Long DOP-PCR of rare archival anthropological samples

The application of molecular DNA technologies to anthropological questions has meant that rare or archival samples of human remains, including blood, hair, and bone, can now be used as a source of material for genetic analysi s. Often, these samples are irreplaceable, and/or yield very small quantiti es of DNA, so methods for preamplifying as much of the whole genome as poss ible would greatly enhance their usefulness. DOP-PCR (degenerate oligonucle otide-primed polymerase chain reaction) is an amplification method that use s a degenerate primer and very low initial annealing temperatures to amplif y the whole genome, We adapted a published DOP-PCR protocol to long PCR enz yme and amplification conditions. The effectiveness of these modifications was tested by PCR amplification of DOP-PCR products at a mixture of genomic targets including 66 different microsatellites, 11 Alu insertion polymorph isms, and variable-length segments of the human lipoprotein lipase gene (LP L). The selected microsatellite markers were chosen to represent every chro mosome, with expected product sizes ranging from 150 base pairs to 8000 bas e pairs in length, while the 22 Alu insertion polymorphisms were selected t o reveal biases in the recovery of alleles of different sizes. To determine nucleotide sequence variation, 2 kilobases (kb) of the LPL gene in 30 Mong olian individuals were sequenced. All gene-specific targets from DOP-PCR pr oduct template were amplified. No unexpected polymorphisms in the sequence results attributable to the DOP-PCR step were found, and 93% to 95% of Alu genotypes that have been amplified from total genomic DNA were replicated. The incorrect typings were all due to the preferential amplification of the shorter of two possible alleles in individuals heterozygous for an Aln ins ertion and were all correctly typed on subsequent reamplification of the ge ne-specific PCR products. This method of whole-genome amplification promise s to be an efficient way to maximize the genetic use of rare anthropologica l samples.