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Allele-Specific Quantitative PCR for Accurate, Rapid, and Cost-Effective Genotyping.

ABSTRACT: Customizable endonucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) enable rapid generation of mutant strains at genomic loci of interest in animal models and cell lines. With the accelerated pace of generating mutant alleles, genotyping has become a rate-limiting step to understanding the effects of genetic perturbation. Unless mutated alleles result in distinct morphological phenotypes, mutant strains need to be genotyped using standard methods in molecular biology. Classic restriction fragment length polymorphism (RFLP) or sequencing is labor-intensive and expensive. Although simpler than RFLP, current versions of allele-specific PCR may still require post-polymerase chain reaction (PCR) handling such as sequencing, or they are more expensive if allele-specific fluorescent probes are used. Commercial genotyping solutions can take weeks from assay design to result, and are often more expensive than assembling reactions in-house. Key components of commercial assay systems are often proprietary, which limits further customization. Therefore, we developed a one-step open-source genotyping method based on quantitative PCR. The allele-specific qPCR (ASQ) does not require post-PCR processing and can genotype germline mutants through either threshold cycle (Ct) or end-point fluorescence reading. ASQ utilizes allele-specific primers, a locus-specific reverse primer, universal fluorescent probes and quenchers, and hot start DNA polymerase. Individual laboratories can further optimize this open-source system as we completely disclose the sequences, reagents, and thermal cycling protocol. We have tested the ASQ protocol to genotype alleles in five different genes. ASQ showed a 98-100% concordance in genotype scoring with RFLP or Sanger sequencing outcomes. ASQ is time-saving because a single qPCR without post-PCR handling suffices to score genotypes. ASQ is cost-effective because universal fluorescent probes negate the necessity of designing expensive probes for each locus.

SUBMITTER: Lee HB

PROVIDER: S-EPMC4931339 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Allele-Specific Quantitative PCR for Accurate, Rapid, and Cost-Effective Genotyping.

Lee Han B HB Schwab Tanya L TL Koleilat Alaa A Ata Hirotaka H Daby Camden L CL Cervera Roberto Lopez RL McNulty Melissa S MS Bostwick Hannah S HS Clark Karl J KJ

Human gene therapy 20160317 6

Customizable endonucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) enable rapid generation of mutant strains at genomic loci of interest in animal models and cell lines. With the accelerated pace of generating mutant alleles, genotyping has become a rate-limiting step to understanding the effects of genetic perturbation. Unless mutated alleles result in distinct morpho ...[more]

PMID: 26986823

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Project description:BackgroundOwing to successful cloning of wheat functional genes in recent years, more traits can be selected by diagnostic markers, and consequently, effective molecular markers will be powerful tools in wheat breeding programs.ResultsThe present study proposed a cost-effective duplex Kompetitive Allele Specific PCR (dKASP) marker system that combined multiplex PCR and KASP™ technology to yield twice the efficiency at half the cost compared with the common KASP™ markers and provide great assistance in breeding selection. Three dKASP markers for the major genes controlling plant height (Rht-B1/Rht-D1), grain hardness (Pina-D1/Pinb-D1), and high-molecular-weight glutenin subunits (Glu-A1/Glu-D1) were successfully developed and applied in approved wheat varieties growing in the middle and lower reaches of the Yangtze River and advanced lines from our breeding program. Three markers were used to test six loci with high efficiency. In the approved wheat varieties, Rht-B1b was the most important dwarfing allele, and the number of accessions carrying Pinb-D1b was much greater than that of the accessions carrying Pina-D1b. Moreover, the number of accessions carrying favorable alleles for weak-gluten wheat (Null/Dx2) was much greater than that of the accessions carrying favorable alleles for strong-gluten wheat (Ax1 or Ax2*/Dx5). In the advanced lines, Rht-B1b and Pinb-D1b showed a significant increase compared with the approved varieties, and the strong-gluten (Ax1 or Ax2*/Dx5) and weak-gluten (Null/Dx2) types also increased.ConclusionA cost-effective dKASP marker system that combined multiplex PCR and KASP™ technology was proposed to achieve double the efficiency at half the cost compared with the common KASP™ markers. Three dKASP markers for the major genes controlling PH (Rht-B1/Rht-D1), GH (Pina-D1/Pinb-D1), and HMW-GS (Glu-A1/Glu-D1) were successfully developed, which would greatly improve the efficiency of marker-assisted selection of wheat.

Project description:BACKGROUND: The JAK2 V617F mutation is the most frequent somatic change in myeloproliferative neoplasms, making it an important tumour-specific marker for diagnostic purposes and for the detection of minimal residual disease. Sensitive quantitative assays are required for both applications, particularly for the monitoring of minimal residual disease, which requires not only high sensitivity but also very high specificity. METHODS: We developed a highly sensitive probe-free quantitative mutant-allele detection method, Quantitative Threefold Allele-Specific PCR (QuanTAS-PCR), that is performed in a closed-tube system, thus eliminating the manipulation of PCR products. QuantTAS-PCR uses a threefold approach to ensure allele-specific amplification of the mutant sequence: (i) a mutant allele-specific primer, (ii) a 3'dideoxy blocker to suppress false-positive amplification from the wild-type template and (iii) a PCR specificity enhancer, also to suppress false-positive amplification from the wild-type template. Mutant alleles were quantified relative to exon 9 of JAK2. RESULTS: We showed that the addition of the 3'dideoxy blocker suppressed but did not eliminate false-positive amplification from the wild-type template. However, the addition of the PCR specificity enhancer near eliminated false-positive amplification from the wild-type allele. Further discrimination between true and false positives was enabled by using the quantification cycle (Cq) value of a single mutant template as a cut-off point, thus enabling robust distinction between true and false positives. As 10,000 JAK2 templates were used per replicate, the assay had a sensitivity of 1/10(-4) per replicate. Greater sensitivity could be reached by increasing the number of replicates analysed. Variation in replicates when low mutant-allele templates were present necessitated the use of a statistics-based approach to estimate the load of mutant JAK2 copies. QuanTAS-PCR showed comparable quantitative results when validated against a commercial assay. CONCLUSIONS: QuanTAS-PCR is a simple, cost-efficient, closed-tube method for JAK2 V617F mutation quantification that can detect very low levels of the mutant allele, thus enabling analysis of minimal residual disease. The approach can be extended to the detection of other recurrent single nucleotide somatic changes in cancer.

Project description:BackgroundInterspecific hybridisation is a powerful tool for increasing genetic diversity in plant breeding programmes. Hexaploid wheat (Triticum aestivum, 2n = 42) × barley (Hordeum vulgare, 2n = 14) intergeneric hybrids can contribute to the transfer of agronomically useful traits by creating chromosome addition or translocation lines as well as full hybrids. Information on the karyotype of hybrid progenies possessing various combinations of wheat and barley chromosomes is thus essential for the subsequent breeding steps. Since the standard technique of chromosome in situ hybridisation is labour-intensive and requires specific skills. a routine, cost-efficient, and technically less demanding approach is beneficial both for research and breeding.ResultsWe developed a Multiplex Polymerase Chain Reaction (MPCR) method to identify individual wheat and barley chromosomes. Chromosome-specific primer pairs were designed based on the whole genome sequences of 'Chinese Spring' wheat and 'Golden Promise' barley as reference cultivars. A pool of potential primers was generated by applying a 20-nucleotide sliding window with consecutive one-nucleotide shifts on the reference genomes. After filtering for optimal primer properties and defined amplicon sizes to produce an ordered ladder-like pattern, the primer pool was manually curated and sorted into four MPCR primer sets for the wheat A, B, and D sub-genomes, and for the barley genome. The designed MPCR primer sets showed high chromosome specificity in silico for the genome sequences of all 18 wheat and barley cultivars tested. The MPCR primers proved experimentally also chromosome-specific for the reference cultivars as well as for 13 additional wheat and four barley genotypes. Analyses of 16 wheat × barley F1 hybrid plants demonstrated that the MPCR primer sets enable the fast and one-step detection of all wheat and barley chromosomes. Finally, the established genotyping system was fully corroborated with the standard genomic in situ hybridisation (GISH) technique.ConclusionsWheat and barley chromosome-specific MPCR offers a fast, labour-friendly, and versatile alternative to molecular cytogenetic detection of individual chromosomes. This method is also suitable for the high-throughput analysis of distinct (sub)genomes, and, in contrast to GISH, can be performed with any tissue type. The designed primer sets proved to be highly chromosome-specific over a wide range of wheat and barley genotypes as well as in wheat × barley hybrids. The described primer design strategy can be extended to many species with precise genome sequence information.

Dataset Information

Allele-Specific Quantitative PCR for Accurate, Rapid, and Cost-Effective Genotyping.

Publications

Allele-Specific Quantitative PCR for Accurate, Rapid, and Cost-Effective Genotyping.

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