Extensive evaluation of DNA polymerase performance for highly degraded human DNA samples.
Highly degraded human DNA is commonly encountered in the forensic studies. Despite many efforts, the poor quality and quantity of the DNA often result in unsuccessful DNA analysis. There has been no extensive evaluation of DNA polymerase performance for the successful PCR of highly degraded DNA samples. We evaluated the most efficient DNA polymerases, based on real-time PCR and agarose gel electrophoresis analyses for a single copy gene amplification, with 200 ancient DNA (aDNA) samples of various origins. Nine commercially available DNA polymerases were tested, which included enzymes that are reportedly effective for PCR-inhibitory samples. The first screening test for the polymerases with 20 aDNA samples showed that Pico Maxx HF, FastStart Taq, and Ex Taq HS DNA polymerases were the most effective. Further tests with 180 aDNA samples showed that AmpliTaq Gold (control) amplified PCR products from 52 aDNA samples, PicoMaxx HF from 62, FastStart Taq from 64, and Ex Taq HS from 65. The use of two or more of Ex Taq HS, FastStart Taq, and PicoMaxx HF resulted in a significantly higher success rate than that of AmpliTaq Gold alone. With 37 positive samples tested in duplicate, Ex Taq HS showed the highest reproducibility (13 samples) and AmpliTaq Gold, the lowest (four samples); this difference was significant. The data also showed preferential amplification by the enzymes; Ex Taq HS exclusively produced amplification from two samples, FastStart Taq from one, and PicoMaxx HF from one. We suggest that the initial use of these three DNA polymerases will increase the probability of successfully amplifying DNA from highly degraded human DNA samples.
Institute for Medical Sciences, College of Medicine, Chung-Ang University, Seoul 156-756, Republic of Korea.
Kijeong
K
Kim
Institute for Medical Sciences, College of Medicine, Chung-Ang University, Seoul 156-756, Republic of Korea.
Munkhtsetseg
M
Bazarragchaa
DNA_VIEW, Oakland, CA 94601, USA; School of Public Health, University of California, Berkeley, CA 94720, USA.
Charles H
CH
Brenner
Institute for Medical Sciences, College of Medicine, Chung-Ang University, Seoul 156-756, Republic of Korea.
Byung-Sun
BS
Choi
Institute for Medical Sciences, College of Medicine, Chung-Ang University, Seoul 156-756, Republic of Korea. Electronic address: skull@cau.ac.kr.
Kyung-Yong
KY
Kim
D053836:DNA Degradation, Necrotic; D004259:DNA-Directed DNA Polymerase / Q000235:genetics*; D004587:Electrophoresis, Agar Gel; D053803:Forensic Genetics; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D004259:DNA-Directed DNA Polymerase
DNA polymerase; Highly degraded DNA; Real-time PCR