Genotyping by apyrase-mediated allele-specific extension

Ahmadian, Afshin; Gharizadeh, Baback; O’Meara, Deirdre; Odeberg, Jacob; Lundeberg, Joakim

doi:10.1093/nar/29.24.e121

Cited by 41 publications

(23 citation statements)

References 16 publications

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“…Among these are: switching strands for analysis; the incorporation of additional mismatches in the allele specific primer near the 3 0 end (ARMS) [Newton et al, 1989]; titration of critical PCR reaction components to near-limiting levels ; the use of truncated Taq DNA polymerase (Stoffel fragment) lacking 5 0 to 3 0 exonuclease activity [Lawyer et al, 1993]; enrichment of target template by PCR prior to adding allele-specific primer [Kaltenbock and Schneider, 1998]; the use of nucleotide analogs to facilitate base conversion at 3 0 mismatched sites [Day et al, 1999]; and the use of apyrase to degrade dNTPs when reaction kinetics are slow at mismatched 3 0 ends (AMASE) [Ahmadian et al, 2001]. Recently, SNP genotyping using bidirectional, real-time allele-specific PCR with SYBR s Green fluorescent melt curve analysis of amplified products was described [Waterfall and Cobb, 2002].…”

Section: Introductionmentioning

confidence: 99%

Enhanced allele-specific PCR discrimination in SNP genotyping using 3? locked nucleic acid (LNA) primers

et al. 2003

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The specificity and reliability of locked nucleic acid (LNA) substitution at the 3' position of allele-specific PCR (AS-PCR) primers for SNP detection was investigated in direct comparison to DNA primers. Both plasmid and human genomic DNA templates were examined in this study. All possible DNA and 3' LNA mismatch combinations were tested in triplicate with the plasmid target. LNA primers yield consistently low amounts of mismatch products with all base combinations, whereas certain mismatches with DNA primers generate strong false positive amplicons. Amplified human SNP alleles within the cystic fibrosis (CFTR) gene were analyzed in AS-PCR by gel analysis and real-time fluorescence generation. A 3' LNA residue in the primer at the SNP site improves allelic discrimination and functions under a wide window of PCR conditions. We demonstrate increased AS-PCR specificity with comparable sensitivity using 3' LNA primers in gel electrophoresis and real-time detection experiments. This increase in AS-PCR discrimination with 3' LNA primers should facilitate the use of this simple, rapid, and inexpensive technique for SNP genotyping applications.

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Section: Introductionmentioning

confidence: 99%

Enhanced allele-specific PCR discrimination in SNP genotyping using 3? locked nucleic acid (LNA) primers

et al. 2003

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“…SNP S01 is a transition G-A, which induces G-T and C-A mismatches. They belong to the difficult mismatches known to yield extension products with Taq DNA polymerase and hinder proper discrimination between genotypes (Day et al 1999;Ahmadian et al 2001). The genotype of the parents, determined from sequence data, is heterozygote (G/A) for parent 1 (P1) and homozygote (G/G) for parent 2 (P2).…”

Section: Pamsa Methods Developmentmentioning

confidence: 99%

“…The advantage of this method is the detection of the amplification products on agarose gel. On the other hand, the single base-pair change at the 3′ primer end is often not sufficient to ensure reliable discrimination between the two SNP alleles (Ahmadian et al 2001), and two reactions are needed, one for each allele (Kim et al 2005). To overcome these disadvantages, some improvement was achieved with PCR amplification of multiple specific alleles (PAMSA), which involves the use of at least two allele-specific primers in the same reaction and allows the detection of all the SNP alleles present in a sample.…”

Section: Introductionmentioning

confidence: 99%

Single-reaction for SNP Genotyping on Agarose Gel by Allele-specific PCR in Black Poplar (Populus nigra L.)

et al. 2007

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The wide development of single nucleotide polymorphism (SNP) markers also in non-model species increases the need for inexpensive methods that do not require sophisticated equipment and time for optimization. This work presents a new method for polymerase chain reaction (PCR) amplification of multiple specific alleles (PAMSA), which allows efficient discrimination of SNP polymorphisms in one reaction tube with standard PCR conditions. This improved PAMSA requires only three unlabeled primers: a common reverse primer and two allele-specific primers having a tail of different length to differentiate the two SNP alleles by the size of amplification products on agarose gel. A destabilizing mismatch within the five bases of the 3′ end is also added to improve the allele specificity. To validate the accuracy of this method, 94 full-sib individuals were genotyped with three SNPs and compared to the genotypes obtained by cleaved amplified polymorphic sequence (CAPS) or derived CAPS. This method is flexible, inexpensive, and well suited for high throughput and automated genotyping.

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“…Single nucleotide polymorphisms (SNPs) genotypes are often determined by scoring technologies that first report the genotypes by one or more quantitative measurements 1,2 . Since the continuous measurements must be reduced to one of three genotypes (in this work, denoted by AA, AB, BB), some values may have ambiguous classification.…”

Section: Introductionmentioning

confidence: 99%

“…Some technologies classify genotypes using a mixture of univariate 2,4 or bivariate normal distributions 1,5 . For example, the Perkin Elmer software SNPscorer 5 uses an ellipsoidal model that they label "Ellipsoidal model of equal dimensions at constant orientation".…”

Section: Introductionmentioning

confidence: 99%

Tradeoff Between No-Call Reduction in Genotyping Error Rate and Loss of Sample Size for Genetic Case/Control Association Studies

Kang

Gordon²,

Brown³

et al. 2003

Biocomputing 2004

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Single nucleotide polymorphisms (SNP) may be genotyped for use in case-control designs to test for association between a SNP marker and a disease using a 2 × 3 chi-squared test of independence. Genotyping is often based on underlying continuous measurements, which are classified into genotypes. A "no-call" procedure is sometimes used in which borderline observations are not classified. This procedure has the simultaneous effect of reducing the genotype error rate and the expected number of genotypes observed. Both quantities affect the power of the statistic. We develop methods for calculating the genotype error rate, the expected number of genotypes observed, and the expected power of the resulting test as a function of the no-call procedure. We examine the statistical properties of the chi-squared test using a no-call procedure when the underlying continuous measure of genotype classification is a three-component mixture of univariate normal distributions under a range of parameter specifications. The genotype error rate decreases as the no-call region is increased. The expected number of observations genotyped also decreases. Our key finding is that the expected power of the chi-squared test is not sensitive to the no-call procedure. That is, the benefits of reduced genotype error rate are almost exactly balanced by the losses due to reduced genotype observations. For an underlying univariate normal mixture of genotype classification to be analyzed with a 2 × 3 chi-squared test, there is little, if any, increase in power using a no-call procedure.

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Genotyping by apyrase-mediated allele-specific extension

Cited by 41 publications

References 16 publications

Enhanced allele-specific PCR discrimination in SNP genotyping using 3? locked nucleic acid (LNA) primers

Enhanced allele-specific PCR discrimination in SNP genotyping using 3? locked nucleic acid (LNA) primers

Single-reaction for SNP Genotyping on Agarose Gel by Allele-specific PCR in Black Poplar (Populus nigra L.)

Tradeoff Between No-Call Reduction in Genotyping Error Rate and Loss of Sample Size for Genetic Case/Control Association Studies

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