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Primer Extension Capture: Targeted Sequence Retrieval from Degraded DNA Sources

发表于 2010-4-14 19:40 | 显示全部楼层 |阅读模式
本帖最后由 王传超 于 2010-4-14 20:38 编辑

Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources

An improved PCR method for endogenous DNA retrieval in contaminated Neandertal samples based on the use of blocking primers

Elena Giglia, 1, Morten Rasmussenb, 1, Sergi Civitc, Antonio Rosasd, Marco de la Rasillae, Javier Forteae, M. Thomas P. Gilbertb, Eske Willerslevb, and Carles Lalueza-Foxa,

a Institut de Biologia Evolutiva, CSIC-UPF, Dr. Aiguader 88, 08003 Barcelona, Spain
b Center for Ancient Genetics, Natural History Museum of Denmark, Universitetsparken 15, DK-2100 Copenhagen Ø, Denmark
c Departament d'Estadística, Universitat de Barcelona, Avda. Diagonal 645, 08003 Barcelona, Spain
d Departamento de Paleobiología, Museo Nacional de Ciencias Naturales, CSIC, José Gutierrez Abascal 2, 28006 Madrid, Spain
e Área de Prehistoria, Departamento de Historia, Universidad de Oviedo, Teniente Alfonso Martínez s/n, 33011 Oviedo, Spain

Received 3 July 2009;  revised 18 August 2009;  accepted 19 August 2009.  Available online 26 August 2009.

AbstractNeandertal skeletal remains are usually contaminated with modern human DNA derived from handling and washing of the specimens during excavation. Despite the fact that the distinct Neandertal haplotypes allow the design of specific primer pairs, for instance in most of the mitochondrial DNA (mtDNA) hypervariable region 1 (HVR1), the human contaminants can often outnumber the endogenous DNA, thus preventing a successful retrieval of Neandertal sequences. We have developed a novel PCR method, based on the use of blocking primers that preferentially bind to modern human contaminant DNA and block their amplification, and greatly improve the efficiency of Neandertal DNA retrieval. We tested the method in four El Sidrón Neandertal samples (two teeth and two bone fragments) with different contamination levels and taphonomic conditions, and we have been able to significantly increase the Neandertal yield from figures around 25.23% (5–69.6%) up to 90.18% (75.3–100%).

Keywords: Neandertals; Blocking primers; PCR; Contamination

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