Poly(A) tailing of ancient DNA: a method for reproducible microsatellite genotyping.
Microsatellites could be of great potential use in the analysis of ancient remains, but so far such analyses have failed to be reproducible mainly because of the high degree of ancient DNA (aDNA) degradation. During PCR, annealing of the primers to the complementary sequences of microsatellites occurs together with cross-annealing of partially degraded repeated sequences. This could create chimeric alleles that do not correspond to the authentic ones. Here we report a simple method for processing aDNA fragments prior to PCR that greatly reduces the production of chimeric alleles. This approach eliminates aDNA molecules broken within the repeats as targets for Taq polymerase by adding poly(A) tails at the 3(') ends of the DNA fragments, which disrupts the homology in the region and thus prevents annealing out of register. We have analyzed one dinucleotide- (D6S337) and two trinucleotide-containing loci (IT15 and SCA1) using poly(A)-tailed and the same untreated aDNA as template. aDNAs were isolated from 28 human remains, 600 and 7000 years of age. In repeated experiments with untreated aDNAs we obtained three to five times more alleles compared to poly(A)-tailed aDNAs. According to our results, modification of aDNA by poly(A) tailing is an efficient pretreatment for accurate genotyping.
Culjković
Biljana
B
Faculty of Biology, University of Belgrade, Studentski trg 16, P.O. Box 52, Belgrade 11000, Yugoslavia.
Savić
Dusanka
D
Stojković
Oliver
O
Romac
Stanka
S
D000818:Animals; D001106:Archaeology / Q000266:history; D004247:DNA / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000266:history; D053938:DNA Fragmentation; D018900:Dinucleotide Repeats / Q000235:genetics; D005838:Genotype; D049690:History, Ancient; D006801:Humans; D018895:Microsatellite Repeats / Q000235:genetics*; D011061:Poly A / Q000032:analysis / Q000737:chemistry* / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D018911:Trinucleotide Repeats / Q000235:genetics; D015022:Yugoslavia
D011061:Poly A; D004247:DNA