Improved Reliability of Allele-Specific PCR

Imyanitov, E. N.; Buslov, Konstantin G.; Suspitsin, Evgeny N.; Kuligina, E.; Belogubova, Evgeniya V.; Grigoriev, M.; Togo, Alexandr V.; Hanson, Kaido P.

doi:10.2144/02333bm04_11841a

Cited by 11 publications

(11 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In contrast, an alternative method to improve the reliability of ASA PCR by use of a competing depository oligonucleotide was not helpful under our rapidcycle PCR conditions [data not shown and Ref. (24 )]. …”

Section: Discussionmentioning

confidence: 97%

Rapid, Long-Range Molecular Haplotyping of Thiopurine S-Methyltransferase (TPMT) 3A, 3B, and 3C

2004

View full text Add to dashboard Cite

Background:Haplotyping is an important technique in molecular diagnostics because haplotypes are often more predictive for individual phenotypes than are the underlying single-nucleotide polymorphisms (SNPs). Until recently, methods for haplotyping SNPs separated by kilobase distances were laborious and not applicable to high-throughput screening. In the case of thiopurine S-methyltransferase (TPMT), differentiating among TPMT*3A, *3B, and *3C alleles is sometimes necessary for predictive genotyping. Methods: The genomic region including the two SNPs that define TPMT*3A, *3B, and *3C alleles was amplified by long-range PCR. The resulting PCR product was circularized by ligation and haplotyped by allele-specific amplification PCR followed by product identification with hybridization probes. Results: Critical points were the long-range PCR conditions, including choice of buffer and primers, optimization of the ligation reaction, and selection of primers that allowed for strict allele-specific amplification in the second-round PCR. Different underlying TPMT haplotypes could then be differentiated. Results from the haplotyping method were in full agreement with those from our standard real-time PCR method: TPMT*1/*3A (n ‫؍‬ 20); TPMT*1/*3C (n ‫؍‬ 4); TPMT*1/*1 (n ‫؍‬ 6); and TPMT*3A/*3A (n ‫؍‬ 6). One TPMT*1/*3A sample failed to amplify, and no whole blood was available for repeat DNA isolation. Conclusions: This method for rapid-cycle real-time, allele-specific amplification PCR-assisted long-range haplotyping has general application for the haplotyping of distant SNPs. The procedure is simpler and more

show abstract

Section: Discussionmentioning

confidence: 97%

Rapid, Long-Range Molecular Haplotyping of Thiopurine S-Methyltransferase (TPMT) 3A, 3B, and 3C

2004

View full text Add to dashboard Cite

show abstract

“…However, standard allele-specific assays can be prone to mispriming. 12,21,22 Therefore, we chose to adopt an ACB-PCR assay to detect the V617F JAK2 mutation. In an ACB-PCR assay, 3 primers are combined in a single tube, a forward primer with a modified 3' end, a second forward primer that targets the mutant allele, and a reverse primer.…”

Section: Discussionmentioning

confidence: 99%

A Simple, Rapid, and Sensitive Method for the Detection of theJAK2V617F Mutation

Tan¹,

Westerman²,

Dobrovic³

2007

Am J Clin Pathol

View full text Add to dashboard Cite

The point mutation 1849 (GT) V617F in the JAK2 gene occurs at high frequency in several chronic myeloproliferative diseases. Although a number of V617F mutation detection methods have been described, few are readily implemented in a diagnostic setting. We developed a simple and sensitive allelespecific competitive blocker polymerase chain reaction (ACB-PCR) assay to detect the V617F mutation. DNA was extracted from peripheral whole blood samples of 26 patients with chronic myeloproliferative disease. The ACB-PCR limit of detection was 1%. All positive samples detected by sequencing were detected by ACB-PCR. In 3 patients with essential thrombocythemia, the V617F mutation was readily detected by ACB-PCR but was near the detection limit of sequencing, confirming that ACB-PCR is more effective at detecting V617F when the mutant cell population is low. Detection of the monomorphic JAK2 V617F mutation using the ACB-PCR assay is easy to perform, rapid, sensitive, and cost-effective, which are key features of an ideal diagnostic method.

show abstract

“…Many of the existing methods (Dutton and Sommer 1991;Okimoto and Dodgson 1996;Imyanitov et al 2002;Hansson and Kawabe 2005;Hinten et al 2007) achieved good results after empirical adjustment of PCR conditions, mainly, the concentration of each primer, of magnesium chloride, and the annealing temperature. On the contrary, the method presented in this work did not need any PCR optimization.…”

Section: Pamsa Methods Developmentmentioning

confidence: 99%

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

et al. 2007

View full text Add to dashboard Cite

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.

show abstract

Improved Reliability of Allele-Specific PCR

Cited by 11 publications

References 0 publications

Rapid, Long-Range Molecular Haplotyping of Thiopurine S-Methyltransferase (TPMT) 3A, 3B, and 3C

Rapid, Long-Range Molecular Haplotyping of Thiopurine S-Methyltransferase (TPMT) 3A, 3B, and 3C

A Simple, Rapid, and Sensitive Method for the Detection of theJAK2V617F Mutation

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

Contact Info

Product

Resources

About

Improved Reliability of Allele-Specific PCR

Cited by 11 publications

References 0 publications

Rapid, Long-Range Molecular Haplotyping of Thiopurine S-Methyltransferase (TPMT*) *3A, *3B, and *3C

Rapid, Long-Range Molecular Haplotyping of Thiopurine S-Methyltransferase (TPMT*) *3A, *3B, and *3C

A Simple, Rapid, and Sensitive Method for the Detection of theJAK2V617F Mutation

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

Contact Info

Product

Resources

About

Rapid, Long-Range Molecular Haplotyping of Thiopurine S-Methyltransferase (TPMT) 3A, 3B, and 3C

Rapid, Long-Range Molecular Haplotyping of Thiopurine S-Methyltransferase (TPMT) 3A, 3B, and 3C