目的以全血为起始模板直接进行PCR多重扩增，建立一种快速、简便的Y染色体微缺失检测方法。方法用作者自行研制的“HpH-Buffer”，以不经基因组DNA提取的抗凝全血为模板直接进行多重PCR扩增。分别在5管中检测无精症因子（azoospermia factor，AZF）区域的a区、b区和c区共12个序列标签位点（sequence tagged sites，SIS）。为保证方法有效性，加做Y染色体性别决定区（sex-determining region Y，SRY）和X／Y连锁锌指蛋白基因（X-linked or Y-linked zinc fingergene，ZFX／Y）作为内控。同时，为考察方法的准确性，对每例血液样本提取基因组DNA平行实验。结果共检测了156例男性血液样本，每组实验均加做阳性对照（正常已生育男性样本）和阴性对照（正常女性样本），以保证实验有效性。结果表明，156例样本中有144例无缺失，AZFa区缺失1例，AZFb区缺失1例，AZFc区缺失7例，AZFb和AZFc区缺失1例，AZFa、AZFb和AZFc区全缺失2例。以全血为模板和以基因组DNA为模板进行扩增所得到的实验结果完全一致。结论所建立的方法省略了DNA提取这一繁琐的步骤，减少了PCR实验过程中可能出现的污染，缩短了操作时间，节约了实验成本。可在2小时内完成所有检测过程，有效地提高了临床检测效率。“HpH-Buffer”的试剂成本很低，全部检测成本与普通PCR扩增基本相同。
To detect multiplex single nucleotide polymorphisms(SNPs)simultaneously,a new method was established by combining ALM-ASA with microfabricated CE-chip.Taking the CYP2D6 gene as an example,six SNPs,100C>T(P34S),1707T>del(frameshift),1758G>T(stop codon),2470T>C,2549A>del(frameshift)and 2613AGA>del(K281del),were typed by four steps consisting of preamplification,digestion and ligation,allele-specific amplification,and amplicon separation by chip-CE.The genotyping results of 20 different genome samples by 6-plexed ALM-ASA were completely consistent with those obtained by polymerase chain reaction-restriction fragment length polymorphism analysis(PCR-RFLP),indicating that the multiplex approach established in this study was accurate and inexpensive.As the small reagent consumption by CE-chip device,a low cost for SNP typing was achieved together with the multiplex PCR technology proposed in this report.Neither modification of microchip channels nor clean-up process of PCR products was required;this greatly shortens the whole time for SNP typing.
