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An efficient and reliable DNA extraction method for preimplantation genetic diagnosis: a comparison of allele drop out and amplification rates using different single cell lysis methods

Cited 9 time in Web of Science Cited 8 time in Scopus
Authors
Kim, Sung Ah; Yoon, Jung Ah; Kang, Moon Joo; Choi, Young Min; Moon, Shin Yong; Chae, Soo Jin
Issue Date
2009-08
Publisher
ELSEVIER SCIENCE INC
Citation
FERTILITY AND STERILITY; Vol.92 2; 814-818
Keywords
Allele drop outpreimplantation genetic diagnosisnested polymerase chain reactionDNA amplificationDNA extraction
Abstract
Objective: To evaluate methods of DNA extraction from single cells for their suitability to amplify and provide a correct diagnosis of target disease genes. Design: Experimental study. Setting: University hospital laboratory. Patient(s): Two normal adult male and female blood donors. Intervention(s): Exon 51 of the dystrophin gene and the ZFX/ZFY gene were amplified from single lymphocytes using nested PCR. Five different methods of DNA extraction were tested (lysis in distilled water with freezing and thawing using liquid nitrogen, lysis in distilled water, alkaline lysis buffer, Proteinase K/sodium dodecyl sulfate (SDS) buffer, and N-lauroylsarcosine salt solution). Main Outcome Measure(s): Allele drop out and amplification rate. Result(s): The amplification efficiency from single unaffected lymphocytes was 89.0% using the liquid nitrogen method, 88.1% with the distilled water lysis method, 97.5% with the alkaline lysis buffer method, 91.5% with the Proteinase K/SDS lysis buffer method, and 84.8% using the N-lauroylsarcosine salt solution method. The mean allele drop out rate was 16.7%, 43.9%, 2.0%, 9.8%, and 18.9%, respectively, for each lysis method using single male lymphocytes as a template. Conclusion(s): Based on these results, DNA extraction using an alkaline lysis buffer results in more efficient rates of DNA amplification and less allele drop out than the other methods of DNA extraction tested. This method is suitable for the lysis of single cells in clinical preimplantation genetic diagnosis. (Fertil Steril (R) 2009;92:814-8. (c) 2009 by American Society for Reproductive Medicine.)
ISSN
0015-0282
Language
English
URI
http://hdl.handle.net/10371/76757
DOI
https://doi.org/10.1016/j.fertnstert.2008.06.033
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College of Medicine/School of Medicine (의과대학/대학원)Obstetrics & Gynecology (산부인과전공)Journal Papers (저널논문_산부인과학전공)
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