WO2009140191A2 - Détection d'une maladie liée à des mutations de gène par une amplification de molécule unique avec des dosages de ligature multiplex - Google Patents

Détection d'une maladie liée à des mutations de gène par une amplification de molécule unique avec des dosages de ligature multiplex Download PDF

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WO2009140191A2
WO2009140191A2 PCT/US2009/043436 US2009043436W WO2009140191A2 WO 2009140191 A2 WO2009140191 A2 WO 2009140191A2 US 2009043436 W US2009043436 W US 2009043436W WO 2009140191 A2 WO2009140191 A2 WO 2009140191A2
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mutations
drug
detection
resistant
resistance
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PCT/US2009/043436
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WO2009140191A3 (fr
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Jun Zhu
Qin Zhang
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Biomedomics, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification

Definitions

  • HIV infection has affected more than 65 million people worldwide during the 27-year epidemic. To date, more than 20 drugs have been approved for treating HIV infections. These drugs typically target three viral genes: protease, reverse transcriptase, and envelope.
  • HAART highly active antiretroviral therapy
  • the resistant viruses When patients develop resistance to antiretroviral therapy, the resistant viruses often do not contain identical resistant mutation(s) and instead, a number of different resistant mutations coexist in an individual's viral genomes (Cai et al., 2007; Martinez-Picado et al., 2000; Palmer et al., 2005). While some of them occur in the major populations, others are relatively low abundant. Major drug-resistant populations are thought to be the cause of treatment failure; the role of co-existing minority resistant populations can also be pivotal.
  • MDR drug-resistant virus carrying multiple drug-resistant mutations
  • Sensitive detection of drug-resistant mutations especially MDR can help predict treatment response and select the most effective drugs to delay the emergence of drug resistance.
  • several technologies have been developed to detect rare resistance mutations. (Cai et al., 2007; Hance et al., 2001 ; Palmer et al., 2006).
  • One of them is parallel allele-specific sequencing (PASS) assay, which is based on single-molecule amplification and detection in a solid phase. Digital in nature, the PASS assay can efficiently detect minor drug-resistant populations at 0.01 %-0.1 %. More importantly, solid-phase detection allows for sequential interrogation of multiple mutations in individual viral genomes; thus, the linkage between mutations can be unambiguously determined.
  • the PASS platform is advantageous because of its high sensitivity and ability to detect mutation linkage.
  • An object of the present invention is to further develop the PASS platform to facilitate its clinical application in HAART treatment. This is achieved by simplifying the amplification strategy and increasing the throughput of mutation detection strategy.
  • taught herein is a method of multiplexing mutation detection strategy of the PASS platform using oligonucleotide ligation assay.
  • oligonucleotide ligation assay replaces single-base extension
  • Bridge PCR is employed to simplify the amplification step of the PASS platform.
  • the clonally amplified PCR products which are covalently linked to a solid support, are analyzed by sequential SBE or OLA to detect drug-resistance mutations. The entire procedure is automated so that a new generation of PASS platform is suitable for routine clinical practice.
  • the present detection method may be used for all HIV drug-resistant mutations including high and low frequency, single and multiple as well as all of other viral drug-resistant mutations such as HBV, HCV, RSV, HPV. Likewise, it may be used for detection for all of bacterial drug-resistant mutations, including TB and other STD. Further, it may be used to detect all cancer drug-resistant mutations and all of causal mutations, i.e., those not related to drug-resistant.
  • the present detention method is cost effective, automatable, rapid and well suited for the clinical setting. It is applicable for detection of mutations in both DNA and RNA, and it is sensitive to low frequency mutation (0.01 - 0.001 %) in single molecules. Further, Bridge-PCR-OLA as well as OLA-polony may use multi-colors for multi-mutation detection in single molecules.
  • rare genetic variations and mutations in genetic material and their linkage are determined by a) single-molecule amplification, bridge PCR, or other clonal amplification, b) detection of genetic variation by means of multiplex ligation products; and c) scanning and repeating steps a and b, so that multiple mutations can be identified and their linkage can be determined.
  • Single-molecule amplification may be achieved by a polymerase colony, wherein the genetic material is selected from DNA, RNA or synthetic polymers.
  • One or more oligonucleotide primer pairs may be included in step b, wherein the primer pairs are labeled for detection by electromagnetic radiation means. Further, the primer pairs may be labeled with one or more fluorophores, wherein the fluorophores are provided a fluorescent dye moiety such as Cy3 or Cy5 moieties. The fluorophores can be added to upstream or downstream primer, or both.
  • Minority resistant viruses could also become dominant quasispecies under drug selection.
  • minority drug-resistant populations were detected at baseline and they soon became dominant virus populations.
  • One group of researchers have suggested that they are the driving force that lead to drug resistance in later antiretroviral treatment under continuous protease inhibitor selection pressure (Charpentier et al., 2004).
  • minority protease inhibitor-resistant viruses were detected in 8 out of 1 1 pre-treated patients; they became majority quasispecies as those patients start to fail the subsequent salvage therapy (Kapoor et al., 2004). These minor resistance populations were only detected by a sensitive RNA/DNA heteroduplex generator-tracking assay.
  • Detection of low-frequency resistant populations is crucial for predicting treatment outcomes and rational selection of treatment regimens.
  • treatment often leads to the generation of resistant vi ruses carrying multiple drug-resistance (M DR) mutations.
  • M DR drug-resistance
  • Fig. 2 very little is known on how individual viruses carrying MDR mutations exert their effects on HAART regi mens.
  • treatment with any combination of drugs would be effective for suppressing viral replication (Fig. 2).
  • MDR mutations are found in individual viral genomes, those viruses, even at low frequencies, are likely to expand into majority populations and quickly develop resistance to the drugs and render them ineffective. Therefore, linkage analysis between drug-resistance mutations is necessary to determine whether they reside in the same viral genome or in different viral genomes. Such information may be especially important for patients who have developed resistance to HAART regimen and will undergo a salvage therapy.
  • the PASS platform With the PASS platform, thousands of individual viral genomes can be simultaneously amplified in a solid phase and known drug-resistance mutations can be subsequently analyzed in a high-parallel fashion. Digital by nature, the PASS assay can reliably detect minority drug-resistant populations at a frequency of 0.01 %-0.1 %. More importantly, solid-phase amplification and detection allows multiple drug-resistant mutations to be sequentially interrogated so that the linkage between mutations can be unambiguously determined. Therefore, the PASS platform is advantageous in that it could offer both highly sensitive mutation detection as well as linkage analysis. From the technology point of view, the PASS platform can be further improved (e.g. multiplexing mutation detection, miniaturization and automation). Therefore, it has the potential to fill the gap in clinically detecting drug-resistance mutations for personalized design of HARRT regimens.
  • the present inventors have developed a parallel allele-specific sequencing (PASS) platform by which tens of thousands of viral genomes can be simultaneously analyzed in a highly cost-effective manner.
  • PASS allele-specific sequencing
  • a 1 .1 kb pol gene fragment containing protease and the partial RT coding regions is amplified, covering all major resistance mutations to NRTIs, NNRTIs, and PIs.
  • Parallel amplification of individual molecules is carried out in an acrylamide gel and one amplification primer is acrydite-modified.
  • rare genetic variations and mutations in genetic material and their linkage are determined by a) single-molecule amplification, bridge PCR, or other clonal amplification, b) detection of genetic variation by means of multiplex ligation products; and c) scanning and repeating steps a and b, so that multiple mutations can be identified and their linkage can be determined.
  • Such genetic variations can be causal for human diseases (e.g. mutations in oncogenic pathways) and/or drug-resistance (antiviral or targeted therapy), wherein the disease may be either inherited, transmitted, or due to somatic mutations.
  • ligation products employed in step b, rehybridization of a different set of oligonucleotides; and ligation and detection of signal in step c.
  • the ligated products may be removed by physical, enzymatic, or chemical means, such as denaturation by increase temperature.
  • the genetic variations detected are not limited to single nucleotide changes; they can be short insertion and deletions. Differentiation of ligate and unligated products may be based on any intrinsic property or extrinsic modifications.
  • Single-molecule amplification may be achieved by a polymerase colony, wherein the genetic material is selected from DNA, RNA or synthetic polymers.
  • One or more oligonucleotide primer pairs may be included in step b, wherein the primer pairs are labeled for detection by electromagnetic radiation means.
  • the primer pairs may be labeled with one or more fluorophores, wherein the fluorophores are provided a fluorescent dye moiety such as Cy3 or Cy5 moieties.
  • the fluorophores can be added to upstream or downstream primer, or both.
  • the modified primers are covalently linked to acrylamide during polymerization and uniformly distributed in the gel matrix.
  • the rest of the PCR reagents (unmodified primer, template, nucleotides, DNA polymerase) are subsequently added into the gel by diffusion.
  • the diffusion of amplicons is restricted by two factors: the immobilized primers and the acrylamide gel itself.
  • the amplification products of each molecule will form a physically distinct sphere.
  • Each sphere (or polony) contains exact copies of the initial template, and thousands to millions of polonies can be amplified in parallel on a slide (Mitra and Church, 1999; Zhu et al., 2003).
  • each amplicon is covalently linked to the acrylamide matrix
  • polony slides can be denatured.
  • the resulting single-stranded amplicons will be detected by hybridization of fluorophore-labeled probes or by single-base extension (SBE) and subsequently visualized by a fluorescence microscope or a conventional microarray scanner (Mitra and Church, 1999; Zhu et al., 2003).
  • WT molecules WEAU-wt
  • mutant WEAU- E44D plasmids
  • PASS platform a ratio of WT molecules
  • Mutations present at 0.1 % or higher (1 :1000) can be reliably detected.
  • the sensitivity can go up to 0.01 % when more molecules are included as input materials although some of wild-type molecules are fused to each other and their exact number can not be accurately counted.
  • the PASS platform allows for identification of multiple drug-resistant mutations on the same viral genomes (linkage analysis) by sequentially SBE with primers specific for different mutation sites. To demonstrate this, a mixture of single- and double-mutation viruses was analyzed by the PASS platform. The results showed that the PASS assay can accurately determine the presence of linked drug-resistant mutations in individual viral genomes.
  • the polony gel can be analyzed by 22 cycles of primer annealing and SBEs so that the linkage of all major drug-resistance mutations to PIs, NTRIs, and NNRTIs in the 1 .1 kb viral genomic region can be determined (Cai et al., 2007)).
  • the overall goal of the invention is to further improve the PASS platform to facilitate its clinical application for detecting drug-resistant mutations in HIV-infected patients. This can be achieved by multiplexing mutation detection procedure and replacing the polony amplification with bridge PCR, which is more convenient to perform and fully automatable.
  • the resulting platform will allow for sensitive mutation detection in highly cost-effective and automated fashion, which will be suitable for effectively monitoring drug-resistant mutations in patient samples to guide rational therapy design for HIV-infected individuals.
  • PASS platform The detection of point mutations by PASS platform are currently achieved by SBE (single-base extension), and the base identity is determined by the fluorescence-nucleotide incorporated.
  • SBE single-base extension
  • the base identity is determined by the fluorescence-nucleotide incorporated.
  • only one mutation site will be queried in each cycle by adding two fluorescently distinct nucleotides.
  • This can in theory be multiplex to two mutation sites if the two sites are consist of different bases (A/G for site 1 and CfT for site 2) and all four nucleotides, which are labeled with distinct fluorophores, will be included in a single reaction.
  • the later scenarios are expected to be rare, and thus, a typical SBE reaction only detect one mutation at a time.
  • each SBE cycle requires 2-3 hours to be completed, it is certainly time-consuming and labor intensive if a large number of mutation sites are interrogated.
  • Relevant to HIV HAART resistance if 22 core mutations are analyzed the average turn-around time is more than 5 days (4 cycles per day).
  • the goal is to increase the throughput in each detection cycle by using a ligation based approach where the fluorescence labels are incorporated into gene-specific sequences.
  • the throughput can be potentially expanded if more fluorophores (e.g. quantum dots) are available in the near future.
  • plasmid samples will be used instead of real patient samples.
  • the plasmids carrying a single mutation or multiple drug-resistance mutations By mixing the plasmids carrying a single mutation or multiple drug-resistance mutations, one can mimic the quasispecies of patient samples. Because the relative abundance of different plasmids in the final mixtures can be precisely controlled, they can serve as a standard to evaluate the performance of the ligation procedure and help optimize its condition.
  • a 1 .1 kb fragment of the pol gene will be used, which covers most of HIV drug-resistant mutations in protease and reverse transcriptase (Table 3; Cai et al., 2007).
  • the concept behinds the oligoligation assay (OLA)-based strategy is that fluorescent signals will be incorporated into the sequence-specific oligos rather than individual nucleotides and different oligos are labeled with distinct fluorophores. Therefore, the presence of a given fluorophore will suggest the existence of the corresponding mutation at a specific position.
  • the overall strategy will be achieved by multiplex OLA with 2 or more primer pairs. For each primer pair, one primer will be fluorescently labeled and a distinct label is unique to the primer pair.
  • Base discrimination is made possible through high-fidelity thermoligase: two primers are juxtaposed at the mutated base, which corresponds to the 3' end of the upstream primer. If the two bases are perfectly complementary to each other, ligation will occur in the presence of thermoligase. However, ligation will not take place if the mutated base does not match the 3' end of the upstream oligo.
  • ligated products have much higher Tm than individual primers, unligated probes can be selectively removed under elevated temperature and the ligated products will be retained in solid phase.
  • a mutated base can be detected by a fluorescence signal and the identity of the base is reflected by its predefined fluorophore.
  • Up to four different primer pairs can be included, which match the current capacity of fluorophores can be detected by a microarray scanner and/or fluorescence microscope.
  • Tests are performed on a 1 .1 kb HIV pol gene fragment, which contained the sites of all major resistant mutations in reverse transcriptase and protease (see preliminary results).
  • the focus is on one primer pairs to determine the specificity of the OLA-based detection. This allows optimization of the primer design (12-15mer, and currently Tm is set at 45 0 C) and ligation condition.
  • the assay is expanded to include 2-4 primer pairs.
  • DNA templates such as those have a single mutation or multiple mutations, are then mixed at different ratios to determine the reliability for capturing the underlying mutation profiles.
  • Linear regression determines the correlation between the observed and expected results especially for the low abundant clones.
  • the advantage of using DNA templates rather than real clinical samples is that the nature of the mutations and their relative abundance are known, which can serve as a good standard to evaluate the performance of the improved PASS assay.
  • the PASS platform is improved by adapting the bridge amplification strategy (Bing et al., 2007; Gingeras et al., 2001 ). Not only is detection throughput improved by at least 10-fold; the subsequent mutation detection assay can also be automated by coupling with microfluidic chambers and a fluorescence microscope. Therefore, guidance to anti-HIV therapies by providing reliable resistance mutation profiles before and/or during treatments.
  • Bridge amplification is carried out on a pre-made Illumina/Solexa flow cell.
  • the amplification primers are coupled onto the solid surface through 5' covalent linkage.
  • the HIV Pol region (1 .1 kb) is amplified using add-on PCR to include specific sequences at each end of the DNA fragments, which match the immobilized primers on the flow-cell.
  • the resulting DNA fragments, together with amplification buffer, polymerase and dNTPs, is added to Illumina/Solexa flow cell.
  • double-stranded templates is denatured as the temperature is raised to 94 0 C (denaturation step).
  • the template DNA hybridizes to the primer (annealing step).
  • the temperature is raised to 72 0 C and polymerase extension occurs (extension step).
  • the amplification products which are covalently linked to the solid surface, serves as templates and hybridize to additional primers.
  • double-stranded bridge is formed with each strand is attached to the flow cell via immobilized primers. Similar to polony amplification, the amplicons derived from the each single-molecule forms a "cluster", and millions of clusters can be formed with a single Illumina/Solexa channel. Double-stranded amplicons is cleaved with Sodium Periodate, which release one DNA strand from the solid phase. The remaining single-strand DNA molecules is suitable for subsequent primer hybridization and mutation detection,
  • Mutation detection is achieved by multiplex OLA. Similar to conventional PASS platform, drug-resistance mutations are interrogated in sequential detection cycles and the linkage between multiple mutations are therefore determined. A initial test is performed on the partial HIV pol gene, for which mutant plasmids are available to validate the system and evaluate its sensitivity/specificity.
  • the flow-cell is connected with microfluidic controller (pump, valves, and heating block) and an epi-fluorescence microscope with an encoded stage, 2 high-speed filter wheels and EM-CCD camera, which also includes software development for image alignment, mutation calling, and linkage analysis.
  • the performance of the improved system is considerably better than the conventional PASS platform. Detection sensitivity (1 in 10 5 ) is 10-fold and 1000-fold better than conventional PASS and Sanger sequencing based assays, respectively (Egger et al., 1997).
  • Bridge Amplificaiton a solid phase PCAr system for the amplificaiton and detection of allelic differences in single copy genes
  • Phenotypic drug susceptibility testing predicts long-term virologic suppression better than treatment history in patients with human immunodeficiency virus infection. J Infect Dis 183, 401 -408.
  • HIV-1 dynamics in vivo virion clearance rate, infected cell life-span, and viral generation time. Science 271, 1582-1586.

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Abstract

L'invention porte sur un procédé de multiplexage de stratégie de détection de mutation de la plateforme PASS à l'aide d'un dosage de ligature d'oligonucléotides. Pour améliorer le rendement d'une procédure de détection de mutation de la plateforme PASS, un dosage de ligature d'oligonucléotides (OLA) remplace une extension à base unique (SBE), qui permet une interrogation de multiples mutations de résistance à un médicament dans chaque cycle de détection de base. L'invention porte également sur le développement d'une nouvelle génération de plateforme PASS à l'aide d'une amplification par PCR en pont. La procédure entière peut être automatisée de telle sorte qu'une nouvelle génération de plateforme PASS est appropriée pour une pratique clinique de routine.
PCT/US2009/043436 2008-05-12 2009-05-11 Détection d'une maladie liée à des mutations de gène par une amplification de molécule unique avec des dosages de ligature multiplex WO2009140191A2 (fr)

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Cited By (2)

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CN107385030A (zh) * 2017-07-14 2017-11-24 广州精科医学检验所有限公司 分子标签、接头及确定含有低频突变核酸序列的方法
CN110468211A (zh) * 2019-10-14 2019-11-19 湖南大地同年生物科技有限公司 膀胱癌肿瘤突变基因特异性引物、试剂盒和文库构建方法

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BENTLY D.R.: 'Whole-genome re-sequencing' CURR. OPIN. GENET. DEV. vol. 16, no. 6, 18 October 2006, pages 545 - 552 *
CAI ET AL.: 'Detection of minor drug-resistant populations by parallel allele-specific sequencing' NAT. METHODS vol. 4, no. 2, 07 January 2007, pages L23 - 125 *
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107385030A (zh) * 2017-07-14 2017-11-24 广州精科医学检验所有限公司 分子标签、接头及确定含有低频突变核酸序列的方法
WO2019010775A1 (fr) * 2017-07-14 2019-01-17 广州精科医学检验所有限公司 Étiquette moléculaire, jonction et procédé de détermination de séquence nucléotidique contenant une mutation à basse fréquence
CN110468211A (zh) * 2019-10-14 2019-11-19 湖南大地同年生物科技有限公司 膀胱癌肿瘤突变基因特异性引物、试剂盒和文库构建方法
CN110468211B (zh) * 2019-10-14 2020-02-14 湖南大地同年生物科技有限公司 膀胱癌肿瘤突变基因特异性引物、试剂盒和文库构建方法

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