Detection Of Rare Alleles Research Articles

High‐throughput genotyping in estimating genetic resources and detecting pathogens in aquaculture

High‐throughput genotyping in estimating genetic resources and detecting pathogens in aquaculture

Modifications of Kompetitive Allele-Specific PCR (KASP) Genotyping for Detection of Rare Alleles.

KASP is commonly used to genotype bi-allelic SNPs and In/Dels, and the standard protocol works well when both alleles are nearly equally prevalent in the DNA template. To detect rare alleles in bulked samples or to distinguish more than three genotypes, such as tri-allelic loci or mutations across orthologous genes in polyploids, adjustments to the protocol and/or data analysis are required. In this chapter, we present modified protocols for these non-traditional applications, including reaction conditions that enhance the fluorophore signal from rare alleles, resulting in increased KASP assay sensitivity. We also describe alternative KASP data analysis approaches that increase statistical certainty of genotyping calls. Furthermore, this increased assay sensitivity enables high-throughput genotyping using KASP, as samples can be pooled and tested in a single reaction. For example, rare alleles can be detected in mixed seed pools when present in ratios as low as 1 in 200. The assay modifications presented here expand the options available for complex genotyping, and retain KASP's advantages of being cheap, fast, and accurate.

VNTR polymorphism in the breakpoint region of ABL1 and susceptibility to bladder cancer

BackgroundABL1 is primarily known as a leukemia-related oncogene due to translocation, but about 2.2% of ABL1 mutations have been identified in bladder cancer, and high expression in solid cancer has also been detected.MethodsHere, we used the NCBI database, UCSC genome browser gateway and Tandem repeat finder program to investigate the structural characterization of the ABL1 breakpoint region and to identify the variable number of tandem repeats (VNTR). To investigate the relationship between ABL1-MS1 and bladder cancer, a case-controlled study was conducted in 207 controls and 197 bladder cancer patients. We also examined the level of transcription of the reporter gene driven by the ABL1 promoter to determine if the VNTR region affects gene expression.ResultsIn our study, one VNTR was identified in the breakpoint region, the intron 1 region of ABL1, and was named ABL1-MS1. In the control group, only two common alleles (TR13, TR15) were detected, but an additional two rare alleles (TR14, TR16) were detected in bladder cancer. A statistically significant association was identified between the rare ABL1-MS1 allele and bladder cancer risk: P = 0.013. Investigating the level of transcription of the reporter gene driven by the ABL1 promoter, VNTR showed inhibition of ABL1 expression in non-cancer cells 293 T, but not in bladder cancer cells. In addition, ABL1-MS1 was accurately passed on to offspring according to Mendelian inheritance through meiosis.ConclusionsTherefore, the ABL1-MS1 region can affect ABL1 expression of bladder cancer. This study provides that ABL1-MS1 can be used as a DNA fingerprinting marker. In addition, rare allele detection can predict susceptibility to bladder cancer.

A needle in a seedstack: an improved method for detection of rare alleles in bulk seed testing through KASP.

Amaranthus palmeri is an aggressive and prolific weed species with major impact on agricultural yield and is a prohibited noxious weed across the Midwest. Morphological identification of A. palmeri from other Amaranthus species is extremely difficult in seeds, which has led to genetic testing for seed identification in commercial seed lots. We created an inexpensive and reliable genetic test based on novel, species-specific, single nucleotide polymorphisms (SNPs) from GBS (Genotyping by Sequencing) data. We report three SNP-based genetic tests for identifying A. palmeri alone or in a mixed pool of Amaranthus spp. Sensitivity ranged from 99.8 to 100%, specificity from 99.59 to 100%. Accuracy for all three tests is > 99.7%. All three are capable of reliably detecting one A. palmeri seed in a pool of 200 Amaranthus spp. seeds. The test was validated across 20 populations of A. palmeri, along with eight other Amaranthus species, the largest and most genetically diverse panel of Amaranthus samples to date. Our work represents a marked improvement over existing commercial assays resulting in an identification assay that is (i) accurate, (ii) robust, (iii) easy to interpret and (iv) applicable to both leaf tissue and pools of up to 200 seeds. Included is a data transformation method for calling of closely grouped competitive fluorescence assays. We also present a comprehensive GBS dataset from the largest geographic panel of Amaranthus populations sequenced. Our approach serves as a model for developing markers for other difficult to identify species. © 2021 Society of Chemical Industry.

Optimization of Chelex 100 resin-based extraction of genomic DNA from dried blood spots.

Dried blood spots (DBS) are widely utilized as part of universal newborn screening and as a means of transporting samples from field sites. We use DBS from African field sites to assess for rare maternal-fetal cell exchange during pregnancy known as microchimerism. We aimed to develop a protocol to maximize the quantity of high-quality genomic DNA (gDNA) extracted from DBS. The total gDNA yield obtained from control DBS utilizing a Qiagen-based protocol and a Chelex® 100 resin-based protocol was first compared. Variations of the Chelex® protocol were subsequently tested to develop an optimized protocol. The gDNA was quantified by qPCR targeting the human beta-globin gene. DNA yield for a given experimental condition was normalized to a Chelex® control performed on the same day, and the total yields were compared using a Student’s t-test. The control Chelex® protocol yielded 590% more DNA than the QIAamp® DNA Blood Mini Kit . The absolute efficiency of the control Chelex® protocol was 54%, compared to an absolute efficiency of 9% for the QIAamp® DNA Blood Mini Kit. Modification of the Chelex® protocol to include a second heat precipitation from the same DBS increased the gDNA yield by 29% (P < 0.001). Our optimized protocol including this modification increased the absolute efficiency of extraction to 68%. The gDNA extracted using the Chelex® protocol was stable through repeated freeze–thaw cycles. In a mock microchimerism experiment, rare donor alleles at a frequency of 10 in 100 000 could be identified in gDNA from DBS extracted using the optimized Chelex® protocol. Our findings may be of significance for a diverse range of applications that utilize DBS and require high-quality DNA, including newborn screening programs, pathogen and drug resistance screening from remote field sites, forensics, and rare allele detection.

Abstract 5119: Customizable all-in-one cancer panels to detect copy number variation, DNA rearrangement and mutations by targeted NGS

Abstract Background: The current standard of genomic profiling of cancer tissues relies upon multiple separate technologies to aid in tumor characterization. Here we describe a customizable NGS design and analysis workflow for cancer samples that can detect single nucleotide polymorphisms (SNPs), insertions and deletions (INDEL), somatic copy number alterations (SCNAs), and translocations (TLs) in a single assay. Methods: We have created a library of strategically placed target-enrichment probes to provide for specific detection of SNP/INDEL, SCNAs, and translocation in genes identified as important in several cancer subtypes. The probes are optimized with the SureSelect XTHS protocol, which enables extremely sensitive detection of rare exonic mutation events within a heterogeneous sample. SCNA detection is further enhanced with additional probes targeting high minor allele frequency SNPs within the target gene boundary. Translocation detection is enabled by targeting intronic regions of fusion gene candidates. The data analysis is performed by the software SureCall that calls SNPs across all regions covered by probes. SCNA detection utilizes either a matched normal or standard control DNA and aberrant regions are identified by jointly segmenting the read depth ratios and SNP allele frequencies. The clonal structure is discerned using two Bayesian cancer models in a hierarchical manner. Translocation are identified by analyzing split reads across putative fusion breakpoints that are supported by both high- and low-confidence reads that pass quality filters. Results: We demonstrate rare allele detection in cancer cells down to 1%. We also show detection of 3 copies of MET at 20% tumor fraction and 16 copies of ERBB2 at 2% tumor fraction. For translocations, DNA level gene fusions were detected for ALK and ROS fusion genes at &amp;lt;=3% tumor fraction. In 39 ERBB2 FISH-positive FFPE samples, ERBB2 CNV detected by AIO panel was 97.4% concordant with FISH (scored according to standard ASCO/CAP convention). Using FISH as the gold standard, the false negative rate was 2.6% and false positive rate was 2.4%. In 43 ALK FISH-positive FFPE samples, ALK fusion was detected using a small AIO lung panel which includes probes spanning introns 18/19 of the ALK gene and is 90.7% concordant with FISH (Agilent SureFISH ALK BA). Using FISH as the gold standard, the false negative rate was 9.3% and false positive rate was 2.3%. Conclusions: This work represents an important advancement in the development of a single assay to detect copy number variation, DNA rearrangement and mutations. Citation Format: Arjun Vadapalli, Ashutosh Ashutosh, Heather Tao, Linus Forsmark, Christian Le Cocq, Akansha Khare, Bahram Arezi, Gilbert Amparo, Michael Ruvolo, Carlos Pabon, Jayati Ghosh, Jimmy Jin, Tracy Liu, Douglas Roberts. Customizable all-in-one cancer panels to detect copy number variation, DNA rearrangement and mutations by targeted NGS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5119.

Development and validation of multiplex-PCR assay for simultaneous detection of rare alleles of crtRB1 and lcyE governing higher accumulation of provitamin A in maize

Vitamin A deficiency is a widely prevalent health disorder among millions of people worldwide. Introgression of crtRB1 and lcyE favourable alleles that enhance concentration of provitamin A in maize endosperm have been employed in maize biofortification programmes. To make marker-assisted selection (MAS) more effective, we have developed rapid and convenient multiplex-polymerase chain reaction (PCR) assay to simultaneously discover the allelic combinations among the segregants. Validation of the multiplex assay was done in two backcross-derived populations developed using elite inbreds viz., HKI193-1 and HKI193-2 carrying unfavourable alleles of crtRB1 (296 bp) and lcyE (300 bp) and HarvestPlus inbreds viz., HP704-22 and HP704-23 possessing favourable alleles of crtRB1 (543 bp) and lcyE (650 bp). We also standardized the uniplex-PCR assays for both the genes that gave robust and reproducible results in sub-tropical populations. Gel profiles of BC1F1, BC2F1 and BC2F2 revealed that these assays identified the backcross progenies homo-or hetero-zygous for the favourable- or unfavourable-alleles. Multiplex-PCR assay also precisely confirmed the results of individual uniplex assays in different backcross generations. Cost and time analyses showed that multiplex-PCR assay has potential to save 41% of cost, and 50% of time compared to two uniplex assays in a MAS programme. It has also saved 50% of the manpower. The multiplex assay possesses significant advantage over uniplex assays and enhances the efficiency of selection. This is the first report of development and validation of multiplex-PCR assay of crtRB1 and lcyE for utilization in maize biofortification programme.

Targeted error-suppressed quantification of circulating tumor DNA using semi-degenerate barcoded adapters and biotinylated baits

Ultrasensitive methods for rare allele detection are critical to leverage the full potential offered by liquid biopsies. Here, we describe a novel molecular barcoding method for the precise detection and quantification of circulating tumor DNA (ctDNA). The major benefits of our design include straightforward and cost-effective production of barcoded adapters to tag individual DNA molecules before PCR and sequencing, and better control over cross-contamination between experiments. We validated our approach in a cohort of 24 patients with a broad spectrum of cancer diagnoses by targeting and quantifying single-nucleotide variants (SNVs), indels and genomic rearrangements in plasma samples. By using personalized panels targeting a priori known mutations, we demonstrate comprehensive error-suppression capabilities for SNVs and detection thresholds for ctDNA below 0.1%. We also show that our semi-degenerate barcoded adapters hold promise for noninvasive genotyping in the absence of tumor biopsies and monitoring of minimal residual disease in longitudinal plasma samples. The benefits demonstrated here include broad applicability, flexibility, affordability and reproducibility in the research and clinical settings.

Rare allele enrichment and detection by allele-specific PCR, competitive probe blocking and melting analysis

Protocol Summary We have combined allele-specific PCR, competitive probe blocking, asymmetric PCR, and melting analysis to enhance rare allele detection in a homogeneous system. Unlabeled, dual hybridization or molecular beacon probes were used for competitive blocking of the wild-type allele at a concentration 10 times that of the allele-specific primer. In each case, rare alleles were detected by probe melting analysis at a sensitivity of &gt;0.001% (1 variant copy within 100,000 wild-type copies), providing single copy detection in typical PCRs. Ninety-one thyroid biopsies were tested for the BRAF mutation p.V600E (c.1799 T &gt; A) by both dual hybridization probes without enrichment and an allele-specific, competitive blocking melting analysis with unlabeled probes. Eighty-seven samples were concordant between methods (43 positive, 44 negative), while 4 samples that were negative by direct analysis became positive after enrichment. Probes that both block wild-type amplification and detect rare variants by melting analysis improve the detection sensitivity of allele-specific PCR for rare alleles. In particular, melting analysis using unlabeled probes and amplification by rapid-cycle PCR provides cost-effective and fast enrichment and detection of rare alleles.

High-specificity detection of rare alleles with Paired-End Low Error Sequencing (PELE-Seq).

BackgroundPolymorphic loci exist throughout the genomes of a population and provide the raw genetic material needed for a species to adapt to changes in the environment. The minor allele frequencies of rare Single Nucleotide Polymorphisms (SNPs) within a population have been difficult to track with Next-Generation Sequencing (NGS), due to the high error rate of standard methods such as Illumina sequencing.ResultsWe have developed a wet-lab protocol and variant-calling method that identifies both sequencing and PCR errors, called Paired-End Low Error Sequencing (PELE-Seq). To test the specificity and sensitivity of the PELE-Seq method, we sequenced control E. coli DNA libraries containing known rare alleles present at frequencies ranging from 0.2–0.4 % of the total reads. PELE-Seq had higher specificity and sensitivity than standard libraries. We then used PELE-Seq to characterize rare alleles in a Caenorhabditis remanei nematode worm population before and after laboratory adaptation, and found that minor and rare alleles can undergo large changes in frequency during lab-adaptation.ConclusionWe have developed a method of rare allele detection that mitigates both sequencing and PCR errors, called PELE-Seq. PELE-Seq was evaluated using control E. coli populations and was then used to compare a wild C. remanei population to a lab-adapted population. The PELE-Seq method is ideal for investigating the dynamics of rare alleles in a broad range of reduced-representation sequencing methods, including targeted amplicon sequencing, RAD-Seq, ddRAD, and GBS. PELE-Seq is also well-suited for whole genome sequencing of mitochondria and viruses, and for high-throughput rare mutation screens.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2669-3) contains supplementary material, which is available to authorized users.

Droplet digital PCR, a new approach to analyze fetal DNA from maternal blood: application to the determination of fetal RHD genotype.

The discovery of free fetal DNA in the maternal circulation has inaugurated the era of non-invasive prenatal diagnosis. The latter has the advantage of avoiding the use of conventional obstetric procedures, such as chorionic villus sampling or aspiration of amniotic fluid, thus limiting the risks of miscarriage they induce. However, as free fetal DNA accounts for about 10% of cell-free DNA in maternal plasma, the presence of ambient maternal DNA can make it difficult to detect fetal alleles of paternal origin. Digital Droplet PCR (ddPCR) is a very sensitive method derived from quantitative real-time PCR (qPCR) for the detection of rare alleles and their absolute quantification by removing the necessity of standards. Here we show that this new technology can be applied in routine prenatal fetal RHD genotyping from maternal blood. In conclusion, the use of quantitative properties of digital PCR, in terms of accuracy, sensitivity and specificity, allows one to consider extending the applications of this new technology in non-invasive prenatal diagnosis of many diseases such as autosomal monogenic diseases, either dominant or recessive.

Abstract P3-06-42: Detection of PIK3CA mutations in cell-free plasma DNA from women with early breast cancer

Abstract Background Activating mutations in the PIK3CA gene represent the most common molecular aberration in luminal breast cancers. Three hot-spot mutations (E542K, E545K, H1047R) account for approximately 70% of the PIK3CA mutations identified. The presence of a mutation is likely to become an important predictive biomarker to select patients for treatment with PIK3CA inhibitors. Tumor-derived DNA can be detected in the systemic circulation in the form of cell-free plasma DNA (cfpDNA), and the ability to interrogate tumor-derived DNA from an easily acquired biospecimen like cfpDNA has practical advantages over analysis of tissue specimens. Furthermore, cfpDNA can be monitored serially and could provide a dynamic measure of tumor progression and response to therapy. Droplet digital PCR (ddPCR) is a high-throughput technique that provides highly precise and sensitive detection of rare alleles in samples with ultra-low abundance of target DNA. Methods A nested case-control study was conducted with plasma samples collected as part of a study to identify blood-based biomarkers. Blood was collected from women scheduled for a breast biopsy at one of seven participating mammography units. All blood samples were collected prior to the biopsy procedure and were processed using a standard protocol. Cases are women diagnosed with invasive ductal carcinoma, stage I-III, that is positive for the estrogen receptor (ER+) and negative for HER2/neu (0 or 1+ by IHC). Controls are women with a benign, non-proliferative lesion, matched by collection site, year of collection and age. cfpDNA was isolated using a commercial kit (Macherey-Nagel, Bethlehem, PA). DNA was extracted from FFPE biopsy specimens by standard techniques. Central pathologic review confirmed at least 10% tumor cellularity in biopsy specimens from cases. A ddPCR assay was developed on a BioRad QX100 Droplet Digital PCR instrument using standard TaqManR assays with probe pairs designed to detect the 3 PIK3CA hotspot mutations and corresponding wild type (wt) alleles. The assay was validated using DNA from four cell lines, each positive for one of the hotspot mutations or wt at all 3 loci (Horizon Diagnostics, Cambridge, UK). ddPCR was performed on cfpDNA and DNA extracted from FFPE biopsy specimens in cases and controls. Next generation DNA sequencing (NGS) of exons 9 and 20 of PIK3CA was performed on biopsy specimens from cases using an Ion Torrent Personal Genome Sequencer. Results Validation of the ddPCR assay with serial dilutions of cell line DNA demonstrated the assay is highly sensitive with mutant alleles detected at an allele frequency down to 25% when input DNA template is as low as 2 ng. With DNA template input of 20 ng (typical recovery of cfpDNA), mutations can be detected down to an allele frequency of 0.78%. We will present data on the rate of hotspot mutation detection with the ddPCR assay performed on cfpDNA and FFPE samples in cases (n=80) and controls (n=80), and we will report performance characteristics of the assay. Mutation status of tumors determined by NGS will serve as the gold standard. Conclusion We have developed a ddPCR assay to detect PIK3CA hotspot mutations in cfpDNA, and will report the performance characteristics of the assay in 80 cases of untreated, ER+ early breast cancer. Citation Format: Kent F Hoskins, Amer Sidani, Tiffany Jones, Stefan Green, Rajyasree Emmadi, Anjen Chenn. Detection of PIK3CA mutations in cell-free plasma DNA from women with early breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-06-42.

Enhanced Ratio of Signals Enables Digital Mutation Scanning for Rare Allele Detection

The use of droplet digital PCR (ddPCR) for low-level DNA mutation detection in cancer, prenatal diagnosis, and infectious diseases is growing rapidly. However, although ddPCR has been implemented successfully for detection of rare mutations at pre-determined positions, no ddPCR adaptation for mutation scanning exists. Yet, frequently, clinically relevant mutations reside on multiple sequence positions in tumor suppressor genes or complex hotspot mutations in oncogenes. Here, we describe a combination of coamplification at lower denaturation temperature PCR (COLD-PCR) with ddPCR that enables digital mutation scanning within approximately 50-bp sections of a target amplicon. Two FAM/HEX-labeled hydrolysis probes matching the wild-type sequence are used during ddPCR. The ratio of FAM/HEX-positive droplets is constant when wild-type amplicons are amplified but deviates when mutations anywhere under the FAM or HEX probes are present. To enhance the change in FAM/HEX ratio, we employed COLD-PCR cycling conditions that enrich mutation-containing amplicons anywhere on the sequence. We validated COLD-ddPCR on multiple mutations in TP53 and in EGFR using serial mutation dilutions and cell-free circulating DNA samples, and demonstrate detection down to approximately 0.2% to 1.2% mutation abundance. COLD-ddPCR enables a simple, rapid, and robust two-fluorophore detection method for the identification of multiple mutations during ddPCR and potentially can identify unknown DNA variants present in the target sequence.

Limiting Dilution Bisulfite Pyrosequencing®: A Method for Methylation Analysis of Individual DNA Molecules in a Single or a Few Cells.

Bisulfite-based methods for DNA methylation analysis of small amounts of DNA from a limited number of cells are technologically challenging. Degradation of genomic DNA by bisulfite treatment, contamination with foreign DNA, and biases in the amplification of individual DNA molecules can generate results, which are not representative of the starting sample. Limiting dilution (LD) bisulfite Pyrosequencing(®) (BSP) is a relatively simple technique to circumvent these problems. The bisulfite-treated DNA of a single or a few cells is diluted to an extent, that only a single DNA target molecule is present in the reaction. Then each individual DNA molecule in the starting sample is separately amplified and analyzed by Pyrosequencing. This allows the detection of rare alleles that are easily masked when pools of DNA target molecules are analyzed. Amplicons containing a heterozygous single nucleotide polymorphism (SNP) allow one to delineate the parental origin of the recovered molecules in addition to their methylation status. The number of cells (DNA target molecules) in the starting sample determines the dilution level and the number of reactions that have to be performed. LD-BSP allows methylation analysis of small cell pools (i.e., 5-10 microdissected cells) and even individual cells. The primers and PCR conditions described here have been successfully employed to analyze the methylation status of up to eight target genes in individual 2-16 cell embryos, germinal vesicle (GV) oocytes, and haploid sperms.

ZCall: a rare variant caller for array-based genotyping

zCall is a variant caller specifically designed for calling rare single-nucleotide polymorphisms from array-based technology. This caller is implemented as a post-processing step after a default calling algorithm has been applied. The algorithm uses the intensity profile of the common allele homozygote cluster to define the location of the other two genotype clusters. We demonstrate improved detection of rare alleles when applying zCall to samples that have both Illumina Infinium HumanExome BeadChip and exome sequencing data available. http://atguweb.mgh.harvard.edu/apps/zcall. bneale@broadinstitute.org Supplementary data are available at Bioinformatics online.

DNA Pooling and Statistical Tests for the Detection of Single Nucleotide Polymorphisms

The development of next generation genome sequencers gives the opportunity of learning more about the genetic make-up of human and other populations. One important question involves the location of sites at which variation occurs within a population. Our focus will be on the detection of rare variants. Such variants will often not be present in smaller samples and are hard to distinguish from sequencing errors in larger samples. This is particularly true for pooled samples which are often used as part of a cost saving strategy. The focus of this article is on experiments that involve DNA pooling. We derive experimental designs that optimize the power of statistical tests for detecting single nucleotide polymorphisms (SNPs, sites at which there is variation within a population). We also present a new simple test that calls a SNP, if the maximum number of reads of a prospective variant across lanes exceeds a certain threshold. The value of this threshold is defined according to the number of available lanes, the parameters of the genome sequencer and a specified probability of accepting that there is variation at a site when no variation is present. On the basis of this test, we derive pool sizes which are optimal for the detection of rare variants. This test is compared with a likelihood ratio test, which takes into account the number of reads of a prospective variant from all the lanes. It is shown that the threshold based rule achieves a comparable power to this likelihood ratio test and may well be a useful tool in determining near optimal pool sizes for the detection of rare alleles in practical applications.

RNase H-dependent PCR (rhPCR): improved specificity and single nucleotide polymorphism detection using blocked cleavable primers

BackgroundThe polymerase chain reaction (PCR) is commonly used to detect the presence of nucleic acid sequences both in research and diagnostic settings. While high specificity is often achieved, biological requirements sometimes necessitate that primers are placed in suboptimal locations which lead to problems with the formation of primer dimers and/or misamplification of homologous sequences.ResultsPyrococcus abyssi (P.a.) RNase H2 was used to enable PCR to be performed using blocked primers containing a single ribonucleotide residue which are activated via cleavage by the enzyme (rhPCR). Cleavage occurs 5'-to the RNA base following primer hybridization to the target DNA. The requirement of the primer to first hybridize with the target sequence to gain activity eliminates the formation of primer-dimers and greatly reduces misamplification of closely related sequences. Mismatches near the scissile linkage decrease the efficiency of cleavage by RNase H2, further increasing the specificity of the assay. When applied to the detection of single nucleotide polymorphisms (SNPs), rhPCR was found to be far more sensitive than standard allele-specific PCR. In general, the best discrimination occurs when the mismatch is placed at the RNA:DNA base pair.ConclusionrhPCR eliminates the formation of primer dimers and markedly improves the specificity of PCR with respect to off-target amplification. These advantages of the assay should find utility in challenging qPCR applications such as genotyping, high level multiplex assays and rare allele detection.

Rare allele enrichment and detection by allele-specific PCR, competitive probe blocking, and melting analysis

Differential amplification of variant and wild-type alleles by PCR is often used for rare allele enrichment. We have combined allele-specific PCR, competitive probe blocking, asymmetric PCR, and melting analysis to enhance rare allele detection in a homogeneous system. Unlabeled, dual hybridization or molecular beacon probes were used for competitive blocking of the wild-type allele at a concentration 10 times that of the allele-specific primer. In each case, rare alleles were detected by probe melting analysis at a sensitivity of &gt;0.001% (1 variant copy within 100,000 wild-type copies), providing single copy detection in typical PCRs. Ninety-one thyroid biopsies were tested for the BRAF mutation p.V600E (c.1799 T &gt; A) by both dual hybridization probes without enrichment and an allele-specific, competitive blocking melting analysis with unlabeled probes. Eighty-seven samples were concordant between methods (43 positive, 44 negative), while 4 samples that were negative by direct analysis became positive after enrichment. Probes that both block wild-type amplification and detect rare variants by melting analysis improve the detection sensitivity of allele-specific PCR for rare alleles. In particular, melting analysis using unlabeled probes and amplification by rapid-cycle PCR provides cost-effective and fast enrichment and detection of rare alleles.

Enrichment and Detection of Rare Alleles by Means of Snapback Primers and Rapid-Cycle PCR

Selective amplification of minority alleles is often necessary to detect cancer mutations in clinical samples. Minor-allele enrichment and detection were performed with snapback primers in the presence of a saturating DNA dye within a closed tube. A 5' tail of nucleotides on 1 PCR primer hybridizes to the variable locus of its extension product to produce a hairpin that selectively enriches mismatched alleles. Genotyping performed after rapid-cycle PCR by melting of the secondary structure identifies different variants by the hairpin melting temperature (T(m)). Needle aspirates of thyroid tissue (n = 47) and paraffin-embedded biopsy samples (n = 44) were analyzed for BRAF (v-raf murine sarcoma viral oncogene homolog B1) variant p.V600E, and the results were compared with those for dual hybridization probe analysis. Needle aspirates of lung tumors (n = 8) were analyzed for EGFR [epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)] exon 19 in-frame deletions. Use of 18-s cycles and momentary extension times of "0 s" with rapid-cycle PCR increased the selective amplification of mismatched alleles. A low Mg(2+) concentration and a higher hairpin T(m) relative to the extension temperature also improved the detection limit of mismatched alleles. The detection limit was 0.1% for BRAF p.V600E and 0.02% for EGFR exon 19 in-frame deletions. Snapback and dual hybridization probe methods for allele quantification of the thyroid samples correlated well (R(2) = 0.93) with 2 more BRAF mutations (45 and 43, respectively, of 91 samples) detected after snapback enrichment. Different EGFR in-frame deletions in the lung samples produced different hairpin T(m)s. Use of snapback primers for enrichment and detection of minority alleles is simple, is inexpensive to perform, and can be completed in a closed tube in <25 min.

An optimized procedure for the design and evaluation of Ecotilling assays

BackgroundSingle nucleotide polymorphisms (SNPs) are the most common form of genetic variability in the human genome and play a prominent role in the heritability of phenotypes. Especially rare alleles with frequencies less than 5% may exhibit a particularly strong influence on the development of complex diseases. The detection of rare alleles by standard DNA sequencing is time-consuming and cost-intensive. Here we discuss an alternative approach for a high throughput detection of rare mutations in large population samples using Ecotilling embedded in a collection of bioinformatic analysis tools. Ecotilling originally was introduced as TILLING for the screening for rare chemically induced mutations in plants and later adopted for human samples, showing an outstanding suitability for the detection of rare alleles in humans. An actual problem in the use of Ecotilling for large mutation screening projects in humans without bioinformatic support is represented by the lack of solutions to quickly yet comprehensively evaluate each newly found variation and place it into the correct genomic context.ResultsWe present an optimized strategy for the design, evaluation and interpretation of Ecotilling results by integrating several mostly freely available bioinformatic tools. A major focus of our investigations was the evaluation and meaningful economical combination of these software tools for the inference of different possible regulatory functions for each newly detected mutation.ConclusionOur streamlined procedure significantly facilitates the experimental design and evaluation of Ecotilling assays and strongly improves the decision process on prioritizing the newly found SNPs for further downstream analysis.