Here we address the issue of enzymatic degradation of carryover contamination in ancient DNA research. We show that UQPCR, that is, the coupling of UPCR and QPCR, and DNA cloning in an E. coli strain deficient in both UNG and dUTPase activities provide a satisfying level of contamination prevention that meets the specific requirements of ancient DNA analyses.Materials and Methods DNA Extraction from Fossil Material
A 103-bp fragment of the tetracycline gene of the plasmid pBR322 was amplified using primers BR1 (5′-ATGCGTTGATGCAATTCT-3′) and BR2 (5′-GTCGATAGTGGCT-CCAAGTA-3′). The sequence of the resulting fragment is 5′-ATGCGTTGATGCAATTTCTATGCGCACC-CGTTCTCGGAGCACTGTCCG-ACCGCTTTGGCCGCCGCC-CAGTCCTGCTCGCTTCGCTACT-TGGAGCCACTATCGA-3′.
The primers used for the analysis of ancient bovine mitochondrial DNA amplify a 153-bp product from nucleotides 16,022-16,175 of the D-loop (BB3, 5′-GCCCCATGCATATAAGAAGT-3′ and BB4, 5′-GCGGCATGGTAATTAAGCTC-3′).
Amplifications were carried out in the LightCycler™ (Roche Applied Science, Penzberg, Germany) in individual glass capillaries using a modified version of the Platinum® Quantitative PCR SuperMix-UDG (Invitrogen, Carlsbad, CA, USA) in a total volume of 20 µL. The final composition was 10 mM Tris-HCl, pH 8.0, 50 mM KCl, 4.5 mM MgCl2, 400 µM dUTP, 200 µM dATP, 200 µM dCTP, 200 µM dGTP, 0.4 U UNG, 0.6 U Platinum Taq DNA polymerase (part of the kit); proprietary stabilizers (part of the kit); 4% glycerol; 100 µg/mL of either bovine serum albumin (BSA; Sigma, St. Louis, MO, USA) or, in the case of bovine fossils, horse albumin (globulin-free Horse Serum Albumin; Sigma); 0.05% polyoxyethylene ether W1 (Sigma); 1/10.000 SYBR® Green I (Molecular Probes, Leiden, The Netherlands) supplemented with 10 pmol of each primer per reaction; and additional 0.8 U Platinum Taq DNA polymerase. The cycling program was as follows: 45 min at 37°C, 2 min at 95°C, and 50 cycles of 5 s at 95°C, 10 s at 58°C or 60°C (for pBR and BB34, respectively), and 12 s at 72°C.Cloning of the PCR Products
The PCR products were cloned using the pGEM®-T Easy Vector System kit (Promega, Madison, WI, USA), according to the manufacturer's protocol. Transformation of the dut, ung E. coli strain RZ1032 (16) was carried out by electroporation in a Gene Pulser® (Bio-Rad Laboratories, Hercules, CA, USA), following the manufacturer's protocol.Results and Discussion QPCR Amplification Using dUTP and Degradation by UNG
We have adapted UNG-coupled quantitative real-time PCR (UQPCR) to the analysis of ancient DNA. This included the assessment and improvement of the efficiencies of both PCR amplification and degradation of carryover contamination to meet the specific requirements of ancient DNA studies.
The replacement of dTTP by dUTP lowers the PCR efficiency (17). Cumulative decrease in efficiency is a major concern with samples containing damaged DNA and inhibitors. We improved a commercially available dUTP- and SYBR Green I-containing QPCR mixture (Platinum Quantitative PCR SuperMix-UDG) by supplementing it with detergents, serum albumin, and extra Taq DNA polymerase. This allowed us to achieve 90% to 100% efficiency of PCR amplification with numerous primers using the Light Cycler. A critical parameter for any DNA amplification starting from a low number of molecules is the sensitivity of the reaction. To test whether the sensitivity of UQPCR is decreased due to the replacement of dUTP by dTTP, we compared PCRs performed with either dUTP or dTTP and very low levels of template molecules (i.e., statistically about one template molecule per reaction). Amplification occurred in 9 PCRs of 18 when dTTP was used and in 10 of 18 when dUTP was used (data not shown). This shows that the use of dUTP in the conditions described here does not affect the sensitivity of the PCR.
We then elaborated conditions for UNG treatment providing sufficient carryover protection even when a short PCR fragment is amplified (103 bp including the primers) because such short fragments can often be amplified from severely degraded ancient DNA when longer fragments cannot. We used as a critical test for the efficiency of the carryover protection a short (103 bp) fragment with a relatively low AT content (41%), thus incorporating a low level of dUTP. This fragment originates from the tetracycline resistance gene in pBR322, which is absent from most modern cloning vectors and is therefore relatively insensitive to the contaminants commonly found in a conventional molecular biology laboratory. Furthermore, this assay does not present a contamination risk of its own for ancient DNA analyses.