molecules immobilized on the membrane, thereby providing higher sensitivity of analysts detection.
The present invention will be described in connection with preferred embodiments, however, it will be understood that this is not intended to limit the invention to the embodiments described. On the contrary, the intent is to cover all alternatives, modifications, and equivalents as may be included within the spirit and scope of the invention as defined by the appended claims.
The following describes a method of the present invention for obtaining the HLA genotyping ASO probes and PCR primers from genomic sequence database, and for performing HLA genotyping analysis:
Select appropriate target sequence(s) to be analyzed by screening data from GenBank and/or by performing population screening by sequencing target genes or DNA segments to obtain SNP or ASO profile appropriate for HLA genotyping.
A method of the present invention for selecting appropriate target sequence(s) is detailed below:
Find all the HLA DNA sequence data from GenBank according to their individual class (i.e., Class I or Class II) and subtypes (i.e., DR or DQ or DP, etc.). Align HLA DNA sequences based on individual classes and subtypes to determine the most polymorphic region(s) that will be appropriate for use in HLA genotyping.
Within polymorphic region(s), identify PCR primer pair(s) that is conserved among the subtypes of interest so that when using these primers, the PCR products can always be amplified for all the subtypes of interest for further analysis using the flow-through hybridization process.
To validate the usefulness of these primers, PCR amplification is performed using a large number of random samples to obtain statistically satisfactory data for positive amplification of the region(s) in question, and to ensure that any drop out of any allele does not result due to false negative amplification process. In addition to statistical validation, internal control (IC) is included to ensure that an inhibitor of PCR is not present in the test sample which will prevent the PCR amplification reaction.
The design of IC (may be 1 or more) will depend on the test required. In the case of HLA and SNP fingerprinting, and genotyping of genes responsible for genetic diseases, the nature of IC sequence will be non-homologous to the human genome in question (i.e., a sequence totally unrelated to sequence in question). Since human cells are mostly diploid (except germline) genome, the concentration issue is much simpler because theoretically the concentration of the test loci should be either 1 (homozygous) or 0.5 (heterozygous). Hence, two different sequence fragments flanked by the same PCR primer sequences can be used for IC. This means that using a fragment different from that of the target fragments to be analyzed, one can use loci of known character of homozygous and heterozygous sequences within the fragment (that is two different probes: 1 homo; 1 hetero in the fragment) as probes so that these IC concentration ratio can be calculated. Since the PCR amplification does not affect the relative concentration, the ratio of signal will be 2:1 theoretically. Any fluctuation will be seen as the hybridization efficiency. The two ICs with a ratio of 2:1 serves as the reference for homozygous and heterozygous, respectively, for the loci in question. If the observed result indicates only one allele, and the concentration in question is equal to or less than 0.5 of the homozygous IC or close to 1 compare to the heterozygous IC, the possibility of loss of heterozygousity should be investigated, meaning: may be one of the two chromosomes has lost or could not amplify. The expected result of ONE dot is homozygous which indicate the two chromosomes are the same, and therefore the concentration should be closer to the homozygous IC than that of the heterozygous IC. Hence, ICs will be important for obtaining accurate results since clinically dependable result is crucial for any diagnostic test.
Additional information on DNA data bank and alignments can be found in the reference section of this application (see Reference No. 5-6).
The procedures described herein for HLA genotyping can also be adopted by one of ordinary skill in the art reading the teaching herein for genotyping of other genes or DNA sequences of interest, or for determining SNP profiles for DNA fingerprinting/identification process performed in combination with flow-through hybridization process.
To maximize the efficiency of PCR amplification, the fragment length of the ASO probes is normally selected or kept as short as possible, preferably to within a few hundred base-pairs. If suitable primer pairs cannot be identified, adjustments on the components in the PCR reaction mixtures and the PCR processes, i.e. the PCR program, can be made to optimize the amplification reaction to ensure the production of amplicons during development of the genotyping process. Such development processes are apparent to persons of ordinary skill in the art after reading the teaching herein. In some cases a totally conserved sequences for PCR Primer cannot be found and degenerate primers or multiple primers for the same loci may have to be use. In these cases, appropriate adjustments to the PCR conditions are made to ensure that all loci will not be missed.
When unique region(s) for successful subtype differentiation cannot be identified, multiplex PCR may be required. The primer pairs are designed to Tm values within the operable range for successful annealing during the amplification process. As used herein, “Tm” means, for example, the temperature of the reaction where the concentration of DNA molecules in double strand as well as single strand is equal. Hence, at higher temperature more double strand DNA will become single strand, and conversely at lower temperature, more single strand molecule will anneal into double strand. For a given population where sequence data is not available, a population screening by direct DNA sequencing is usually performed. For example, a screening of a sample Chinese population was performed as follows: (a) Random samples of over a hundred subjects were amplified by PCR using two pairs of primers, followed by hybridization using the ASO Probes designed based the procedures described in the following sections; (b) The results were confirmed by DNA sequencing.
Using the data obtained above, the ASO sites to be used for genotyping are determined and selected. Determine whether the sites selected are indeed unique for HLA typing within a population using data obtained from GenBank or generated by sequencing random samples. The procedures for designing ASO probes and for validating the uniqueness of the probes are as follows:
From the selected region(s) of the alignments for which satisfactory polymorphism among the subtypes are found, further search for a unique 20-30 nucleotide sequence or fragment is performed. The unique sequence or fragment is used as an allele specific oligonucleotide (ASO) probe to capture the amplified target by hybridization process in order to detect the presence of such unique HLA subtype. As used herein, “unique sequence or fragment” means, for example, that the sequence or fragment is completely homologous to only one sequence, among these subtype sequences.
To verify that the ASO probe(s) is unique, the sequence is matched with all human DNA sequence data which are available in the GenBank or in its European equivalent to determine if indeed the ASO probe has 100% match with ONLY the HLA subtype of interest. This will ensure that at least (until new sequences are discovered) among the available data, the ASO probe(s) is unique for a given region.
Since PCR fragment length is kept short to improve the efficiency of amplification, one ASO probe may not be sufficient to provide a definitive differentiation to determine the unique subtype. Hence, a set of multiple ASO probes within one PCR fragment and/or in different fragments using the multiplex PCR may have to be used to give a definitive genotyping classification. In this case, a specific pattern of the ASO array will be generated by the hybridization process for each of the given HLA subtypes. After thorough analysis of the data, the ASO sequences undergo validation using random human DNA samples.
Similar procedures can be used for other genes and genetic materials from other organisms. The primer sequences and the number of ASO probes vary for different genes and for different applications. For example, human identification may require a 50 or more ASO array to obtain definitive identification. The detection format can include an array of dots or linear lines depending on the configuration of the flow-through hybridization device.
ASO oligonucleotides are immobilized on a membrane or any suitable matrix for capturing the target loci. As used herein, “membrane” means, for example, any suitable matrix material capable of immobilizing ASO oligonucleotide probe(s) and porous enough for test solution containing the target nucleic acid molecules to pass through freely. In an embodiment, the membrane or matrix may be constructed of Nylon, N.C., Biodyne, Porex, and porous metal or durable gel matrixes.
Immobilization of the target sequence (or loci) can be achieved by covalent bonding, non-covalent (i.e., electrostatic, hydrophobic or any other interactions including UV cross-linking) interactions, or interactions through mediators such as receptors or antibodies. Avidin-biotin linkage or ASO poly-T tailing for UV cross-linking is equally effective.
Target sequences are amplified using appropriate primers to generate enough amplicons to enable adequate analysis. The target molecules can be appropriately labeled for signal generation according to the method of signal detection used for final analysis. Labeling can be performed on one or both of the primers by covalent linkage of the label molecule at the 5′ end or using one of the four dNTP labeled nucleotides during the PCR amplification process for extension labeling of the newly generated amplicons. The label molecule can be of any kind as long as the signal can be monitored and developed. Biotin coupled with avidin-enyzme conjugate can be used for color detection. Other suitable labeling systems include, but are not limited to, colloidal gold conjugate and fluorescence label, magnetic particles conjugate, quantum dots, chemiluminescence label molecules, or other suitable systems already developed or to be developed may be used as well.
ASO profile analyses are performed using flow-through hybridization process such as the method described in U.S. Pat. No. 5,741,647. Hybridization is performed in the hybridization chamber with ASO capture probes coupled to the membrane. An ASO profile analysis is described below:
(a) Denaturing and contacting solution containing target DNA or sequence with membrane;
(b) Washing membrane with washing (or SSC or blocking) solution, preferably three times; and
(c) Developing color for visual inspection or spectrometric measurements. For quantitative measurements, one can use a scanner and imaging software to perform the analysis. Alternatively, target DNA or sequence can be labeled with fluorescence dye and analyzed using a spectrometric imager immediately after the washing step.
The results are compared with known sequence data to ensure accuracy of genotyping assay. Probes and testing conditions can be further modified to improve the accuracy of genotyping assay, and RDB-ASO data is verified by DNA sequencing.
Validation is performed using random samples. A validation procedure of the present invention is described below:
As mentioned above, once the primers pair(s) and ASO capturing probe(s) are immobilized on a membrane, random DNA samples of sufficient number are used to perform PCR amplification. Hybridization of target sequence(s) or amplified and labeled DNA products or molecules is performed to generate an ASO array pattern.
HLA subtype and corresponding DNA sequence(s) are determined from ASO probes. Corresponding PCR samples are prepared for direct DNA sequencing.
Agreement of the results obtained from DNA sequencing and from flow-through array is needed to validate the HLA genotyping assay. Samples for validating genotyping assay can be obtained from any randomly selected individuals. Once the samples have been obtained, true genotyping can be performed by DNA sequencing as stated above. If sequencing data agree with those obtained from flow-through array, the validity of the data is confirmed. Further validation of genotyping assay can be performed through field tests, such as testing random samples, analyzing data and comparing statistically values such as sensitivity, specificity, positive predicted value or negative predicted value, at independent laboratories to evaluate the accuracy of genotyping assay.
The following is a method of the present invention for constructing a SNP database and developing a SNP genotyping assay:
Select SNP sites and determine the power of exclusion. As used herein, “power of exclusion” means, for example, the accuracy of the method in differentiating people or organisms. For instance, power of exclusion of 1 in 10 billion or 100 billion means that when using a selected number of SNP Sites one can only expect to find two identical individuals in screening 10 or 100 billion individuals, respectively.
Select appropriate SNP oligonucleotide probes for capturing specific target sequences to be analyzed either by screening data from GenBank or by performing population screening, i.e., direct DNA sequencing of target genes or target DNA segments, to obtain SNP profile and population frequencies.
From these data, identify the SNP sites to be used for fingerprinting based on polymorphic frequency, and determine whether or not the selected sites are indeed hot spots for mutation within a population by sequencing the samples obtained from random population screening.
Determine the number of SNP probes required, and calculate total heterozygosity to determine the power of exclusion with the given number of SNP points used for the analysis profile pattern (i.e., the SNP dot array as shown in
Power of exclusion depends on the number of SNP in a given loci. For example, in a given loci, if 2 different bases, e.g., G and A, are found in a sample population at 50% each. The probability is ½. If 50 of such sites can be found (these sites are randomly distributed and are not linked to each other in the chromosome) the differentiation power will be ½ to the power of 50.
Perform SNP profile pattern (a combination of SNP array showing the array of corresponding genotypes in each loci of that individual) detection.
Design appropriate primers for amplification, and select appropriate SNP-probes for hybridization detection after the loci are screened and selected as described above.
Amplify target sequence(s) and perform SNP profile analyses using flow-through hybridization process such as the method described in U.S. Pat. No. 5,741,647.
Compare the data obtained with known sequence data to evaluate the accuracy of the SNP genotyping analysis.
Modify SNP probes and testing conditions to improve the accuracy of the SNP genotyping analysis. RDB SNP data are verified by DNA sequencing.
It should be understood that under the general term of SNP genotyping analysis, the invention described herein should not be considered limited to human genome; nor the SNP be limited to Single Nucleotide Polymorphisms (or single base mutations). The term “SNP” shall include short deletions and insertion of nucleotides. “Genotyping” shall apply to any genetic sequences of any organism where variation of sequences would enable any skilled professional in the art to differentiate the variation among different sequences in the case of nucleic acids and variation of structures
Conventional hybridization involves many solution changes and transfers steps and therefore is a labor intensive and time consuming process. The Flow-through inventions disclosed in U.S. Pat. Nos. 5,471,547 and 6,020,187 eliminate all transfer steps and substantially reduce the reagents volume to make the hybridization a very efficient process. Nonetheless, the number of steps in the hybridization protocol remained basically similar. The present invention describes further significant improvements in the hybridization protocols which include:
Elimination of the pre-hybridization step in which the blocking and hybridization steps are combined using an improved reagent mixture i.e., DNA samples are placed in hybridization solution and flow-through detection membrane without pre-hybridization).
A single-step hybridization process in which the target sequences or molecules are labeled with fluorescence tags, quantum dot, colloidal gold particles, magnetic particles or other appropriate labeling tags to eliminate the enzyme-link conjugate substrate color development step. These improvements will enable a technician to complete the entire hybridization process in 5 minutes or less. Hence, the method of the present invention should provide further savings in terms of time and reagent cost.
New embodiments described herein take advantages of the physical dimensions of the relative length and area of the membrane used to capture the target molecules whereby increasing the sensitivity and lowering the detection limit to achieve the best results. Examples given below illustrate such principle. Other embodiments can be constructed to optimize for complete capture of the target molecules. Thus recirculation of the hybridization mixture is an alternative. This and automation process can be incorporated into the examples described herein.
The invention being generally described, will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.
The present invention provides a method of performing rapid nucleic acid detection, comprising the steps of: (a) obtaining a sample comprising a target nucleic acid molecule; (b) mixing the target nucleic acid molecule with (i) a first probe that will bind to the nucleic acid molecule, and (ii) a second agent that will bind to the first probe, thereby forming a nucleic acid molecule complex in solution; (c) applying in a flow through manner the solution comprising the nucleic acid molecule complex to an array comprising a third probe that will bind to the complex, thereby capturing the nucleic acid molecule complex on the array; and (d) detecting the captured nucleic acid molecule on the array.
In general, the target nucleic acid molecule is of human, bacterial, or viral origin. In one embodiment, the human is having or is suspected to have cancer. In another embodiment, the target nucleic acid molecule is obtained without amplification.
Preferably, the first probe and the third probe bind to different regions of the target nucleic acid molecule. In one embodiment, the third probe is an allelic-specific oligonucleotide probe. The allelic-specific oligonucleotide probe can be generated, for example, from nucleic acid sequence data bank. In another embodiment, the third probe can identify single nucleotide polymorphism of the target nucleic acid molecule. For the detection of the captured nucleic acid molecule, it can be generally performed by fluorescence tags, quantum dot labeling, colloidal gold particle labeling, magnetic particle labeling, or enzyme-linked substrate assay.
The present invention also provides a method of performing rapid nucleic acid detection, comprising the steps of: (a) obtaining a sample comprising a target nucleic acid molecule; (b) mixing the target nucleic acid molecule with (i) a first probe that will bind to the nucleic acid molecule, (ii) a first antibody, and (iii) a labeling agent, wherein the first probe will form a complex with the target nucleic acid molecule, and the first antibody will bind to the resulting complex; (c) applying in a flow through manner a solution comprising the nucleic acid molecule complex to an array comprising a second antibody that will bind to the first antibody, thereby capturing the nucleic acid molecule complex on the array; and (d) detecting the captured nucleic acid molecule on the array.
In general, the target nucleic acid molecule is of human, bacterial, or viral origin. In one embodiment, the human is having or is suspected to have cancer. In another embodiment, the target nucleic acid molecule is obtained without amplification. For the detection of the captured nucleic acid molecule, it can be generally performed by fluorescence tags, quantum dot labeling, colloidal gold particle labeling, magnetic particle labeling, or enzyme-linked substrate assay.
The present invention also provides a device capable of practicing the detection methods described above.
The present invention also provides a lateral flow hybridization device comprising: (a) a membrane for immobilizing capture molecules capable of capturing target analysts, wherein the membrane is positioned in an reaction chamber where it can be maintained in controlled conditions; (b) controlling elements that can be regulated to maintain the reaction chamber in the controlled conditions; (c) connecting elements for connection to a power supply and control unit that can regulate and maintain the controlled conditions; and (d) liquid delivery elements capable of accepting and removing solution to and from the reaction chamber, wherein the solution is maintained in a lateral flow direction that allows the solution flows through the membrane pores in cross-section so that all target analysts pass through the capture molecules immobilized on the membrane, thereby providing higher sensitivity of analysts detection.
In general, the power supply and control unit is capable of supplying energy and providing regulatory control to maintain the reaction chamber in the controlled conditions. In one embodiment, the controlled conditions comprise accurate control of temperature within 0.5 degree C.
In one embodiment, the controlling elements of the hybridization device are heating and cooling elements such as Peltier heating and cooling elements, or elements for circulating liquid or air, or any other means known in the art for controlling temperature for the reaction chamber. In another embodiment, the heating and cooling elements comprise heat conduction plate, heating and cooling sensor, heat sink, and heating and cooling fan.
In general, the membrane in the reaction chamber is formed of material containing nitrocellulose, nylon, Nytron, Biodyne, or Porex, or other porous materials known in the art.
In one embodiment, solution containing target analysts is delivered to the reaction chamber and the membrane by regulated liquid pumping. For example, liquid pumping can be used to recirculate solution containing the target analysts through the membrane.
The present invention also provides a lateral flow hybridization system comprising more than one of the hybridization devices described herein, wherein each of the hybridization devices is connected to a power supply and control unit capable of supplying energy and providing regulatory control to the hybridization devices. Hence, in one embodiment, each of the hybridization devices can be controlled independently by the power supply and control unit so that each hybridization device can perform different analysis under different conditions.
The present invention also provides a lateral flow hybridization device comprising: (a) more than one reaction chambers, each of which comprises a membrane for immobilizing capture molecules capable of capturing target analysts; (b) controlling elements that can be regulated to maintain the reaction chamber in controlled conditions; (c) connecting elements for connection to a power supply and control unit that can regulate and maintain the controlled conditions; and (d) liquid delivery elements capable of accepting and removing solution to and from the reaction chambers, wherein the solution is maintained in a lateral flow direction that allows the solution flows through the membrane pores in cross-section so that all target analysts pass through the capture molecules immobilized on the membrane, thereby providing higher sensitivity of analysts detection. General features for the membrane, controlling elements, and liquid delivery have been described above. In one embodiment, the reaction chambers of this hybridization device are disposable. In another embodiment, each of the reaction chambers is a separate unit.
The following procedures were used to determine HLA genotypes of 140 human samples obtained from a sample Chinese population. The results were used to develop a kit for rapid classification of HLA subtypes of the first 2 digit codes corresponding to WHO nomenclature. In one embodiment, a kit can be designed to cover HLA-DR, -DQ and -DP alleles which are typically used for tissue matching between a donor and a recipient prior to organ transplantation.
The following protocols are recommended, but alternative procedures which are readily apparent to one of ordinary skill in the art and which are equally effective may be employed. Nucleated cells such as WBC or tissues are washed with PBS, centrifuged, and the supernatant is removed. The pellet is re-suspended in 200.mu.l PBS. DNA extraction is performed with QIAamp DNA mini kit (QIAGEN) following Blood and Body Fluid Spin protocol as recommended by the manufacturer. Other commercially available kits for isolating DNA may also be used. However, DNA isolation procedures that produce purified DNA that do not contain DNA polymerase inhibitor are crucial to ensure efficient amplification. Elute DNA in 50-200.mu.l Buffer AE and store at −20.degree. C. until use.
Since PCR is extremely sensitive process, special care must be taken to prevent cross contamination and/or to prevent false positive results. Hence, the following guidelines should be observed: Wear surgical gloves at ALL TIMES when performing PCR procedures. Preferably, prepare PCR reaction mixtures in a pre-PCR area or a clean PCR preparation station where no amplification products are present. PCR reaction and hybridization should be performed in separate areas. For example, hybridization should be performed in a post-PCR area.
Use 70% ethanol and paper towel to clean the working bench/area. Use 70% ethanol and paper towel to clean all the pipettes before starting PCR amplification. Use filtered/sterile tips for ALL pipetting steps. NEVER reuse any tips.
PCR is just one of the many techniques which can be used for amplification. Other amplification techniques, which are readily apparent to one of ordinary skill in the art, such as various methods for isothermal amplification using appropriate primers to amplify target sequence(s) to obtain sufficient quantity of target sequence(s) (or DNA molecules) to carry out flow-through array detection may be used.
PCR reactions are performed using, for example, commercially available polymerase AmpliTaq™ Gold (Applied Biosystem). Five primers pairs (1 pair used for amplifying the DR genes, i.e., forward primer DRB-F1: 5′-ATCCTTCGTGTCCCCACAGCACG-3′ (SEQ ID No. 97) and the reversed primer DRB-R1: 5′-GCCGCTGCACTGTGAAGCTCTC-3′ (SEQ ID No. 98); 1 pair used for amplifying the DQ genes, i.e., the forward primer is DQB-E2-F2: 5′CGGTGATTCCCCGCAGAGGAT-3′ (SEQ ID No. 99) and the reversed primer is DQB-E2-R2: 5′-CCACCTCGTAGTTGTGTCTGC-3′ (SEQ ID No. 100); and 3 pairs used for amplifying the DP gene, the forward primers (SEQ ID NOs. 101-103) and the reversed primers (SEQ ID NOs. 104-106) listed in
TABLE-US-00001 Each reaction (.mu.l) 10 reactions PCR-Mix 19.00 190.0 DNA Taq Polymerase 1 ul (1 unit) 10 ul (10 units) DNA Template 5 (˜100 ng)—Total 25 200
Amplification program was optimized using PE 9700 thermal cycler. For PE 9700 (or MJ thermal cycler):
When using different primers or other thermal cycler, modifications to the cycling program may be required.
It is important to have a positive control and a negative control in every PCR analysis. Positive control is needed to demonstrate the efficiency and specificity of PCR, and negative control is needed to determine whether PCR reagents are contaminated. Internal controls are also needed to provide proper interpretation, and to ensure the dependability and accuracy of the data. The type and the number of IC depend on the type or nature of the test needed.
IC can be used to track each step of the hybridization reaction or procedure. For example, IC can be used to determine whether a sample was added or whether any inhibitors are present in a sample which will prevent PCR reaction from working properly. IC can also be used to track the efficiency of the reaction, or to determine the concentration of target molecule in a semi-quantitative or quantitative manner.
If a quantitative or semi-quantitative measurement is needed, the detection array or membrane can be prepared with a signal generating internal control (IC) onto the membrane to provide a detectable signal at predetermined concentrations when a preprogrammed or predetermined condition has been met. In such embodiment, the IC is developed simultaneously with the test samples after hybridization. In another embodiment, additional IC can be added into the PCR reaction mix to indicate the intrinsic PCR efficiency. This can serve also as an internal control to indicate the presence or absence of inhibitor within the PCR reaction.
The IC system in the FOR reaction mix of the present invention provides several advantages. Since the PCR reaction is carried out in the same reaction tube, the absence of IC and test sample signal indicates the presence of an inhibitor. If the same primer sequences for IC and test samples are used for the FOR amplification, it can serve as an intrinsic control for PCR reaction efficiency. IC can also be used to determine the detection limits or cut off value of the PCR reaction and/or the efficiency of the hybridization process, and to provide an indication of whether hybridization was successfully completed. In addition, IC can be used to determine whether reagents for signal development are acceptable or prepared properly, and whether proper procedures were followed during hybridization.
Pre-warm hybridization solution (i.e., 2.times.SSC or any commercially available solution/product) to 42.degree. C. in a water bath before use. If precipitate is present in solution B (SSC+0.5% SDS), dissolve the precipitate by incubating solution B at 42.degree. C. until the precipitate is dissolved. Keep the temperature at 42.degree. C. throughout the hybridization process to maintain set stringency.
Prepare NBT/BCIP working solution by dissolving a tablet in 10 ml of solution C or PBS buffer (phosphate buffer saline). Protect the diluted working NBT/BCIP solution from light and store any unused solution at 4.degree. C.
Equilibrate hybridization solution (2.times.SSC+0.05% tween 20) to room temperature.
Denature all biotinylated PCR products by heating at 95.degree. C. for 5 min, and then chill on ice immediately for at least 2 min.
For the flow through hybridization studies, one can use the direct flow through device described in U.S. Pat. No. 6,020,187 or a lateral flow device described in this application. For the genotyping of HLA in this example, a direct flow device is used. Switch on hybridization device to preheat at 42.degree. C. filled with distilled water.
Place the detection membrane(s) which are embedded with capture probe(s), such as those listed in the sequence listing below, in the hybridization chamber. Secure the membrane(s) with, for example, a cover lid.
When the temperature reaches 42.degree. C. (+/−0.5.degree. C.), deliver 1 ml of the pre-warmed hybridization solution for pre-hybridization to cover the membrane (NOTE: The temperature stated here is the optimal annealing temperature for the given probe sequence. In general, to skill in the art may design the probe sequence to fit this temperature. However if required, different temperature can be used to fit other sequences.) Incubate for at least 2 minutes with the cover closed to prevent heat loss during pre-hybridization. This is to ensure temperature equilibrium at the set temperature.
Add 0.5 ml of the pre-warmed hybridization solution to each denatured PCR products separately for testing, and add the DNA samples into designated well. Contacting DNA samples which contain the target sequence(s) with membrane surface and incubate at 42.degree. C. for 5 minutes, and then allow DNA samples which to flow through the membrane. Hybridization is normally completed within 30 seconds. The 2-5 minutes incubation period ensures that the temperature of the DNA samples will reach the set temperature.
Wash the membrane with 3.times.0.8 ml of hybridization solution.
Set temperature to 37.degree. C. Start pump, and immediately begin dispensing 0.5 ml of blocking solution. Stop pump. Add another 0.5 ml of blocking solution and incubate for 5 min, then pump out the solution.
Turn off pump and add 0.5 ml of the enzyme conjugate. Let the membrane sit for 3 min. Color development works well between 25-37.degree. C. Start pump. Wash the membrane thoroughly, preferably four times, with 0.8 ml of buffered saline solution at pH 7.4.
Turn off pump and add 0.5 ml of the NBT/BCIP solution (from Roche). Cover lid. Incubate for about 5 minutes or until color develops. Note: DO NOT incubate for over 10 minutes.
Turn pump on to remove NBT/BCIP solution. Wash the membrane, preferably three to four times, with 1 ml of solution B after the color has completely developed. Rinse membrane with 2 ml of demonized H.sub.2O once.
Inspect results as soon as possible, preferably within 1 hour, by direct visualization, or scan the image for semi-quantitative detection.
The presence of a clearly visible dot indicates a positive result. A total of 96 ASO probes (DRB: 29; DQB1: 24 and DPB1: 43), corresponding to SEQ ID 1-96 in the sequence listing for the HLA cluster, and five PCR primer pairs, corresponding to SEQ ID NO 97-106 in the sequence listing for amplification had been designed according to the scheme in
The flow-through DNA hybridization method and device as described in the U.S. Pat. Nos. 5,741,647 and 6,020,187, respectively, reduces hybridization time from many hours or days to minutes (the whole hybridization assay can be completed in 5-minutes depending the method used to generate detection signal). The device is also inexpensive to manufacture, and uses 10 times less reagents than convention hybridization devices which will lead to more affordable DNA diagnosis technology.
The present invention provides an inexpensive platform for studying the nucleic acids interactions using a low-density array format. This invention further provides a method of genotyping complex HLA systems. As illustrated above, the genotyping method of the present invention has been shown to provide significant improvements over conventional hybridization processes, even the hybridization process as described in U.S. Pat. Nos. 5,741,647 and 6,020,187.
The present invention further provides additional improvements over existing flow-through hybridization techniques. For example, the hybridization device of the present invention includes a detection membrane, such as the membrane shown in
The array format may be adapted by one of ordinary skill in the art to accommodate additional wells for analyzing a larger set of nucleotide sequences rapidly and inexpensively. The hybridization device of the present invention is a breakthrough in rapid DNA diagnostics. A lateral flow and miniature embodiment of the device of the present invention is depicted in
Significant improvements on this lateral device are described in
To identify the DRB genotypes, the PCR were carried out with the primer pair of DRB-F1: 5′-ATCCTTCGTGTCCCCACAGCACG-3′ (SEQ ID No. 97) and DRB-R1: 5′-GCCGCTGCACTGTGAAGCTCTC-3′ (SEQ ID No. 98) and a 29 ASO probes (as listed in
In the case of DQB1 genotypes, PCR is carried out using DQB-E2-F2: 5′-CGGTGATTCCCCGCAGAGGAT-3′ (SEQ ID No. 99) and DQB-E2-R2: 5′-CCACCTCGTAGTTGTGTCTGC-3′ (SEQ ID No. 100) as primers (see
To identify DPB1 genotypes, a total of 43 ASO probes were tested of which a set of 35 SSO Probes are shown in
HLA class A and B genotype identification can be done the same way as above when appropriate primers pairs and a number of probes all of which having the unique sequences corresponding to the HLA class A or B are used. Such sequences can be found either from the literature as reported in which their identities have been proven. Alternatively one can design these sequences using the scheme disclosed in this invention depicted in
The flow-through array format can be used to identify different organisms of different sequences or different genotypes.
Other panels identifying pathogens such as STD, hepatitis or bird flu such as H5N1 or related influenza viruses can also be developed for detection in similar manner.
The cause of cancer is undoubtedly genetic in origin. Therefore if any genetic trait is identified, early detection is possible before tumorgenesis progresses into disease stages, which normally takes many years. The majority of cases in cancers are sporadic cases, due to either somatic mutations or structural modification of genes during one's life span. The longer one lives, the higher the concentration of mutated gene(s) and/or regulatory sequences will accumulate in an individual. Consequently, functionally defective phenotype (by either reducing the level of expression or producing defective gene product) shall occur leading to the onset of tumorgenesis. Thus in principle, genetic profile of a group of genes can be generated in the form of (i) expression level; (ii) the accumulation of defective products or (iii) the presence of genetic alterations such as (a) DNA mutation(s) in the responsible gene(s) and/or (b) alteration of the controlling region(s)—the promoter or enhancer. Such profile should provide insights into when and how tumorgenesis may have occurred and present diagnostic and prognostic assays.
Indeed mutation(s) of various genes have been identified to have definitive association with certain cancers such as the BRCAs gene for breast cancer; APC gene for colon cancer; p53 gene and Kras for cancers of multiple origins in human. Moreover, hypermethylation in the promoter region mostly located in the CpG islands will lead to silencing of tumor suppressor genes and induction of sporadic cancer. Since alteration(s) in the DNA is the prerequisite condition for cancers, detection of such mutation(s) and/or methylation status would be the earliest possible assay for preventive healthcare.
Recently, activities on epigenetic studies have gained a lot of momentum. Hypermethylation of a number of genes have been reported and that hypermethylation on these genes was found to be closely linked to cancer progression. These have led to the development of Methylation Specific Quantitative Real Time PCR (MSQ-PCR) in an attempt to give early diagnosis (Lo et. al., 1999; Facker et. al., 2006). However, despite its sensitivity, MSQ-PCR has its limitations. Firstly, the maximum number of color dyes is 6 and therefore only 3 genes' CpG islands can be tested simultaneously in a single reaction mixture. This is far less than the number of genes required to produce accurate and meaningful result. Secondly, since sample is often limited in quantity, it is not possible to repeat tests on other genes to confirm the presence or absence of cancer and to identify the type and site of cancer present.
In contrast, examples shown here in this invention can be done by multiplex amplification and array assay which can screen a high number of genes or sequences simultaneously, giving a much higher probability for early cancer diagnosis.
12 shows an alternative embodiment for Lateral flow-through assay disclosed herein. This new embodiment can be made as a new device that includes the essential elements, namely (i) the electronic control unit for operations; (ii) temperature block; (iii) the reaction chamber; and (iv) liquid delivery system. However, as depicted the reaction chamber where the membrane is located can be designed as a new separate unit, a disposable unit that can be fitted into the existing Flow-through Device by modifying its temperature block to accept such unit to achieve the required flow-through operation, manual or automation. The flow direction, the speed of flow and the sequence of solution reagents flow can be controlled accurately by the controller. This disposable reaction chamber can be made as single or multiple cassettes for separate or parallel reactions to provide increase in throughput. Evidently this embodiment can provide optimal conditions for effective detection with increase in sensitivity and specificity. The disposable as well as the totally enclosed setting in this embodiment can prevent any possibility of cross contamination. This system is well suited for clinical diagnoses that involve target amplification process and would reduce the risk of false positive results. Hence this new and novel embodiment disclosed herein is unique and is a very significant improvement for rapid flow-through assays.
Conventional hybridization and related assays are very time consuming because of complicated procedures that involve many separate steps. The Flow-through process and the device described herein have simplified the operation and resulted in substantial reduction of time and reagents cost without scarifying sensitivity and specificity for detection. The invention disclosed herein would provide further procedural improvements and expand the scope of testing.
Other than those outlined in
Besides fluorescence tags, a single-Step hybridization process can label target sequences or molecules with quantum dot, colloidal gold particles, magnetic particles or other appropriate labeling tags to eliminate the enzyme-linked conjugate substrate color development step. These improvements will enable a technician to complete the entire hybridization process and signal production in 5 minutes or less. Hence, the method of the present invention should provide further savings in terms of time and reagent cost.
Contamination can be a serious problem for PCR reactions. Hence, instead of product amplification by PCR, molecular biology or DNA-based diagnosis can be enhanced by an alternative strategy of signal amplification. Branch DNA (b-DNA) has been and still is being used with certain success. DNA super molecular complexes are also under investigation. Recently the hybrid capture technology for HPV detection is another example. Unfortunately, these methods are neither suitable nor applied to membrane-based assays.
The present invention presents examples of membrane-based application using the flow-through platform. As shown in
As shown in
In principle, single step is possible by putting in appropriate components together in the sample solution (i.e. after deproteinization to exclude cell debris and non-nucleic acid complexes) and flow into the membrane for hybridization capture. The present invention employs detection assay direct from the neat solution sample (the starting sample) without amplification. It is applicable and enabled using the flow-through system because there is no limitation on the solution volume used. The sensitivity of detection varies according to the signal generating system used. For example, using the AP-AV and color development system we had achieved 0.3 fetomole/label. At least ten-fold increase in sensitivity can be achieved by chemiluminescence assay. By increasing the number of label molecule in the signal DNA tag, detection in the range of attomole is achievable in principle. At this concentration many of the viral infections can be readily detected.
The disclosures in connection with preferred embodiments are not intent to limit the invention to the procedures and embodiments described. On the contrary, the intent is to cover all alternatives, modifications, and equivalents as may be included within the spirit and scope of the invention as defined by the appended claims.
Those Skilled in the Art Will Recognize, or be Able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
This application is a divisional of U.S. Ser. No. 12/295,747 filed Oct. 2, 2008, which is a National Stage of PCT/CN2007/001108 filed Apr. 4, 2007, which claims the benefit of priority of U.S. Ser. No. 11/398,433, filed Apr. 4, 2006, now U.S. Pat. No. 7,732,138, which is a continuation in-part of U.S. Ser. No. 10/291,168 filed Nov. 7, 2002, now abandoned, and U.S. Ser. No. 10/291,168 claims the benefit of U.S. Ser. No. 60/345,948 filed on Nov. 7, 2011, and U.S. Ser. No. 11/398,433 is also a continuation in-part of U.S. Ser. No. 10/293,248 filed on Nov. 9, 2002, the contents of which are hereby incorporated in their entirety by reference into this application.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12295747 | Oct 2008 | US |
| Child | 13341128 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13341128 | Dec 2011 | US |
| Child | 14025064 | US |
