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

Fertility and Sterility
Sung Ah KimShin Yong Moon

Abstract

To evaluate methods of DNA extraction from single cells for their suitability to amplify and provide a correct diagnosis of target disease genes. Experimental study. University hospital laboratory. Two normal adult male and female blood donors. 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). Allele drop out and amplification rate. 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. Based on these results, DNA extraction using an alkaline lysis buffer results in more efficient rates of DNA amplification ...Continue Reading

References

Feb 1, 1996·Journal of Assisted Reproduction and Genetics·P F RayA H Handyside
Aug 1, 1997·Human Reproduction·W Lissens, K Sermon
Oct 7, 2004·Molecular Human Reproduction·Ali HellaniPinar Ozand
Dec 7, 2005·Journal of Assisted Reproduction and Genetics·Shinichi TsuchiyaYasunori Yoshimura
Aug 10, 2006·Molecular Human Reproduction·P BurletJ Steffann
Oct 10, 2006·Fertility and Sterility·Belén LledóRafael Bernabeu

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Citations

Mar 4, 2010·Journal of Assisted Reproduction and Genetics·Keren DotanLeah Peleg
Jan 18, 2014·Forensic Science, Medicine, and Pathology·Thitika KitpipitPhuvadol Thanakiatkrai
Nov 17, 2015·Analytical Chemistry·Sanjin HosicAbigail N Koppes
Jan 5, 2011·Journal of Zhejiang University. Science. B·Ying-ming ZhengFan Jin
Aug 17, 2021·Journal of Clinical Laboratory Analysis·Xiao-Xia ChenSu-Mei Cao

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