DNA sequences from multiple amplifications reveal artifacts induced by cytosine deamination in ancient DNA

Abstract

We show that DNA molecules amplified by PCR from DNA extracted from animal bones and teeth that vary in age between 25 000 and over 50 000 years carry C-->T and G-->A substitutions. These substitutions can reach high proportions among the molecules amplified and are due to the occurrence of modified deoxycytidine residues in the template DNA. If the template DNA is treated with uracil N-glycosylase, these substitutions are dramatically reduced. They are thus likely to result from deamination of deoxycytidine residues. In addition, 'jumping PCR', i.e. the occurrence of template switching during PCR, may contribute to these substitutions. When DNA sequences are amplified from ancient DNA extracts where few template molecules initiate the PCR, precautions such as DNA sequence determination of multiple clones derived from more than one independent amplification are necessary in order to reduce the risk of determination of incorrect DNA sequences. When such precautionary measures are taken, errors induced by damage to the DNA template are unlikely to be more frequent than approximately 0.1% even under the unlikely scenario where each amplification starts from a single template molecule.

Figure 1

DNA sequences from five different amplifications from one cave bear sample. Dots indicate identity to the consensus sequence, given at the top of the alignment. The first number in front of a sequence gives the number of the amplification, the second the number of the sequenced clone. Four of the five amplifications carry consistent substitutions (indicated by an asterisk) relative to the consensus sequence at one to five positions. Clone 4-6 represents a jumping PCR artifact.

Figure 2

Proposed mechanisms explaining the incorporation of deoxyadenosine residues opposite to deoxycytidine residues in the template molecule. (A) Cytosine deamination. The U in the ancient DNA template represents a deoxyuridine residue, a 5-hydroxydeoxyuridine residue or any other modified deoxycytidine residue read as T by Taq polymerase. (B) Jumping PCR. In the first step, the primer is extended on a template that does not include the second primer site and a terminal deoxyadenosine residue is added at the end of the extension product. In the second step, this product is extended on a second template. If the terminal A primes where the template carries a C, the result is incorporation of a deoxyadenosine residue opposite a deoxycytidine residue.

Figure 3

Schematic illustration of the strategy where two or three amplifications are used to determine an ancient DNA sequence. The correct base (C) is blue and the incorrect base (T) red. An error rate of 2% is assumed and the probability of determining an incorrect base is the sum of the three paths that arrive at the incorrect base after two or three amplifications (see text for details).