Kim Kijeong, Bazarragchaa Munkhtsetseg, Brenner Charles H, Choi Byung-Sun, Kim Kyung-Yong
Institute for Medical Sciences, College of Medicine, Chung-Ang University, Seoul 156-756, Republic of Korea.
DNA_VIEW, Oakland, CA 94601, USA; School of Public Health, University of California, Berkeley, CA 94720, USA.
Forensic Sci Int. 2015 Jun;251:171-8. doi: 10.1016/j.forsciint.2015.04.001. Epub 2015 Apr 13.
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.
在法医研究中经常会遇到高度降解的人类DNA。尽管付出了诸多努力，但DNA的质量和数量不佳往往导致DNA分析失败。对于高度降解的DNA样本进行成功的聚合酶链式反应（PCR），尚未有对DNA聚合酶性能的广泛评估。我们基于实时PCR和琼脂糖凝胶电泳分析，针对单拷贝基因扩增，用200份来自不同来源的古代DNA（aDNA）样本，评估了最有效的DNA聚合酶。测试了九种市售的DNA聚合酶，其中包括据报道对PCR抑制样本有效的酶。用20份aDNA样本对这些聚合酶进行的首次筛选测试表明，Pico Maxx HF、FastStart Taq和Ex Taq HS DNA聚合酶最为有效。用180份aDNA样本进行的进一步测试表明，AmpliTaq Gold（对照）从52份aDNA样本中扩增出PCR产物，PicoMaxx HF从62份中扩增出，FastStart Taq从64份中扩增出，Ex Taq HS从65份中扩增出。同时使用Ex Taq HS、FastStart Taq和PicoMaxx HF中的两种或更多种，其成功率显著高于单独使用AmpliTaq Gold。对37份阳性样本进行重复测试时，Ex Taq HS显示出最高的重复性（13份样本），而AmpliTaq Gold最低（4份样本）；这种差异具有显著性。数据还显示了这些酶的优先扩增情况；Ex Taq HS仅从两份样本中产生扩增，FastStart Taq从一份样本中产生扩增，PicoMaxx HF从一份样本中产生扩增。我们建议，最初使用这三种DNA聚合酶将增加从高度降解的人类DNA样本中成功扩增DNA的概率。
