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.
OBJECTIVE
To evaluate methods of DNA extraction from single cells for their suitability to amplify and provide a correct diagnosis of target disease genes.


METHODS
Experimental study.


METHODS
University hospital laboratory.


METHODS
Two normal adult male and female blood donors.


METHODS
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).


METHODS
Allele drop out and amplification rate.


RESULTS
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.


CONCLUSIONS
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.
Kim
Sung Ah
SA
The Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University College of Medicine, Seoul, South Korea.
Yoon
Jung Ah
JA
Kang
Moon Joo
MJ
Choi
Young Min
YM
Chae
Soo Jin
SJ
Moon
Shin Yong
SY
D002458:Cell Fractionation / Q000379:methods*; D005591:Chemical Fractionation / Q000379:methods*; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification*; D005787:Gene Frequency / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D019836:Preimplantation Diagnosis / Q000379:methods*
D004247:DNA