[Direct multiplex-PCR from whole blood for rapid detection of Y chromosome microdeletions].
OBJECTIVE
To establish a rapid and simple method to detect Y chromosome microdeletions directly using whole blood as starting material for multiplex-PCR.


METHODS
Using a self-prepared pHpH-Bufferq, multiplex-PCR amplification was directly carried out from the anticoagulant whole blood sample without DNA extraction step. Twelve sequence tagged sites (STS), namely SY84, SY86, SY127, SY134, SY124, SY132, SY152, SY157, SY239, SY242, SY254 and SY255, in AZFa, AZFb, and AZFc gene regions were detected in 5 different tubes. In order to ensure the validity of the experiments, sex-determining region Y (SRY) and X-linked or Y-linked zinc finger gene (ZFX/Y) were used as internal controls. Furthermore, conventional PCR using genomic DNA extracted from each blood sample was performed in parallel for evaluating the accuracy of the experiments.


RESULTS
A total of 156 male samples were detected, and a normal male sample and a female sample were used as a positive and a negative control respectively. The results showed that 144 samples had no deletion; one sample was AZF-deleted; one sample was AZFb-deleted; seven samples were AZFc-deleted; one sample was both AZFb- and AZFc- deleted; and two samples were all AZFa-, AZFb- and AZFc- deleted. The observed results from two kinds of starting material (whole blood and purified DNA) are completely consistent.


CONCLUSIONS
In our method, PCR amplification was directly carried out from whole blood without any DNA extraction step. So it has the advantages of low cost, simple process, time-saving operation and less cross-contamination. The whole process can be completed within 2 hours. Thus the efficiency of clinical detection is improved greatly.
Bu
Ying
Y
Huadong Research Institute for Medicine and Biotechnology, Nanjing, Jiangsu, 210002 People's Republic of China.
Huang
Huan
H
Wu
Hai-ping
HP
Zhang
Xiao-dan
XD
Zhou
Guo-hua
GH
Cui
Ying-xia
YX
Yao
Bing
B
Lu
Hong-yong
HY
Xiang
Jing-ying
JY
D053713:Azoospermia / Q000235:genetics*; D002478:Cells, Cultured; D002872:Chromosome Deletion; D041322:Chromosomes, Human, Y; D005260:Female; D006801:Humans; D008297:Male; D009845:Oligospermia / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012729:Sex Chromosome Aberrations