Developing multiplexed SNP assays with special reference to degraded DNA templates

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This protocol describes a single nucleotide polymorphism (SNP) genotyping strategy for highly degraded DNA, using a two-stage multiplex whereby multiple fragments are first amplified in a single exponential reaction and the products of this PCR are added to a linear single-base-extension reaction. It utilizes the analytical power of a capillary electrophoresis system to simultaneously type all the target sites. The protocol is specifically written for use with severely fragmented templates, typical of ancient DNA, and can be adapted to widely used detection platforms. The addition of the single-phase genotyping step avoids the need for the re-amplification and cloning of PCR products, while providing its own controls for the detection of contamination and allelic drop-out. This protocol can facilitate the routine analysis of up to 52 SNP markers (haploid or diploid) in 96 samples in a single day, and is recommended for the authentication of data in all areas of DNA research (population and medical genetics, forensics, ancient DNA).

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Figure 1: Workflow diagram describing the steps of the multiplex protocol.
Figure 2: Examples of unacceptable primer-dimer formations that drastically reduce the primer concentration available for hybridization to the target sequence.
Figure 3: Analysis of 26 mtDNA SNPs by multiplex assay using 1,000 copies of ancient mtDNA from a typically degraded historic Asian sample (not able to generate amplicons above 161 bp in length).


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Correspondence to Juan J Sanchez.

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