Genotyping and haplotyping of polymorphisms directly from genomic DNA via coupled amplification and sequencing(CAS)
Open Access
- 25 December 1991
- journal article
- research article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 19 (24) , 6877-6882
- https://doi.org/10.1093/nar/19.24.6877
Abstract
Coupled amplification and sequencing (CAS) allows a segment of DNA to be sequenced directly from genomic DNA. An initial PCR amplification stage selects and amplifies the target. During a subsequent stage both strands of the target segment are sequenced simultaneously and amplified further. We show that CAS can readily identify variant base pairs. Genotyping of a population for known sequence variation can be achieved simply and directly from genomic DNA of each organism by performing CAS only for the variant bases. The procedure supercedes development and optimization of alternative typing assays based on oligonucleotide hybridization or ligation. In addition, we show that competitive oligonucleotide priming with allelic primers can be readily performed in concert with the second stage of CAS. The combination of techniques allows sequencing of a single chromosome from aheterozygous genomic sample and direct haplotying of the polymorphism at the priming site with any others encompassed within the amplified segment.Keywords
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