Repair of degraded duplex DNA from prehistoric samples using Escherichia coli DNA polymerase I and T4 DNA ligase
Repair of degraded duplex DNA from prehistoric samples using Escherichia coli DNA polymerase I and T4 DNA ligaseCarsten M. Pusch*, Ian Giddings and Michael Scholz1
Molecular Genetics Laboratory, University Eye Hospital, University of Tübingen, D-72076 Tübingen, Auf der Morgenstelle 15, Germany and 1Institute of Prehistoric Research, Department of Archeobiology, University of Tübingen, D-72072 Tübingen, Eugenstrasse 40, Germany
Received October 13, 1997;Revised and Accepted December 5, 1997
ABSTRACT
The most notable feature of DNA extracted from prehistoric material is that it is of poor quality. Amplification of PCR products from such DNA is consequently an exception. Here we present a simple method for the repair of degraded duplex DNA using the enzymes Escherichia coli DNA polymerase I and T4 DNA ligase. Adjacent sequences separated by nicks do not split up into intact strands during the denaturation step of PCR. Thus the target DNA is refractory to amplification. The proposed repair of nicked, fragmented ancient DNA results in an increase of amplification efficiency, such that the correct base order of the respective nuclear DNA segment can be obtained.
While scientific approaches to investigating prehistoric material such as bones, teeth or mummified tissue are feasible, they are highly complex. Three factors commonly prevent PCR-mediated amplification of ancient DNA (aDNA); the presence of contamination, a low copy number for the desired DNA segment or that the isolated DNA is heavily degraded (i.e. present in small fragments).
A number of the contaminants found in aDNA extracts have been shown to have a proven record as PCR inhibitors (e.g. humic acid, proteins and sugar remnants) (1-5). Even highly purified extracts (e.g. cleaned by silica, proteolytic enzymes, phenol extractions and/or repeated ethanol precipitations) still result in a very low rate of nuclear-specific amplification products. Therefore, we focused on the state of preservation of the chemically altered DNA.
A straightforward and reliable model for explaining these alterations, posits damaged DNA consisting of nicked double strands due to the influence of DNase activity, mechanical and/or physical actions (e.g. by hydrolysis, oxidation or enzymatic destruction) (3). The typically small fragmented duplex aDNA does not separate into two intact strands during the initial denaturation of PCR, but rather into a number of smaller fragments. This greatly minimises the chance of two complementary-oriented PCR primers amplifying a common DNA segment.
In order to assess this `damage' hypothesis, we performed repair reactions (RRs) with the DNA polymerase I enzyme from Escherichia coli (Stratagene) and T4 DNA ligase (Boehringer Mannheim). This allows an increase in the number of informative PCR-based amplifications of single-copy DNA segments of the human nuclear genome.
Escherichia coli DNA polymerase I translates the nicks in the DNA, and the remaining gaps are closed by the subsequent use of T4 DNA ligase (Boehringer Mannheim).
This strategy was successfully applied to aDNA samples where an amplification product could reasonably be expected, but from which no PCR products were obtained. It was also used when highly purified isolates remained non-informative throughout 6-10 independent PCR assays, or when the total yield of isolated DNA was too low (<50 ng).
The individuals from whom samples were taken were buried at the Alamannic burial site at Neresheim (FRG) between ~450 and 700 AD (6). For bone sampling and DNA extraction, the Mix and Clean (MCM) guidelines were followed (7). All possible precautions to exclude contamination with modern DNA were observed (3,7). For each sample, contamination monitoring was performed by the use of two to three extraction blank controls and one water control. In addition PCR amplifications were carried out upon all samples, including negative controls, using mitochondrial specific primers. Due to the high copy number of mitochondrial genome, this is currently the most sensitive contamination monitoring protocol available.
A typical repair reaction contained 2.5 U E.coli DNA polymerase I, 5 µl 10× nick translation buffer, 50-1000 ng aDNA isolate, 0.4 mM each dNTP and H2Odd to a total volume of 50 µl. The reaction was carried out for 60-90 min at 37°C, and terminated with a 20 min incubation at 70°C. Subsequently, 18 µl of the polymerase-treated aDNA (50-300 ng) were mixed with 2 µl of 10× ligase buffer and 0.5 U ligase enzyme. A 1 h ligation reaction was then performed using a temperature-cycle ligation (TCL) protocol on a trio-thermocycler (Biometra) (8). The ligation reaction cycled between 10 and 30°C, holding at each temperature for 10 s. Following enzymatic treatment, the aDNA isolates were purified three times with glass wool/Sephadex G-50 columns, then subjected to a phenol/chloroform/isoamylalcohol (25:24:1) extraction and finally precipitated with 2.5 vol EtOHabs and 1/10 vol 3 M NaOAc, pH 5.3. The pellet was washed twice with 300 µl 70% EtOH, air dried and subsequently dissolved in 10-15 µl of TE buffer, pH 8.0 prior to amplification by PCR.
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