摘要
目的为适应大规模基因组扫描技术的需要，建立一种以ＴａｑＳｔａｒｔ抗体为酶保护剂、退火温度在一定范围内递降的多重ＰＣＲ体系。方法在５μｌ的总体积内，加５～１５对ＰＣＲ引物，用不同颜色的荧光标记（或相同荧光标记但其扩增片段相差５０ｂｐ以上），多个微卫星位点可同时得到扩增，并可通过ＤＮＡ测序仪进行电泳及数据分析。结果多个微卫星位点在５μｌ的体积内同时得到扩增。电泳条带图象清晰，获得了满意的基因分型结果。结论多重ＰＣＲ反应高产量、低成本，适用于人类基因组计划，尤其是基因作图、基因定位。
Objective To develop a high output and low cost multiplex PCR approach to facilitating large scale gene scan and gene typing with microsatellite markers.Methods 5-15 pairs of primers of microsatellite loci were added in one single tube with 5μl reation volume,containing 10mol/L Tris HCl (pH8.3),50mmol/L KCl,0.1mg/ml gelatin,3.0mmol/L MgCl 2,200μmol/L dNTPs (each),0.25U Taq DNA polymerase,Taq Start antibody and DNA templates.PCR thermocycles were carried out on Perkin Elmer Gene Amp PCR System 9600 with touch down algorithm(the annealing temperature was decreased by 0.5℃ in each cycle) to meet the different demands of different primers.Results Up to 10 15 loci were labeled by different fluorescents or by the same fluorescent,but their PCR amplification fragments did not overlap in size.Almost all target microsatellite loci were successfully co amplified in a 5 microliter reaction volume, electrophoresized and analyzed in a single gel lane with Perkin Elmer ABI 373A DNA sequencer,and 9 microsatellite loci were well genotyped.Conclusion This high output and low cost genotyping protocol is applicable to gene mapping,human evolution and forensic analysis.
出处
《中华医学遗传学杂志》
EI
CAS
CSCD
北大核心
1998年第2期104-107,共4页
Chinese Journal of Medical Genetics
基金
国家自然科学基金
上海市科委资助
上海市教委曙光计划及卫生部科研基金
