Overlapping PCR for bidirectional PCR amplification of specific alleles: A rapid one-tube method for simultaneously differentiating homozygotes and heterozygotes

Qiang Liu, Erik C Thorland, John A. Heit, Steve S. Sommer

Research output: Contribution to journalArticle

138 Citations (Scopus)

Abstract

Rapid detection of single-base changes is fundamental to molecular medicine. PASA (PCR amplification of specific 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.

Original languageEnglish (US)
Pages (from-to)389-398
Number of pages10
JournalGenome Research
Volume7
Issue number4
StatePublished - Apr 1997

Fingerprint

Homozygote
Heterozygote
Alleles
Polymerase Chain Reaction
Tail
Guidelines
Molecular Medicine
Catechol O-Methyltransferase
Mutation
Factor V
DNA
Nucleotides
Prospective Studies

ASJC Scopus subject areas

  • Genetics

Cite this

Overlapping PCR for bidirectional PCR amplification of specific alleles : A rapid one-tube method for simultaneously differentiating homozygotes and heterozygotes. / Liu, Qiang; Thorland, Erik C; Heit, John A.; Sommer, Steve S.

In: Genome Research, Vol. 7, No. 4, 04.1997, p. 389-398.

Research output: Contribution to journalArticle

@article{a47593edc7dd475fa266b7be7f9b2141,
title = "Overlapping PCR for bidirectional PCR amplification of specific alleles: A rapid one-tube method for simultaneously differentiating homozygotes and heterozygotes",
abstract = "Rapid detection of single-base changes is fundamental to molecular medicine. PASA (PCR amplification of specific 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.",
author = "Qiang Liu and Thorland, {Erik C} and Heit, {John A.} and Sommer, {Steve S.}",
year = "1997",
month = "4",
language = "English (US)",
volume = "7",
pages = "389--398",
journal = "Genome Research",
issn = "1088-9051",
publisher = "Cold Spring Harbor Laboratory Press",
number = "4",

}

TY - JOUR

T1 - Overlapping PCR for bidirectional PCR amplification of specific alleles

T2 - A rapid one-tube method for simultaneously differentiating homozygotes and heterozygotes

AU - Liu, Qiang

AU - Thorland, Erik C

AU - Heit, John A.

AU - Sommer, Steve S.

PY - 1997/4

Y1 - 1997/4

N2 - Rapid detection of single-base changes is fundamental to molecular medicine. PASA (PCR amplification of specific 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.

AB - Rapid detection of single-base changes is fundamental to molecular medicine. PASA (PCR amplification of specific 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.

UR - http://www.scopus.com/inward/record.url?scp=0030977752&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030977752&partnerID=8YFLogxK

M3 - Article

C2 - 9110178

AN - SCOPUS:0030977752

VL - 7

SP - 389

EP - 398

JO - Genome Research

JF - Genome Research

SN - 1088-9051

IS - 4

ER -