[Sex determination of human preimplantation embryo using nested polymerase chain reaction].
OBJECTIVE
Using nested polymerase chain reaction (PCR) to perform preimplantation gender diagnosis.


METHODS
One (or two) lymphocyte and blastomere (n=50/group) were collected and prepared under the following conditions: (1) water only (H(2)O); (2) freeze-thaw liquid nitrogen, then boiling; (3) potassium hydroxide/dithiotheriol, heated to 65 degree centigrade, followed by acid neutralization (KOH). Cells were analyzed by PCR using nested primers amplification with amelogenin gene.


RESULTS
The amplification rate and allele dropout (ADO) rate for male lymphocytes by the three methods were 83%, 94%, 95% and 24%, 12%, 4%, respectively. Using two cells per reaction did not increase the amplification rate for the KOH method.


CONCLUSIONS
The KOH method for DNA preparation is superior to the other methods evaluated. Dual blastomere biopsy and independent blastomere analysis may improve preimplantation diagnostic reliability.
Jiao
Zhe-xu
ZX
Reproductive Medical Center, First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, Guangdong, PR China. jiaozexu@yahoo.com
Zhuang
Guang-lun
GL
Zhou
Can-quan
CQ
Zhang
Min-fang
MF
Li
Li-lin
LL
D053523:Amelogenin; D001755:Blastocyst / Q000166:cytology / Q000378:metabolism*; D001757:Blastomeres / Q000166:cytology / Q000378:metabolism; D004247:DNA / Q000235:genetics; D003746:Dental Enamel Proteins / Q000235:genetics; D005260:Female; D005838:Genotype; D006801:Humans; D008214:Lymphocytes / Q000166:cytology / Q000378:metabolism; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods; D012732:Sex Determination Analysis / Q000379:methods*
D053523:Amelogenin; D003746:Dental Enamel Proteins; D004247:DNA