CN101421418A

CN101421418A - Method of detecting genetic mutations

Info

Publication number: CN101421418A
Application number: CNA200780013086XA
Authority: CN
Inventors: 高峰; 朱军
Original assignee: Duke University
Current assignee: Duke University
Priority date: 2006-04-10
Filing date: 2007-04-10
Publication date: 2009-04-29

Abstract

The present invention relates to a method of detecting genetic variants, including genetic variants associated with drug resistance, cancer and genetic dysfunctional diseases.

Description

Detect the method for genetic mutation

The application requires the U.S. Provisional Application No.60/790 of submission on April 10th, 2006, the U.S. Provisional Application No.60/878 that on January 5th, 535 and 2007 submitted to, and 700 right of priority is incorporated the full content of these two applications into by reference in this.

The present invention is subjected to government-funded with the ROI GM065057 that NIH authorizes.Government has some right of the present invention.

Technical field

The present invention relates to a kind of method that detects genetic variant, comprise method with resistance, cancer, genetic variant that hereditary functional disorder disease is relevant.

Background of invention

Bound drug treatment or high reactivity antiretroviral therapy (HAART) are the main method of treatment human immunodeficiency virus (HIV) infected individuals.Since HAART the virus replication in the blood samples of patients can be suppressed to detect less than level, therefore oneself has significantly reduced M ﹠ M (the Egger et al of HIV infected patient, BMJ 315:1194-9 (1997), Palella et al, N Engl J Med 338:853-60 (1998)).Yet, often be variable for the reaction of HAART.Some patient reacts well, can successfully suppress duplicating of virus, and other patient has failed with regard to treatment after HAART soon.Show resistance (Condra et al from HAART treatment failure patient's proteolytic enzyme and ThermoScript II (RT) gene sequencing to multiple medicine, J Virol 70:8270-6 (1996), Larder and Kemp, Science246:1155-8 (1989), Johnson et al, Top.HIV Med.13:125-131 (2005)).The biological function of these mutant strains confirms by analyzed in vitro that soon these mutant strains import in the molecular cloning that infects and measure the susceptibility (Kellam and Larder, J Virol 69:669-74 (1995)) of these clones to medicine.These data presentation hereditary changes are the bases that produce multiple resistance (MDR).

The HIV-1 genome is a height change, and the individuality of each infection comprises a large amount of viral variants (quasispecies) similar but different in heredity.When in the treatment patient body resistance taking place, oneself shows that some viral genome does not comprise the resistance sudden change at least.In containing the virus of resistant mutation, the number of these sudden changes is different in each genome.Therefore, the mutual relationship between resistance sudden change and the result of treatment can only fully be understood by measuring from identity, character and the number of the sudden change of the resistance in each viral genome in a large amount of viral colony of infected individuals.To advance for the understanding of resistance mechanism and more effectively to develop antiretroviral drugs, better treatment plan and outcome prediction more accurately.

At present, two kinds of methods can be used for detecting the existence of resistance sudden change and the resistance of possibility (Petropoulos et al prediction produces to(for) new HAART scheme, Antimicrob Agents Chemother44:920-8 (2000), Van Laethem et al, J Acquir Immune Defic Syndr 22:107-18 (1999)).Yet these two kinds of testing method are very low to the sensitivity that detects less drug-resistant virus colony, and the shared per-cent of drug-resistant virus in can not the reliable detection wild-type virus.In addition, because resistance sudden change is based on viral colony and detects, so these two kinds of testing method all are unsuitable for linkage analysis, and this linkage analysis can disclose for understanding resistance mechanism and the required critical information of predicted treatment effect.Recently, developed real-time PCR method (the Charpentier etal of the less resistance colony of multiple detection, J Virol 78:4234-47 (2004), Hance et al, J Virol 75:6410-7 (2001), Metzner et al, JInfect Dis 188:1433-43 (2003), Metzner et al, Aids 19:1819-25 (2005), Mohey etal, J Clin Virol 34:257-67 (2005), Thelwell et al, Nucleic Acids Res 28:3752-61 (2000), Whitcombe et al, Nat Biotechnol 17:804-7 (1999)).But these testing method only can detect a kind of resistance sudden change at every turn, and this makes them lower for the practicality of the analysis of whole resistance sudden changes.And for the height hereditary variability in the HIV-1 genome, very difficult design can be used for detecting whole resistance sudden changes of most of genetic variants.

Oneself is used to study that the sudden change of resistance on less drug-resistant virus colony, each viral genome distributes and the linkage analysis of a large amount of virus clones the cloning and sequencing method.Though can obtain the sequence of whole amplicons, this method consumption power, time-consuming.The sensitivity of this testing method is subjected to that available clone's quantity limits in the analytic process, and this method is influenced by the reorganization between different virus genome in the pcr amplification process or viral genome amplicon and the repeated sampling of same molecular.Limiting dilution PCR or single-gene group TRAP (SGA) can reduce the possibility of above related problem significantly, hindered its application (Palmer et al, J Clin Microbial 43:406-13 (2005)) in the conventional sense of resistance sudden change but it is expensive.

Oneself is through having developed the high-flux sequence method and might being used to detect the resistance sudden change.Skin rises scale and multiple polysaccharase cloning and sequencing method (picoliter-scale and multiplex polony sequencingmethod) and can be used in the good mode of cost benefit viral genome (the Margulies et al that checks order, Nature437:376-80 (2005), Shendure et al, Science 309:1728-32 (2005)).Viral genome is to be assembled by short dna fragmentation (being respectively 200bp and 20bp).Therefore, can not obtain whole resistant mutations, and linkage analysis can not use by the sequence that arbitrary method obtained in two kinds of methods and undertaken from the single virus genome.Because high-caliber hereditary variability is difficult to the design primer collection and covers the pol gene that can comprise whole resistance sudden changes.In these two kinds of methods, the sample very complicated (Rogers et al, Nature 437:326-7 (2005)) of preparation a small amount of RNA molecule from patient's blood plasma.

In view of above-mentioned viewpoint, need badly exploitation can be used in detect less resistance colony (≤0.01%), simultaneously analyze several ten thousand viral genome, use limited sample to identify the method that all mainly suddenly change, determine the per-cent of the different resistance sudden changes that exist in the viral genome and carry out linkage analysis at single molecules level.The present invention relates to a kind of novel parallel allele-specific sequencing method (PASS) that can satisfy above all requirements.It can be used for studying the mechanism of multiple drug resistance, and the method for predicted treatment effect of being used for can be provided.

Summary of the invention

The present invention relates to a kind of method that detects genetic variant, comprise method with resistance, genetic variant that cancer is relevant with hereditary functional disorder disease.In a special embodiment, the present invention relates to a kind of testing method that can be used for detecting less resistance colony, as viral colony.

Purpose of the present invention and advantage will be from the clear embodiments of following description.

Description of drawings

Figure 1A and 1B.The diagram of parallel allele-specific sequencing method (PASS).(Figure 1A) 3 of 5 of unmodified ' end primer, Acryditeization ' end primer, dna profiling and PCR pack are embedded in 6% the acrylamide gel.Because in polymerization process, the primer of Acryditeization and polyacrylamide gel covalent coupling and be fixed are so amplification PCR products accumulates in around the dna profiling and form separately point (polysaccharase is cloned (polony)) in the amplification site.After the double-stranded DNA sex change, wash-out free DNA chain, and be retained in the gel from the DNA chain of Acryditeization primer extension.Then sequencing primer is annealed on the single-stranded template and also extends with fluorescently-labeled wild-type or resistance base.Then gel is obtained image with microarray scanner scanning.(Figure 1B) with the 3 ' end and the drug resistance mutant site of primer and put.After extending primer, can determine the base identity at place, mutational site by the fluorophore that mixes with fluorescently-labeled Nucleotide.Template sequence shows that in the bottom M184V primer sequence indicates with underscore.Primer sequence shows on top.Wild-type (WT) base that adds is represented with the Cy3 mark and with green.The mutating alkali yl that mixes is represented with the Cy5 mark and with redness.

Fig. 2 A-2C.Detect little ratio drug resistance gene mutational site (minordrug-resistance mutation) by PASS.According to the PASS sequence measurement of standard, wild-type (WT) and mutant clone are mixed.The clone carries out sequencing with the M184V primer with these polysaccharases, for wild-type base Cy3-dATP mark, for mutant base Cy5-dCTP mark.At first obtain Cy3 wild-type channel image (Fig. 2 A), be Cy5 sudden change passage (Fig. 2 B) then.At last, thus two image mutual superposition are shown the colony (Fig. 2 C) of mutant and wild-type virus in same image.

Fig. 3 A-3C.Detect little ratio resistance colony by PASS.(Fig. 3 A) mixes E44D mutant clon (WEAU-E44D) and wild-type clone (WEAU-wt) by the 1:10 serial dilution.Behind the pcr amplification, the clone checks order with the E44D primer with these polysaccharases, for wild-type base Cy3-dATP mark (green), for mutating alkali yl Cy5-dCTP mark (redness).Show that as Fig. 3 B ratio is that the molecule number of mutant/wild-type mixture of 1:1000 has increased by 10 times, so that in bigger wild-type virus colony, detect rare resistant mutation better.Behind the pcr amplification, the clone checks order with the E44D primer with these polysaccharases, with Cy3-dATP mark (green), uses Cy5-dCTP mark (redness) for the wild-type base for suddenling change.Detect the sensitivity (Fig. 3 C) that the PASS of little ratio resistance colony analyzes.To in Fig. 3 A, show for every kind of wild-type: the identical experiment of mutant ratio repeats 6 times.Carry out linear regression analysis and drawing.Error bars is represented mean value ± SD.

Fig. 4 A-4C.The detection of a plurality of resistance sudden changes on the differing molecular.Three resistance sudden changes (L90M, E44D and M184V) are introduced in the wild-type WEAU pol gene.In PASS measures, wild-type clone (WEAU-wt) and three kinds of resistance clones (WEAU-E44D, WEAU-L90M and WEAU-M184V) are mixed by 1:1:1:1.Use E44D (Fig. 4 A), M184V (Fig. 4 B) and L90M (Fig. 4 C) primer to carry out the continuity order-checking these polysaccharases clone.Following extension mutational site: the E44D site uses Cy5-dCTP (mutant) and Cy3-dATP (wild-type) to extend, the M184V site uses Cy5-dGTP (mutant) and Cy3-dATP (wild-type) to extend, and the L90M site uses Cy3-dATP (mutant) and Cy5-dUTP (wild-type) to extend.Be better observation, demonstrate the zonule in each image.Indicate the representative polysaccharase among each clone to clone with the numbering arrow.E44D, M184M, L90M and wild-type clone's color mode is respectively redness-green-redness (1), green-redness-redness (2) and green-green-green (3) in three images, and green-green-redness (4).

Fig. 5 A-5C.By the main resistance in the identical viral genome of sequence PASS analysis (sequential PASS assay) mensuration.In PASS measures, be template and the polysaccharase clone checked order with the dNTP of Cy3 or Cy5 mark with the WEAU-wt clone.For with a kind of polysaccharase clone, use different sequencing primers to carry out continuous probe for 22 main drug resistance mutant sites.In same polysaccharase clone, before a new primer annealing is attached on this template, in 45 ℃ in methane amide sex change remove each sequencing primer.According to medicament categories the result is divided into groups.The number on image top shows the sequence of PASS order-checking.The bottom of image shows each mutational site.

Fig. 6 A-6G.The detection of single base mutation in different HIV-1 hypotype and the recombinant chou.Analyze 27 HIV-1 hypotypes or recombinant chou molecular cloning by PASS.They are other recombinant chou of 2 A hypotypes, 8 B hypotypes, 5 inferior C types, 3 D hypotypes, 1 F hypotype, 2 G hypotypes, 1 H hypotype, 2 CRF01_AE, 1 CRF04_cpx and 2.These polysaccharases clone is checked order with the E44D primer of Cy3-dCTP.

Fig. 7 A and 7B.Determine the susceptibility that PASS analyzes by the serial dilution target molecule.WEAU-wt molecule (100,000,10,000,1,000,100 and 10) is embedded in (Fig. 7 A) in the every gel.The number of the actual gene group that PASS detects shows in bracket.Behind the pcr amplification, the clone uses the E44D primer of Cy5-dATP to carry out sequencing with these polysaccharases.When the molecule number of using is 10,000 or more for a long time, many points have the trend that merges each other and are difficult to counting; And the molecule number of working as use was put and is easy to accurate counting less than 10,000 o'clock.In test, use 1 copy or not copy add fashionablely, do not detect polysaccharase clone (data not shown).Arrow indicates detected 2 kinds of polysaccharases clone in the image of 10 molecules.This test repeats 6 times.Carry out linear regression analysis and draw (Fig. 7 B).Error bars is represented mean value ± SD.When analyzing 100,000 or more during polymolecular, be difficult to the quantity of accurate counting point.Therefore, they are not included in the estimation to analytical method sensitivity.Because the primer that Acrydite modifies is fixed in the acrylamide gel, and PCR carries out under solid state shape in acrylamide gel, therefore do not detect the molecule of each expection not astonishing in PASS analyzes.

Fig. 8 A and 8B.The detection of multiple drug resistance sudden change in patient's plasma sample.Never the patient (not treatment group (naive)) who treated before patient's plasma sample that (Fig. 8 A) PASS analyzes comprises, once treated but do not carry out patient's (treatment experience group) of any treatment or patient's (ineffective group) of treatment plan failure at present at present.From 140 μ l blood plasma, extract RNA, get the cDNA that is equal to 14 μ l blood plasma and be used for each PASS mensuration.Measure this M184V mutational site with Cy5-dGTP (wild-type, redness) and Cy3-dATP (mutant, green).Arrow is indicated little ratio drug-resistant virus or wild-type virus.(Fig. 8 B) analyzes from scatter 12 drug resistance mutant sites of plasma sample of patient 200372 of treatment with PASS.Using 12 different sequencing primers to carry out continuity with a kind of polysaccharase clone detects.Before new primer annealing is attached on the identical template, in methane amide, each sequencing primer is removed by sex change in 45 ℃.In 4 PI (proteinase inhibitor) mutational site (M461, G48V, D30N and 184V), 3 RT sites (V118I, E44D and K65R) relevant with the nucleosides resistance and 2 RT sites (V106A and K103N) relevant with NNRTI (non-nucleoside reverse transcriptase inhibitor) do not detect the resistance sudden change.Yet the M184V site in 2 PI resistant mutation sites (L90M and V82A) and RT detects resistance.Do not find wild-type virus in all mutational sites.This patient once accepted other treatment plan, comprised the proteinase inhibitor scheme, and this might explain the existence of PI sudden change.The result divides into groups according to medicament categories.Each mutational site shows in the image bottom.

Fig. 9 A-9D.Detect resistance sudden change in patient's plasma sample by PASS.From 140 μ l blood plasma, extract RNA, get the cDNA that is equal to 14 μ l blood plasma and be used for each PASS mensuration.With 12 drug resistance mutant sites of PASS analysis from treatment failure patient's 200369 plasma sample.Detect use 12 different sequencing primers to carry out continuity with a slice gel.The image that shows comprises resistant mutation (L100I, K103N and M184V) and a representative K65R site wild-type image.The top of image shows the shared per-cent of little ratio resistant mutation colony.Each mutational site shows in the image bottom.

Figure 10.The distribution of sudden change.

Detailed Description Of The Invention

The present invention relates to for detection of the genes of individuals group (as, virus or cancer cell genome) in sudden change (as, The relevant sudden change of MDR sudden change or cancer) parallel allele-specific sequence measurement (PASS). The method bag Draw together application (Mitra et al, the Proc.Natl.Acad.Sci.USA 100:5926-5931 of polymerase clone technology (2003); Zhu et al, Science 301:836-838 (2003)). The sensitivity of the method is suitable at the sudden change base Because of group number very low (when only existing as 6 so few mutator groups copies) or in sample, suddenly change Genome only accounts for when the full gene group copies total minor proportion (for example, so low as about 0.01%) and examines Survey sudden change. For example, can analyze simultaneously the viral genome of hundreds of thousands and can pass through identical according to the present invention The inquiring after continuously of a plurality of mutational sites of molecule (sequential interrogation) and determining in the individual molecular Main mutational site.

In a preferred embodiment, the present invention relates to a kind of a large amount of nucleic acid-templated in (as, strand or double-stranded DNA) (as, from the oncogene of resistance to the action of a drug pathogen or sudden change group) detect the method for sudden change or nucleotide modification, the method comprises:

I) (for example be fixed at the amplimer that makes modification, be covalently attached to) under the condition in the semi-solid upholder, a plurality of 5 ' amplimers nucleic acid-templated and 3 ' amplimer modified or modification are embedded in (as, record (casting) in) in this semisolid upholder;

Ii) semi-solid upholder that will obtain by step (i) and PCR composition (as 5 of, polysaccharase, dNTPs, unmodified ' or 3 ' amplimer and damping fluid) contact (as, cover with it);

Iii) make under the extremely nucleic acid-templated condition of amplimer hybridization of modification and/or unmodified with formation and primer bonded template (primed template), processing from step (ii) composition and in semi-solid upholder, carry out the pcr amplification of pre-treatment template, wherein the two strands of this amplification thing of expanding production is collected in around the template;

Iv) with double-stranded amplified production sex change and remove (as, wash-out) and be not fixed to the strand in the semi-solid upholder;

V) sequencing primer is hybridized to step (iv) on the own fixed strand of gained, and in the presence of at least 2 kinds of Nucleotide that have different detectable marker things, carry out the extension of sequencing by hybridization primer, the Nucleotide of one of them is attached in the extension products when the immobilization strand contains sudden change or nucleotide modification, and other Nucleotide the immobilization strand do not contain sudden change or be attached to during modified nucleotide in the extension products (as, the Nucleotide complementation of labeled nucleotide of one of them and sudden change or modification, and other labeled nucleotide and the Nucleotide complementation that is present in the wild-type); And

Vi) detect step (the v) extension products of the sequencing primer of gained, thus can determine to comprise the per-cent (with the per-cent of template) of extension products of the sequencing primer of sudden change or modified nucleotide.

According to present method, for example can use Standard operation procedure SOP to extract viral RNA or DNA from the viral organism sample (tissue or body fluid (as blood plasma, saliva or blood cell sample)) that contains of taking from the patient.RNA reverse transcription and the separating obtained cDNA that extracts for example, can be used Standard operation procedure SOP.This cDNA or the DNA that directly extracts can be carried out PASS mensuration by following description then.

5 ' primer of unmodified, 3 ' primer, dna profiling and the amplifing reagent of modification (as PCR reagent) are embedded in the semi-solid upholder (as, acrylamide gel)-(perhaps, can use 5 ' primer of modification and 3 ' primer of unmodified).Because in polymerization process, the primer of modifying (as the primer of Acryditeization) can be attached in the upholder and fix, this amplified production (as the PCR product) can accumulate in the cDNA template around, and form independent point (polysaccharase clone) in the site of this amplification.After the amplification, remove (as wash-out) with double-stranded DNA sex change (for example, according to the flow process of standard, methane amide) and with free DNA chain as 70%.Only the DNA chain of grappling keeps still being connected with upholder.Subsequently can be on single-stranded template with the sequencing primer annealed combination, and (as the fluorescently-labeled) wild-type or the resistance base of applying marking are extended.Can scan upholder then and obtain image.For example, when using fluorescently-labeled Nucleotide to extend primer, the discriminating of mutational site base can be measured by the Nucleotide and the final fluorophore (or other marks) that forms that embed.Although in following examples because employed instrument, only used two kinds of fluorescence dyes to measure wild-type and mutant base, but the interpolation of blue laser allows to detect with the whole four kinds of possible bases of different fluorochrome labels (Shendure et al, Science 309:1728-1732 (2005)).Described in following examples, after sex change, can remove previous sequencing primer, can use different sequencing primers up to determining all expection sudden changes then continuously.

Sequence measurement of the present invention can height than the susceptibility of conventional genotype and phenotype analytical 200-2 for example, 000 times (according to detection) to little ratio resistance virus colony (as, 0.1%～0.01% to 20%).Therefore, it can the more accurate prediction result of treatment.Owing to use the PASS assay method to determine virus genomic actual quantity, the polysaccharase clone sum in each mensuration also can be used to assess the carrying capacity of virus, therefore makes the doctor can assess the effectiveness and the prognosis of pharmacological agent.

In the method, viral cDNA or viral DNA molecule directly can be imported in the upholder (as, semi-solid as acrylamide gel), and on the single chain molecule level, increase.Therefore can eliminate the artificial sequence (artifact sequence) that produces by reorganization or repeated sampling in the PCR reaction process fully.

On having measured each independent viral genome after the sudden change of all resistance, the quantity of the sudden change that comprises of the resistant mutation that exists in each viral genome, each viral genome and contain the shared per-cent of viral genome (number in each order-checking with 100,000 viral genome in) of resistant mutation as can be known.Owing on single chain molecule, obtained all resistant mutations, can use the data of acquisition to carry out linkage analysis (Fig. 4 and 10) and determine MDR mechanism, and determine the effect of in resistance, recombinating between single or simple MDR virus by the continuity sample of analyzing the patient.

The intravital average virus load of patient that HIV infects is approximately every milliliter 10 ⁵Individual RNA copy.For example, if use the normal blood plasma of 140 μ l (280 μ l blood plasma are used for RNA and extract, and wherein half is used for PASS mensuration), in each mensuration, there is about 14,000 RNA copy.Because the verification and measurement ratio that PASS measures is 16% (Fig. 5), each analysis can detect 2,520 polysaccharase clones.Therefore, the PASS measuring method is suitable for the analysis of the plasma sample of Most patients well.Even when virus load up to every milliliter 10 ⁶When individual RNA copies, once analyze the detection that to carry out 25,200 resistance sudden changes in the virus copy, because this analysis can detect 1-10 molecule (Fig. 7) in 50,000 molecules.When virus load is too low, can concentrate viral sample, as concentrate from being used for a large amount of blood plasma that PASS measures (as, pass through ultracentrifugation).

Although the present invention will be described in detail the embodiment relevant with HIV following, but this PASS assay method also can be used for the existing of infectious agent in the detection of biological sample (as, virus or bacterial infection agent) and/or detect resistance sudden change in HBV, HCV or other the infectious agent ((TB)) as mycobacterium tuberculosis (Mycobacterium tuberculosis).And the PASS assay method can be used to detect CTL (cytotoxic T lymphocyte) and neutralizing antibody escape sudden change (neutralizing antibody escape mutation).

Remove the above, the present invention also can be applicable to detect from sudden change in cancer patients's the sample or methylated nucleosides.Genetic expression preface type analysis (gene-expression profiling) can be used for tumor grade and measures cancer patients's prognosis (Hoheisel, Nat.Rev.Genet.7:200-210 (2006); Schena et al, Science 270:467-470 (1995); Endoh et al, J.Clin.Oncol.22:811-819 (2004); Lossos et al, N.Engl.J.Med.350:1828-1837 (2004); Chen et al, N.Engl.J.Med356:11-20 (2007)).Some sudden change can cause the generation of cancer in the host gene.Up to now, with many sudden changes and the not controlled cell growth phase related (having reported the sudden change that is associated with mammary cancer as oneself) that can cause tumour to form.Also reported development to the energy remarkable influence cancer that methylates of some gene or promotor.The tumour cell that carries relevant sudden change of cancer or methylated nucleosides only accounts for the part of total cell count in the biopsy; The high per-cent of cancer cells has illustrated higher aggressive or bad prognosis.Therefore, key is to detect the sudden change or the methylated nucleosides of low per-cent in the allos cancer sample.Recently, use large-scale parallel skin to rise the reactor sequences determination techniques, detected 0.2% sudden change (Thomas et al, Nat.Med.12:852-855 (2006)) in the epidermal growth factor receptor gene in nonsmall-cell lung cancer (EGFR).This method helps selecting downpayment methods of treatment and target inhibitor for the recurrence patient.PASS assay method described herein is 0.01% to the detection sensitivity of rare mutant strain, and it can more effectively detect the sudden change or the methylated nucleoside of lower frequency in the allos cancer sample.Can be used for the extraction of genomic dna from biopsy, blood or the ejecta (as urine, ight soil or mucus or serous secretion thing (saliva) etc.) of individuality.These DNA can be digested to then to be used to detect the relevant sudden change of cancer or to handle to modify and be used to detect methylated nucleoside (Fisher et al, LungCancer Epub ahead of print December 28,2006 by sodium bisulfite than small segment; Tomii et al, Int.J.Cancer120:566-573 (2007)).As described herein, measure for standard P ASS, the DNA that handled can be embedded in the polyacrylamide gel, and carry out PASS and analyze sudden change of cancer dependency or the methylated nucleoside that detects rareness.This method can be used for the diagnosis and the prognosis of cancer, and selects downpayment methods of treatment for better nursing the patient.

Some aspect of the present invention will be described in more detail in following non-limiting example (also can be referring to USPs6,432,360,6,485,944 and 6,511,803 and U.S. Patent application Nos.20030207265,20030124594,20020127552,20060014167,20020120127,20030215856 and 20020120126 in to the detailed description of polysaccharase clonal expansion).(also can be referring to Butz et al, BMC Biotechnology 3:11 (2003); Mitra and Church, Nucleic AcidsResearch 27 (24): e34 (1999); USP 5,958, and 698, USP 5,616,478; Samatov et al, Nucleic Acids Research 33 (17): e145 (2005); Samatov et al, AnalyticalBiochemistry 356:300-302 (2006); Chetverina et al, BioTechniques 33:150-156 (2002); Chetverina et al, Analytical Biochemistry 334:376-381 (2004).)

Embodiment

Experimental detail

The preparation of resistance mutant clon.The part pol gene that will comprise maximum drug resistance mutant sites is from almost total length HIV-1 clone WEAU.A1 amplification.By overlapping PCR method, E44D, M90L and M184V sudden change are introduced.Then this wild-type (WEAU.wt) and mutant PCR product (WEAU.E44D, WEAU.M90L and WEAU.M184M) directly are cloned into the pSTBBlue carrier (Novagen, Madison, WI) in.Confirm these resistance sudden changes by sequencing.For tracer experiment, mutant (is higher than 10 with the wild-type clone with different ratios ⁴) mix.By the concentration of NanoDrop spectrophotometric determination plasmid DNA, calculate the number of molecule in each dna sample based on DNA concentration and plasmid length (bp).

Slide treatment.With teflon-coating glass slide (Eriescientific, Portsmouth, NH) be exposed to UV-light 15 minutes in the PCR stink cupboard (PCR hood), (0.02% Glacial acetic acid and 0.4% is in conjunction with salt solution { Amersham Biosciences then to use binding soln, Piscataway NJ}) handled 1 hour.Then with slide glass water flushing 3 times, alcohol flushing 2 times, and in the PCR stink cupboard dry 40 minutes.The slide glass of handling is stored in the vacuum drier.

Gel casting.Slide glass and cover plate are exposed to UV-light 15 minutes in the PCR stink cupboard.Fill 20 μ l gel mixtures in the oval space on the slide glass, this mixture contains in 6% polyacrylamide gel: reverse primer, template (1-18 μ l), 0.3% diallyl tartramide, 5% Rhinohide, 0.1% APS, 0.1% TEMED, 0.2% BSA (bovine serum albumin) that the Acrydite of 1 μ M modifies).Make this gel mixture polyase 13 0 minute.With blade remove cover plate thereafter.The tween 20 solution flushing slide glass of use 0.05% 15 minutes, complete drying then.

Pcr amplification in the gel.The Jumpstart Taq archaeal dna polymerase (Sigma that contains 1 μ M forward primer, 0.1% polysorbas20,0.2% bovine serum albumin, 1xPCR damping fluid, 250 μ M dNTP mixtures, 3.3 units in the diffusive mixing thing of PCR reagent, St.Louis, MO) and water (supplying 20 μ l).In case mixture is added to the central authorities of gel, covers gel with cover plate immediately, and placed 10 minutes in room temperature.With gel and cover plate SecurSeal groove (Grace Bio-Labs, Bend, OR) sealing.Each groove is filled with the mineral oil of 640 μ l.To contain proteolytic enzyme and part RT gene about 1.1kb the pol gene fragment with primer PAF15 '-CTTTGGCAACGACCCCTCGTCACA-3 ' (2265-2288, HXB2) and PAR25 '-CCTGCATAAATCTGACTTGCCCAATTCAA-3 ' (3367-3339) increase.This PCR product comprises all the main resistant mutations for efabirenz (NRTI), non-nucleoside reverse transcriptase inhibitor (NNRTI) and proteinase inhibitor (PI).With PTC-200 thermal cycler (PTC-200 Thermal Cycler; MJ Research, Hercules, CA), carry out the polysaccharase clone PCR according to following reaction conditions: 94 ℃ were reacted 3 minutes; Totally 65 of 30 seconds, 56 ℃ reactions of 94 ℃ of reactions 45 seconds and 3 minutes circulations of 72 ℃ of reactions; 72 ℃ were reacted 6 minutes.

Single-basic extension (SBE).After the pcr amplification, slide glass was soaked 15 minutes with hexane in Coplin jar (Coplin jar), and wash 4 times with washing lotion 1E (10mM Tris-HCI pH7.5,50mM KCl, 2mM EDTApH8.0 and 0.01% Triton X-100).Then with slide glass (70% methane amide and 1xSSC) sex change 15 minutes in the methane amide denaturing soln.Water flushing 1 time, washing lotion 1E uses FrameSeal groove (Bio-Rad to every slide glass after washing 2 times, Hercules, and add the annealing mixture (6xSSPE, the sequencing primer of the 100 μ M of 0.01%Triton X-100 and 75 μ l) of 125 μ l to every slide glass CA).In the PTC-200 thermal cycler, carry out the hybridization of sequencing primer according to following reaction conditions: 94 ℃ of reactions were reacted 20 minutes in 6 minutes and 53 ℃.After primer annealing is attached on the template, slide glass with washing lotion 1E flushing 2 times, is used Klenow damping fluid (50mM Tris-HCI pH7.4,5mM MgCl again ₂With 0.01%Triton X-100) in equilibrium at room temperature 3 minutes.45 microlitres are extended mixture (deoxynucleotide { wild-type or mutant } of 0.5 μ M Cy5 mark in the Klenow damping fluid, different deoxynucleotide { mutant or wild-type }, 1% single-stranded DNA binding protein { the USB corporation of 0.5 μ M Cy3 mark, Cleveland, the Klenow enzyme of OH} and 10 units) be added on the every gel.With slide glass incubated at room 3 minutes, with washing lotion 1E washing 2 times, and with ScanArray Express (PerkinElmer, Wellesley MA) scan.

The extra anti-medicine that checks order suddenlys change.For measuring the main resistance sudden change in each proteolytic enzyme/RT amplicon, polysaccharase to be cloned slide glass use the methane amide denaturing soln in 45 ℃ of processing 15 minutes, then water flushing is washed 2 times with washing lotion 1E.Scanned slide is annealed them then to guarantee to remove fully the sequencing primer from previous SBE (extension of single base) with different sequencing primers.Repeat the SBE step until the sudden change that determines all expectations (referring to table 1 and Fig. 5).

The primer that uses in the table 1:PASS order-checking

Viral RNA extracts and reverse transcription.(Valencia CA) extracts viral RNA (vRNA) from 280 μ l plasma samples from the HIV-1 infected individuals for QIAamp Viral RNA Mini Kit, Qiagen Inc. to use the QIAamp viral RNA to extract test kit in a small amount.The vRNA elution is gone in the 60 μ l AVE damping fluids, and it is synthetic that the vRNA of 17 μ l is used for cDNA, described synthesizing used Superscript III (Invitrogen Corp in the reaction volume of 40 μ l, Carl sbad is CA) with primer RTunil5 '-CCAATCCCCCCTTTTCTTTTA AAATTGTG-3 '.To make an appointment with half cDNA (18 μ l) to be used for a PASS measures.

The result

The parallel detection of single resistance sudden change.Recently, oneself is through having developed polysaccharase clone (polymerasecolony; Polony) technology is to detect the single base mutation (Mitra et al, ProcNatl Acad Sci USA 100:5926-31 (2003), Mitra et al, Anal Biochem 320:55-65 (2003)) of single molecules level in solid phase.Oneself is used to describe the montage (combinatorialalternative pre-mRNA splicing) and parallel sequencing (Shendure et al of combination selection precursor-mRNA this method, Science309:1728-32 (2005), Zhu et al, Science 301:836-8 (2003)).For detecting the viral colony of little ratio resistance, in once measuring, analyze hundreds thousand of kinds of viral genome by using the polysaccharase clone technology simultaneously, developed parallel allele-specific sequencing (PASS) method.The pol gene fragment of the 1.1kb that contains proteolytic enzyme and Partial Inverse transcriptase (RT) gene has increased.This amplicon comprises all the main resistant mutations at reversed transcriptive enzyme and proteolytic enzyme.In acrylamide gel, carry out polysaccharase clone's amplified reaction.Because in polymerization process, the primer that Acrydite modifies fix in polyacrylamide gel by covalent attachment, the PCR product accumulate in dna profiling around and at the position of amplification formation point (polysaccharase clone) separately (Figure 1A).After the amplification, negative DNA chain and the hybridization of complementary sequencing primer, primer 3 ' end and resistant mutation are also put.After primer extended by single-basic extension (SBE), the identity of mutational site base was measured (Figure 1B) by the Nucleotide of the different fluorophore marks of usefulness of embedding.Obtain image by microarray scanner, this image is used for the analysis of wild-type and mutant colony.

In order to measure the sensitivity that PASS analyzes, wild type molecule (WEAU-wt) is mixed (1:1,1:10,1:100 and 1:1,000) with mutant molecules (WEAU-E44D) according to different ratios.Detect this mixture with the E44D primer then.With 0.1% or the sudden change that exists of higher value be easy to detect (Fig. 3 A).With identical 1:1, under 000 mutant and the wild-type ratio, virus genomic input is increased at 10,000 o'clock, although the part wild type molecule merges each other and can not count exactly, still can detect resistant mutation (Fig. 3 B) easily.Therefore, being used to detect the sensitivity of method of the resistant mutation of little ratio can be up to about 0.01%.Linear regression analysis has disclosed between the mutant/wild-type ratio of detected and expection and has had good linear relationship (R ²=0.9945) (Fig. 3 C).

By multiple single-basic extension (SBE), 22 main sudden changes on independent molecule, have been detected, this explanation is when the circulation time that more checks order, and the PASS assay method can be used for detecting the main sudden change of the great majority of analyzing for interlock type and other less important sudden change (Fig. 5) of potential.27 viral genome have also been measured from many hypotypes and recombinant form (2 A hypotypes, 8 B hypotypes, 5 C hypotypes, 3 D hypotypes, 1 F hypotype, 2 G hypotypes, 1 H hypotype, 2 CRF01_AE recombinant types, 1 CRF04_cpx recombinant type and 2 other recombinant chou).All oneself hypotype and recombinant chous of test have all obtained equal amplification, and this explanation PASS assay method can be used in most hereditary hypotype and recombinant chou (Fig. 6).This assay method is about 6 viral genome copies for the sensitivity of wild-type and the detection of mutant colony.Linear regression analysis is presented between 10 to 10,000 viral genome copies and has good linearity test dependency (R ²=0.9958) (Fig. 7).When analyzing greater than 200,000 wild type molecule, do not detect the base of sudden change, this shows that under present experiment condition the mutation rate of introducing by the Taq polysaccharase is lower than 5x10 ⁶Therefore, will unlikely be the PCR mistake by the detected low frequency sudden change of PASS.

For detection is present in each intramolecular different resistant mutation in the virus population, prepared 3 independent resistance clones (WEAU-E44D, WEAU-L90M and WEAU-M184V), and the mixed that (WEAU-wt) presses 1:1:1:1 has been cloned in they and wild-type.Then these polysaccharases clone is carried out the succession order-checking with E44D, M184V and L90M primer.As expection, in each reaction, about 25% polysaccharase clone contains resistant mutation, and other polymkeric substance of 75% clone is wild-type (Fig. 4 A-4C).When from all 3 image analysis polysaccharase clones, the pattern of the shown in green-green-redness of wild-type clone, and the pattern of the shown in red-green-redness of E44D clone, M184V clones the pattern of shown in green-redness-redness, and L90M shown in green-pattern of green-green.These results have proved when a large amount of viral genome is carried out parallel analysis, and the PASS assay method can be measured the existence of the different resistant mutation of every kind of viral genome being used for linkage analysis exactly.

In order to utilize the PASS assay method to measure, 13 plasma samples have been analyzed: the patient who did not treat (not treatment group), once treated but do not carry out patient's (treatment experience group) of antiretroviral therapy and patient's (ineffective group) of treatment plan failure at present at present from 3 different patients' groups from the resistance colony in the sample of HIV infected patient.At first measure the M184V sudden change, and in the patient who did not treat, do not measure this sudden change; In 6 patients that before treated, detect little ratio resistance colony (＜2%) in 2 patient's bodies; And in the patient of treatment failure, detect height ratio (major) resistance colony (36%-100%) (Fig. 8 and table 2).

The detection of the resistance sudden change of A → G in the table 2.M184V codon

For detecting the resistant mutation that whether has other in the virus population of being studied, 7 patient's samples from three groups have been carried out 12 possible resistant mutation sites analyzed (table 3).In the patient of 1 treatment experience group patient and all 3 treatment ineffective group, detect other resistant mutation.In No. 200372 patients, detect L90M and V82A sudden change, detect K70R and T215Y sudden change (table 3 and Fig. 7) among No. 200371 patients.In these two patients, all do not detect wild-type virus.In other two patients, all detect resistance and wild-type virus (Fig. 9) in a plurality of resistant mutations site.This makes and carries out detailed linkage analysis and become possibility.In No. 200369 patient's bodies in the treatment ineffective group, there is the L100I sudden change in 90.32% the virus, and has the K103N sudden change in 89.64% the virus.Utilize the genotype detection method of standard to fail detected 36.26% M184V mutated viruses and can detect (Fig. 9) with the PASS assay method at an easy rate.The linkage analysis demonstration is carried L100I and K103N sudden change simultaneously more than the virus (52.14%) of half, and 35.10% virus contains 3 kinds of resistant mutations (L100I, K103M and M184V).There is (L100I0.66%, K103N 0.07%, M184V 0.07%, M100I/M1184V 0.07%) (table 3) in other the virus that contains single or two resistant mutations as little ratio colony.No. 200362 patients that carried out treatment in the past also demonstrate the chain preface type of similar resistant mutation (profile).Although most of viruses contain one or more resistant mutations, two patients still have a spot of wild-type virus population (1.67% and 9.22%).

The linkage analysis of resistance sudden change in the individual viral genome of table 3.

Still unconfirmed in the patient of retrovirus treatment failure as the continuous improvement on the HIV treatment result of genotype or phenotype test result (Johnson et al, Top.HIV Med.13:125-131 (2005)).This result's partly cause may be owing to be used to detect the assay method sensitivity of little ratio resistance colony too low (exist〉20% mutated viruses) (Petropoulos et al in these patients, Antimicrob.AgentsChemother.44:920-928 (2000), Van Laethem et al, J.Acquir.Immune Defic.Syn dr.22:107-118 (1999)).Compare with genotype or phenotype test method, the mutation determination method all has significantly higher sensitivity (Van Laethem et al, Acquir.Immune Defic.Syndr.22:107-118 (1999)).Because the resistance sudden change that detects by these methods is based on colony, they all can not carry out linkage analysis.Yet oneself is used for studying the little ratio resistance colony of every kind of viral genome and the linkage analysis of resistant mutation (Condra et al, Nature 374:569-571 (1995)) the cloned sequence assay method.Though the PASS assay method need spend a couple of days and analyze all main sudden changes, to compare with the cloned sequence assay method, it can be more effective in itself, quicker, more sensitive detects little ratio resistance colony.Need spend the long time and go to finish although compare the PASS assay method with the genotype detection method of standard, for detecting little ratio resistant mutation and he provides the sensitivity much outstanding for linkage analysis.Though the clinical meaning of little ratio resistance virus population is also not definite fully, but these colonies are remarkably influenced result of treatment (Charpentier et al afterwards possibly, J.Virol.78:4234-4247 (2004), Metzner et al, J.Infect.Dis.188:1433-1443 (2003), Winters et al, J.Virol.72:5303-5306 (1998)).Because for detecting the viral colony of little ratio resistance, the PASS assay method is higher about 1,000 times than the sensitivity of genotype or phenotype test method (0.01% pair 20%), it can the more accurate prediction result of treatment.The PASS assay method allows the detailed linkage analysis to a plurality of sudden changes, makes research difference sudden change combination become possible (table 3) for the influence of little ratio and the viral colony of big ratio.Except these improvement clinically, the technical superiority of PASS assay method merits attention.For example, in PASS operation with the direct embedding of viral cDNA molecule to polyacrylamide gel, and in the enterprising performing PCR amplification of single molecular level.Therefore, the artificial sequence that produces by reorganization or repeated sampling in the conventional PCR reaction process is eliminated (Fang et al, J.Virol.Methods 76:139-148 (1998), Liu et al, Science273:415-416 (1996)).

To sum up, described above is a kind of have highly sensitive, high specific and high-throughout PASS assay method that can detect little ratio resistant population in the individuality of infected by HIV.This method has been used the polysaccharase clone technology, oneself is used to detect the montage of combination selectivity precursor mRNA this technology, parallel detection single base mutation and parallel sequencing (Zhu et al, Science 301:836-838 (2003), Merrit et al, Biotechnol.Bioeng.92:519-531 (2005), Butz et al, BMC Genet.5:3 (2004), Shendure et al, Science 309:1728-1732 (2005)).This new technology also can be full of HIV that potentiality ground is used for the antigenic determinant that detects at T and B cell-targeting escape sudden change, other human pathogen (as B-mode and hepatitis C virus, TB etc.) and sudden change or the nucleosides modification relevant with cancer.

All Files cited above and other information resources are all incorporated reference in this.

Claims

1. one kind in a large amount of methods that detect or measure the frequency of sudden change or nucleotide modification in nucleic acid-templated, and this method comprises:

I) will be a large amount of 5 ' amplimer of nucleic acid-templated and the 3 ' amplimer modified or modification under the amplimer that makes described modification is fixed in condition in the semi-solid upholder, be embedded in the described semi-solid upholder;

Ii) the described semi-solid upholder that step (i) is obtained and 5 of polysaccharase, triphosphate deoxy-nucleotide (dNTPs) and unmodified ' or 3 ' amplimer contact;

Iii) nucleic acid-templatedly handle the composition that (ii) obtains from step to described to form under the condition with primer bonded template in the amplimer hybridization that makes described modification and/or unmodified;

Iv) carry out described in described semi-solid upholder and amplification primer bonded template, the double-stranded product of wherein said amplification concentrates on described with around the primer bonded template;

V) with described double-stranded amplified production sex change and remove to obtain be not fixed to strand in the described semi-solid upholder;

Vi) with sequencing primer and (the immobilized strand hybridization that v) obtains of described step, and having in the presence of at least 2 kinds of Nucleotide of different detectable labels, the sequencing primer of described hybridization is extended forming extension products, and a kind of in the wherein said Nucleotide is attached in the described extension products when described immobilization strand contains described sudden change or nucleotide modification; Described Nucleotide in addition is not attached in the described extension products when described immobilization strand does not contain described sudden change or modified nucleotide; And

Vii) detect from step (the vi) extension products of the sequencing primer of Huo Deing, and determine to comprise the described nucleic acid-templated existence or the frequency of described sudden change Nucleotide or modified nucleotide.

2. according to the process of claim 1 wherein that described method is a kind of method of measuring mutation frequency.

3. according to the process of claim 1 wherein described a large amount of nucleic acid-templated double-stranded DNA that comprises.

4. according to the process of claim 1 wherein described a large amount of nucleic acid-templated pathogenic agent that derives from.

5. according to the method for claim 4, wherein said pathogenic agent is viral pathogen or bacterial pathogens.

6. according to the method for claim 5, wherein said pathogenic agent is human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV) or mycobacterium tuberculosis (TB).

7. according to the method for claim 5, wherein said antigen is the resistance pathogenic agent.

8. according to the process of claim 1 wherein described a large amount of nucleic acid-templated genome that derives from cancer cells.

9. method according to Claim 8, wherein said cancer cells is the human cancer cell.

10. according to the process of claim 1 wherein that described cancer cells derives from biopsy, blood or ejecta.

11. according to the method for claim 10, wherein said ejecta comprises urine, ight soil, mucus or serous secretion thing.

12. according to the process of claim 1 wherein that the amplimer covalency of described modification attaches to described semi-solid upholder.

13. according to the method for claim 12, wherein said semi-solid upholder is an acrylamide gel.

14. according to the process of claim 1 wherein that 5 ' primer of unmodified and 3 ' primer of modification are embedded in the described semi-solid upholder.

15. according to the process of claim 1 wherein that 3 ' primer of unmodified and 5 ' primer of modification are embedded in the described semi-solid upholder.

16. according to the process of claim 1 wherein that described Mdification primer is the primer of Acrydite mark.

17. according to the process of claim 1 wherein that described detectable label is a fluorescent mark.

18. a method of measuring patient's body inner virus carrying capacity, this method comprises:

I) from biological specimen, extract viral DNA from described patient;

Ii) 5 ' amplimer of 3 ' amplimer of described DNA and modification or modification under being fixed in condition in the semi-solid upholder, the amplimer that makes described modification is embedded in the described semi-solid upholder;

Iii) the described semi-solid upholder that step (i) is obtained and 5 of polysaccharase, triphosphate deoxy-nucleotide (dNTPs) and unmodified ' or 3 ' amplimer contact;

Iv) under the condition with formation and primer bonded template, handle the composition that (iii) obtains from step at amplimer that makes described modification and/or unmodified and described DNA hybridization;

V) carry out described in described semi-solid upholder and amplification primer bonded template, the double-stranded product of wherein said amplification concentrates on described with around the primer bonded template;

Vi) with described double-stranded amplified production sex change and remove strand in the described semi-solid upholder of not being fixed to of gained;

Vii) with sequencing primer and described from step (the immobilized strand hybridization that vi) obtains, and having the sequencing primer to described hybridization extends with the formation extension products in the presence of at least a Nucleotide of detectable label; And

Viii) detect from step (the vii) extension products of the mark of Huo Deing, and definite thus described virus load.

19. according to the method for claim 18, wherein step (ii) before, shear described DNA.

20. according to the method for claim 19, the length of the DNA after the wherein said shearing is that about 100 bases are to about 1000kb.

21. according to the method for claim 20, the length of the DNA after the wherein said shearing is that about 1kb is to about 100kb.

22. a method of measuring patient's body inner virus carrying capacity, this method comprises:

I) from biological specimen, extract viral RNA from described patient;

Ii) the described RNA of reverse transcription to be generating cDNA, and 5 ' amplimer of 3 ' amplimer of described cDNA and modification or modification is embedded in the described semi-solid upholder under the amplimer that makes described modification is fixed in condition in the semi-solid upholder;

Iv) handle the composition that (iii) obtains from step to form under the condition with primer bonded template in the amplimer that makes described modification and/or unmodified and described cDNA hybridization;

Vii) with (the immobilized strand hybridization that vi) obtains, and of sequencing primer and described step at the extension products of the extension that has the sequencing primer that carries out described hybridization in the presence of at least a Nucleotide of detectable label with the formation mark; And

23. a method of measuring patient's body inner virus carrying capacity, this method comprises:

I) from biological specimen, extract viral DNA from described patient;

Iii) the semi-solid upholder that step (i) is obtained and 5 of polysaccharase, triphosphate deoxy-nucleotide (dNTPs) and unmodified ' or 3 ' amplimer contact;

Iv) handle the composition that (iii) obtains from step to form under the condition with primer bonded template in the amplimer that makes described modification and/or unmodified and described DNA hybridization;

(the immobilized strand hybridization that vi) obtains is to form the duplex of mark vii) will to have the probe of specific mark and described step for described virus; And

Viii) detect the existence of the duplex of described mark, and determine described virus load thus.

24. according to the method for claim 23, wherein step (ii) before, shear described DNA.

25. a method of measuring patient's body inner virus carrying capacity, this method comprises:

I) from biological specimen, extract viral RNA from described patient;

26. a method that detects sudden change or nucleotide modification in being derived from from biological specimen separated DNA a large amount of nucleic acid-templated, this method comprises:

I) 5 ' amplimer of described a large amount of nucleic acid-templated and the 3 ' amplimer modified or modifications under being fixed in condition in the semi-solid upholder, the amplimer that makes described modification is embedded in the described semi-solid upholder;

Vi) with sequencing primer and described from step (the immobilized strand hybridization that v) obtains, and having in the presence of the Nucleotide of detectable label, sequencing primer to described hybridization extends, the described Nucleotide that wherein has described detectable label is attached in the described extension products when described immobilization strand contains described sudden change or nucleotide modification, thereby forms the extension products of mark; And

Viii) detect from step (the vii) extension products of the mark of Huo Deing, and the existence of definite thus described sudden change or nucleotide modification.

27. according to the method for claim 26, wherein said biological specimen comprises the DNA from cancer cells.