摘要
目的(1)研究消除PCR反应非特异性扩增新技术。(2)研究全血直接PCR能否用于葡萄糖6-磷酸脱氢酶基因突变检测。方法(1)将新鲜全血混合基因组DNA进行PCR扩增,看全血能否抑制基因组DNA非特异性扩增,将新鲜人血清混合基因组DNA进行PCR扩增,看新鲜人血清能否抑制基因组DNA非特异性扩增。(2)将1μl全血加入PCR反应液,98℃变性后转入PCR循环,使用Taq(Fermentas)扩增G6PD基因外显子3-4,取扩增产物50μl进行DNA双向自动测序,使用ClustalX软件将测序结果与基因库中标准G6PD基因外显子3-4序列进行比对分析。结果(1)发现新鲜全血能抑制基因组DNA非特异性扩增,进一步研究发现是新鲜人血清抑制了基因组DNA非特异性扩增。(2)全血直接PCR加上DNA测序找到一例G6PD突变g.13503A>G。结论(1)新鲜人血清中存在PCR非特异性扩增抑制因子。(2)使用Taq DNA聚合酶(Fermentas)能进行全血直接PCR扩增,加上DNA测序可用于G6PD基因突变研究。
Objective ： （ 1 ） To investigate the new technology of eliminating PCR non - specific amplification. （2） To investi- gate whether whole blood direct PCR can be used in G6PD gene mutational detection. Methods： （ 1 ） To mix fresh whole blood with genomic DNA to proceed PCR amplification, to detect whether whole blood can inhibit genomic DNA non- specific amplification, to mix fresh human serum with genomic DNA to proceed PCR amplification, to detect whether fresh human serum can inhibit genomic DNA non - specific amplification. （2） To add 1μl whole blood into PCR liquid, after denaturing in 98% then turning to PCR cycling, using Taq （Fermentas） to ampli（y G6PD gene exort3 -4, using 50μl PCR products to proceed DNA two - direction autosequencing, using ClustalX software to compdre the PCR product sequence with standard G6PD exon3 - 4 sequence in gene bank. Results ： （ 1 ） Discovering that fresh whole blood can inhibit genomic DNA non - specific amplification, additional investigation show that it is fresh human serum which inhibit genomic DNA non- specific amplification. （2） Using whole blood direct PCR and DNA sequencing the author find a G6PD mutation g. 13503A 〉 G. Conclusion： （ 1 ） There is PCR non - specific amplification inhibition factor in fresh human serum. （2） Whole blood direct PCR can be successfμlly proceed by Taq DNA polymerase （Fermentas）, plus DNA sequen- cing it can be used in G6PD gene mutational research.
出处
《中国优生与遗传杂志》
2010年第3期22-25,共4页
Chinese Journal of Birth Health & Heredity
基金
国家自然科学基金(30571025)
国家科技部基金(2006BAI05A08)
