Multiplex amplification of ancient DNA.
This method is designed to assemble long, continuous DNA sequences using minimal amounts of fragmented ancient DNA as template. This is achieved by a two-step approach. In the first step, multiple fragments are simultaneously amplified in a single multiplex reaction. Subsequently, each of the generated fragments is amplified individually using a single primer pair, in a standard simplex (monoplex) PCR. The ability to amplify multiple fragments simultaneously in the first step allows the generation of large amounts of sequence from rare template DNA, whereas the second nested step increases specificity and decreases amplification of contaminating DNA. In contrast to current protocols using many template-consuming simplex PCRs, the method described allows amplification of several kilobases of sequence in just one reaction. It thus combines optimal template usage with a high specificity and can be performed within a day.
Römpler
Holger
H
Molecular Biochemistry, Institute of Biochemistry, Medical Faculty, University of Leipzig, Johannisallee 30, 04103 Leipzig, Germany.
Dear
Paul H
PH
Krause
Johannes
J
Meyer
Matthias
M
Rohland
Nadin
N
Schöneberg
Torsten
T
Spriggs
Helen
H
Stiller
Mathias
M
Hofreiter
Michael
M
D001106:Archaeology / Q000379:methods; D004247:DNA / Q000032:analysis* / Q000235:genetics*; D007753:Laboratories; D010163:Paleontology / Q000379:methods; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D015203:Reproducibility of Results; D013698:Templates, Genetic; D013997:Time Factors
D004247:DNA