Present address: Department of Biology, New Mexico State University, Las Cruces, NM 88003, USA.
Allele-specific PCR can improve the efficiency of experimental resolution of heterozygotes in resequencing studies
Article first published online: 21 DEC 2009
© 2009 Blackwell Publishing Ltd
Molecular Ecology Resources
Volume 10, Issue 4, pages 647–658, July 2010
How to Cite
CHEN, H., MORRELL, P. L., TOLENO, D. M., LUNDY, K. E. and CLEGG, M. T. (2010), Allele-specific PCR can improve the efficiency of experimental resolution of heterozygotes in resequencing studies. Molecular Ecology Resources, 10: 647–658. doi: 10.1111/j.1755-0998.2009.02804.x
- Issue published online: 11 JUN 2010
- Article first published online: 21 DEC 2009
- Received 3 July 2009; revision received 20 October 2009; accepted 27 October 2009
- allele-specific PCR;
- computational phasing;
- experimental phasing;
Resolution of the two haplotypes present in an individual that is heterozygous at a locus has been a difficult problem for nucleotide sequence-based population genetic studies. Here, we demonstrate a method in which allele-specific polymerase chain reaction (AS-PCR) and computational phasing are combined for relatively high-throughput, efficient resolution of phase in resequencing studies. Using data from multiple loci that were fully experimentally phased, we demonstrate that the popular computational tool PHASE can accurately phase heterozygous individuals with common SNPs (single nucleotide polymorphisms) and/or common haplotypes. However, we also demonstrate that experimental phasing with AS-PCR can efficiently supplement computational phasing, providing a rapid means to phase individuals with rare SNPs or haplotypes and with heterozygous insertion/deletion polymorphisms. By following simple stepwise procedures, AS-PCR can result in much more efficient and accurate experimental phasing of haplotypes than is possible with traditional methods such as cloning.