[HLA-A site genotyping on single blastomeres is studied by nest-PCR-SSP method].
OBJECTIVE
To assess the accuracy and reliability of the nest-PCR-sequence specific primer(SSP) method in HLA-A site genotyping of single blastomeres retrieved from human pre-implantation embryos.


METHODS
By nest PCR on HLA-A exon 2, the success rate of first-round amplification was estimated for single blastomeres. Based on the first-round amplification, the HLA-A genotype of every single blastomeres was analyzed by commercially available PCR-SSP kits.


RESULTS
The amplification of HLA-A exon 2 were performed to 120 blasotmeres retrieved from in vitro fertilization(IVF) surplus embryos donated by 10 couples. The average success rate of family 1-5 and 6-10 was 78.2%(43/55) and 93.8%(61/65), respectively. And 86.7%(104/120) in total. Eighty blastomeres were further tested by nest-PCR-SSP, among which 11 blastomeres failed to HLA-A exon 2 amplification and then failed to genotyping while the other 69 blastomeres succeed in HLA-A exon 2 amplification and succeed in genotyping. Except for 6 blastomeres that were uncertain for allele lost because of parents' homozygosity, the left 63 blastomeres had accurate HLA genotyping. Among these 63 blastomeres, 59 blastomeres had genotypes confirmed from their parents(93.6%), 3 blastomeres lost one of parents' alleles(4.8%), and only one blastomere had two more than parents' alleles(1.6%).


CONCLUSIONS
The above research results indicated that based on the successful first round amplification of single blastomeres, nest-PCR-SSP strategy offers a convenient and reliable option for HLA genotyping on single blastomeres, which is a key process in pre-selecting HLA-identical sibling for allogeneic cord blood cell transplantation.
Xu
Bing-sen
BS
Department of Reproductive Medicine Centre, First Affiliated Hospital, Wenzhou Medical College, Wenzhou, Zhejiang, 325000 P.R.China.
Hu
Yong-wu
YW
Huang
Xue-feng
XF
Lin
Jin-ju
JJ
Zhou
Yin
Y
Ye
Bi-lu
BL
Xu
Li-xin
LX
Xu
Kan-pu
KP
Yang
Huan-ming
HM
D001483:Base Sequence; D001757:Blastomeres / Q000378:metabolism*; D004247:DNA / Q000032:analysis; D016172:DNA Fingerprinting / Q000379:methods; D004252:DNA Mutational Analysis; D005260:Female; D006680:HLA Antigens / Q000032:analysis; D015234:HLA-A Antigens / Q000032:analysis / Q000235:genetics*; D015395:Histocompatibility Antigens Class I / Q000032:analysis / Q000235:genetics; D006801:Humans; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods*; D012847:Single Person
D006680:HLA Antigens; D015234:HLA-A Antigens; D015395:Histocompatibility Antigens Class I; D004247:DNA