A Novel Method for Detecting Point Mutations or Polymorphisms and Its Application to Population Screening for Carriers of Phenylketonuria

Sommer, Steve S.; Cassady, Joslyn D.; Sobell, Janet L.; Bottema, Cynthia D.K.

doi:10.1016/s0025-6196(12)65378-6

Cited by 120 publications

(52 citation statements)

References 24 publications

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“…The specificity of the PCR amplification is conferred by placing the 3Ј end of one of the primers directly over and matching one or the other of the variant nucleotides (Newton et al 1989;Sommer et al 1989;Wu et al 1989). This specificity can be enhanced particularly by using the Stoffel fragment of Taq DNA polymerase (Lawyer et al 1993;Tada et al 1993;Germer and Higuchi 1999).…”

Section: Principle Of the Methodsmentioning

confidence: 99%

High-Throughput SNP Allele-Frequency Determination in Pooled DNA Samples by Kinetic PCR

Germer

Holland²,

Higuchi³

2000

Genome Res.

397

295

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We have developed an accurate, yet inexpensive and high-throughput, method for determining the allele frequency of biallelic polymorphisms in pools of DNA samples. The assay combines kinetic (real-time quantitative) PCR with allele-specific amplification and requires no post-PCR processing. The relative amounts of each allele in a sample are quantified. This is performed by dividing equal aliquots of the pooled DNA between two separate PCR reactions, each of which contains a primer pair specific to one or the other allelic SNP variant. For pools with equal amounts of the two alleles, the two amplifications should reach a detectable level of fluorescence at the same cycle number. For pools that contain unequal ratios of the two alleles, the difference in cycle number between the two amplification reactions can be used to calculate the relative allele amounts. We demonstrate the accuracy and reliability of the assay on samples with known predetermined SNP allele frequencies from 5% to 95%, including pools of both human and mouse DNAs using eight different SNPs altogether. The accuracy of measuring known allele frequencies is very high, with the strength of correlation between measured and known frequencies having an r 2 = 0.997. The loss of sensitivity as a result of measurement error is typically minimal, compared with that due to sampling error alone, for population samples up to 1000. We believe that by providing a means for SNP genotyping up to thousands of samples simultaneously, inexpensively, and reproducibly, this method is a powerful strategy for detecting meaningful polymorphic differences in candidate gene association studies and genome-wide linkage disequilibrium scans.

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Section: Principle Of the Methodsmentioning

confidence: 99%

High-Throughput SNP Allele-Frequency Determination in Pooled DNA Samples by Kinetic PCR

Germer

Holland²,

Higuchi³

2000

Genome Res.

397

295

View full text Add to dashboard Cite

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“…PCR can be adapted for the rapid detection of singlebase changes in genomic DNA by using specifically designed oligonucleotides in a method called PCR amplification of specific alleles (PASA) (Sommer et al 1989;Sarkar et al 1990). This rapid method is also known as allele-specific amplification (ASA), allele-specific PCR, and amplification refractory mutation system (ARMS) (Newton et al 1989;Nichols et al 1989;Wu et al 1989).…”

mentioning

confidence: 99%

Overlapping PCR for Bidirectional PCR Amplification of Specific Alleles: A Rapid One-Tube Method for Simultaneously Differentiating Homozygotes and Heterozygotes

Liu¹,

Thorland²,

Heit³

et al. 1997

Genome Res.

Self Cite

137

100

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Rapid detection of single-base changes is fundamental to molecular medicine. PASA (PCR amplification ofspecific alleles) is a rapid method of genotyping single-base changes, but one reaction is required for each allele. Bidirectional PASA (Bi-PASA) was developed to distinguish between homozygotes and heterozygotes in one PCR reaction by utilizing novel primer design with appropriate cycling conditions. In Bi-PASA, one of the alleles is amplified by a PASA reaction in one direction while the second allele is amplified by a PASA reaction in the opposite direction. Two outer (P and Q) and two inner allele-specific (A and B) primers are required. In heterozygotes, three segments are amplified: a segment of size AQ resulting from one allele, another segment of size PB resulting from the second allele, and a combined segment of size PQ. In homozygotes, segment PQ and either segments AQ or PB amplify. The two inner primers (A and B) contain a relatively short complementary region and a 10-nucleotide G+C-rich 5′ tail. The inner primers “switch” from low-efficiency to high-efficiency amplification when genomic DNA is replaced by previously amplified template DNA. In addition, the 5′ tails prevent “megapriming”. The parameters for optimizing Bi-PASA were investigated in detail for common mutations in the human factor V and catechol–O-methyltransferase genes. Guidelines for optimization of Bi-PASA also were developed and tested in a prospective study. Three additional Bi-PASA assays were optimized rapidly by utilizing these guidelines. In conclusion, Bi-PASA is a simple and rapid method for detecting the zygosity of known mutations in a single PCR reaction.

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“…Amplification of an HLA-11 unique sequence (524 -560 of HLA-A exon 3) was performed by first amplifying region 456 -617 using HLA-A11 primer ϩ 5'-GGACCTGCG-CTCTTGGAC-3' and HLA-A11 primer Ϫ 5'-GTGCG-CTGCAGCGTCTCC-3'. The HLA-A11-specific sequence was determined by the bidirectional PCR amplification of specific alleles method, as previously described (Bottema and Sommer, 1993;Liu et al, 1997;Sommer et al, 1999).…”

Section: Bidirectional Pcr Amplification Of Specific Alleles For the mentioning

confidence: 99%

Epstein-Barr Virus Nuclear Antigen (EBNA)-1 Carboxy-Terminal and EBNA-4 Sequence Polymorphisms in Nasal Natural Killer/T-Cell Lymphoma in the United States

Gaal

Weiss

Chen

et al. 2002

Laboratory Investigation

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SUMMARY:Epstein-Barr virus (EBV) polymorphisms were examined in 12 cases of nasal natural killer (NK)/T-cell lymphoma diagnosed in the United States (U.S.-NL) with respect to the EBV-associated nuclear antigen (EBNA)-1 carboxy (C)-terminal region and the EBNA-4 region. A single dominant EBV strain was found in all cases. EBNA-1 sequences were remarkably homogeneous, showing either a P-ala (2/12) or P-ala variant (9/12) sequence. Other EBNA-1 subtypes known to be common in U.S.-reactive samples, such as P-thr or V-leu, were not identified. The final case had a base deletion with frame shift and premature stop codon. EBNA-1 C-terminal amino acid substitutions were common at codons 499 (10/12 cases), 502 (7/12), 524 (9/12), and 528 (6/12), all previously reported "hot spots." However, unlike previous reports of other EBV-associated neoplastic and reactive tissues, mutations were absent at residues 487 and 492. Mutations within HLA-A11-restricted immunogenic EBNA-4 epitopes 399 -408 and 416 -424 occurred in 3 of 12 cases but were not associated with HLA-A11 status. In summary, the exclusive finding of P-ala variant or P-ala EBNA-1 sequences in U.S.-NL cases differs from that reported in U.S.-reactive and non-U.S.-NL cases. Although the significance of this difference is not known for certain, it may be related to geographic and/or site-specific variations, rather than oncogenicity per se. (Lab Invest 2002, 82:957-962).

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A Novel Method for Detecting Point Mutations or Polymorphisms and Its Application to Population Screening for Carriers of Phenylketonuria

Cited by 120 publications

References 24 publications

High-Throughput SNP Allele-Frequency Determination in Pooled DNA Samples by Kinetic PCR

High-Throughput SNP Allele-Frequency Determination in Pooled DNA Samples by Kinetic PCR

Overlapping PCR for Bidirectional PCR Amplification of Specific Alleles: A Rapid One-Tube Method for Simultaneously Differentiating Homozygotes and Heterozygotes

Epstein-Barr Virus Nuclear Antigen (EBNA)-1 Carboxy-Terminal and EBNA-4 Sequence Polymorphisms in Nasal Natural Killer/T-Cell Lymphoma in the United States

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