Abstract
While standard DNA-sequencing approaches readily yield genotypic sequence data, haplotype information is often of greater utility for population genetic analyses. However, obtaining individual haplotype sequences can be costly and time-consuming and sometimes requires statistical reconstruction approaches that are subject to bias and error. Advancements have recently been made in determining individual chromosomal sequences in large-scale genomic studies, yet few options exist for obtaining this information from large numbers of highly polymorphic individuals in a cost-effective manner. As a solution, we developed a simple PCR-based method for obtaining sequence information from individual DNA strands using standard laboratory equipment. The method employs a water-in-oil emulsion to separate the PCR mixture into thousands of individual microreactors. PCR within these small vesicles results in amplification from only a single starting DNA template molecule and thus a single haplotype. We improved upon previous approaches by including SYBR Green I and a melted agarose solution in the PCR, allowing easy identification and separation of individually amplified DNA molecules. We demonstrate the use of this method on a highly polymorphic estuarine population of the copepod Eurytemora affinis for which current molecular and computational methods for haplotype determination have been inadequate.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.