A comparison of different lysis buffers to assess allele dropout from single cells for preimplantation genetic diagnosis.
Single cell polymerase chain reaction (PCR) for preimplantation genetic diagnosis (PGD) requires high efficiency and accuracy. Allele dropout (ADO), the random amplification failure of one of the two parental alleles, remains the most significant problem in PCR-based PGD testing since it can result in serious misdiagnosis for compound heterozygous or autosomal dominant conditions. A number of different strategies (including the use of lysis buffers to break down the cell and make the DNA accessible) have been employed to combat ADO with varying degrees of success, yet there is still no consensus among PGD centres over which lysis buffer should be used (ESHRE PGD Consortium, 1999). To address this issue, PCR amplification of three genes (CFTR, LAMA3 and PKP1) at different chromosomal loci was investigated. Single lymphocytes from individuals heterozygous for mutations within each of the three genes were collected and lysed in either alkaline lysis buffer (ALB) or proteinase K/SDS lysis buffer (PK). PCR amplification efficiencies were comparable between alkaline lysis and proteinase K lysis for PCR products spanning each of the three mutated loci (DeltaF508 in CFTR 90% vs 88%; R650X in LAMA3 82% vs 78%; and Y71X in PKP1 91% vs 87%). While there was no appreciable difference between ADO rates between the two lysis buffers for the LAMA3 PCR product (25% vs 26%), there were significant differences in ADO rates between ALB and PK for the CFTR PCR product (0% vs 23%) and the PKP1 PCR product (8% vs 56%). Based on these results, we are currently using ALB in preference to PK/SDS buffer for the lysis of cells in clinical PGD.
Thornhill
A R
AR
Guy's and St Thomas' Centre for Preimplantation Genetic Diagnosis, London SE1 7EH, UK. thornhill.alan@mayo.edu
McGrath
J A
JA
Eady
R A
RA
Braude
P R
PR
Handyside
A H
AH
D000483:Alleles; D002021:Buffers; D017931:DNA Primers; D005260:Female; D006801:Humans; D008214:Lymphocytes; D008297:Male; D009154:Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D011247:Pregnancy; D019836:Preimplantation Diagnosis / Q000379:methods
D002021:Buffers; D017931:DNA Primers