[Study of single cell PCR for HLA typing].
OBJECTIVE
To apply the single cell nested multiplex polymerase chain reaction (PCR) to HLA typing, and analyze the influence factors on the amplification results.


METHODS
Single cell DNA templates were prepared with different methods. The exon 2, 3 and intron 2 of HLA-A, B, and exon 2 of DRBI were amplified using multiplex PCR. The second round of SSP-PCR HLA typing was carried out according to the large scale routine HLA typing results.


RESULTS
Enzyme lysis method was the most efficient procedure for preparing the single cell DNA template, with a success rate (SR) of 93.3%, while the SRs of alkali lysis and freezing-thaw lysis methods were 83.3% and 73.3%, respectively. The second round amplification using enzyme lysis and SSP-PCR in 20 samples obtained a 95% success rate and a 15% allele drop out rate. The time for performing the whole procedure was less than 6 hours.


CONCLUSIONS
The modified nested multiplex PCR technique is efficient for single cell HLA typing and might be applied to clinical preimplantation genetic diagnosis.
Li
Dong
D
Lab of Cryomedicine, Qilu Hospital, Shandong University, Jinan 250012, China.
Zhang
Le-ling
LL
Ju
Xiu-li
XL
Hou
Huai-shui
HS
Shi
Qing
Q
Shen
Bai-jun
BJ
D006650:Histocompatibility Testing / Q000379:methods*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*