Single nucleotide polymorphism genotyping using allele-specific PCR and fluorescence melting curves.
We present a PCR method for identification of single nucleotide polymorphisms (SNPs), using allele-specific primers designed for selective amplification of each allele. Matching the SNP at the 3' end of the forward or reverse primer, and additionally incorporating a 3' mismatch to prevent amplification of the incorrect allele, results in selectivity of the allele-specific primers. DNA melting analysis with fluorescent SYBR Green affords detection of the PCR products. By incorporating a GC-rich sequence into one of the two allele-specific primers to increase the melting temperature, both alleles can be measured simultaneously at their respective melting temperatures. Applying the DNA melting analysis to SNPs in ApoE and ABCA1 yielded results identical to those obtained with other genotyping methods. This provides a cost-effective, high-throughput method for amplification and scoring of SNPs.
Papp
Audrey C
AC
Department of Pharmacology 5068, Graves Hall, 333 W. 10th Avenue, Ohio State University, Columbus, OH 43210, USA. papp.2@osu.edu
Pinsonneault
Julia K
JK
Cooke
Glen
G
Sadée
Wolfgang
W
D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics*; D000483:Alleles; D001057:Apolipoproteins E / Q000737:chemistry / Q000235:genetics*; D020869:Gene Expression Profiling / Q000379:methods*; D005838:Genotype; D006358:Hot Temperature; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D044366:Transition Temperature
D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters; D001057:Apolipoproteins E