Alternative PCR method for diagnosis of mutation causing activated protein C resistant Gln506-factor V.
Alternative PCR method for diagnosis of mutation causing activated protein C resistant Gln506-factor V.
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用于诊断导致活化蛋白 C 抗性 Gln506 因子 V 突变的替代 PCR 方法。
DOI：
10.1016/0049-3848(95)00197-y
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发表时间：
1995
影响因子：
7.5
通讯作者：
Griffin,JH
中科院分区：
文献类型：
--
作者：
Greengard,JS;Xu,X;Gandrille,S;Griffin,JH
用Mnl I酶切(2)分型后的基因组DNA用不同的引物进行PCR扩增。这些序列包括FV7(3，8)(图1，通道L-3)或FV7X(SCATGATCAGAGCAG‘ITCAAC3’，nt1500-1519)(图1，通道5-7)和FV506tst2(5‘TTACTTCAAGGACAAAATACCTGTAaagCT3’，nt1692-1701加上20个核苷酸的内含子序列)。FV506tst2从外显子10延伸到内含子10(9，10)。小写碱基表示当使用的模板基因组DNA包含因子V基因时，因子V(9，10)的外显子10的序列发生了变化，该外显子在扩增子中引入了Hind III位点，其中A位于1691处，这会导致精氨酸-506到谷氨酰胺的突变。含有G的正常DNA在nt1691处没有产生这样的Hind III位点。进行30个循环的PCR扩增(94℃下1分钟，60℃下2分钟，72℃下3分钟)，50~1反应包括1pg基因组DNA和50pmoL每个引物，每个dNTP 200 PM，1.5 mM氯化镁，50 mM氯化钾，10 mM Tris-HCl(pH 9.0)，0.1%Triton X-100和1单位Taq聚合酶(Promega，Madison，WI)。10~1反应混合物经Hind III(5个单位)消化，产物在琼脂糖凝胶上溶解(图1)。
Genomic DNA that had been genotyped using Mnl I digestion (2) was subjected to PCR amplification with various primers. These included either FV7 (3, 8)(Fig. 1, lanes l-3) or FV7X (SCATGATCAGAGCAG’ITCAAC3’, nt 1500-1519)(Fig. 1, lanes 5-7) and FV506tst2 (5’TTACTTCAAGGACAAAATACCTGTAaagCT3’, nt 1692-1701 plus 20 bp of intronic sequence). The FV506tst2 primer extends from exon 10 into intron 10 (9, 10). The lower case bases indicate alterations from the sequence of exon 10 of factor V (9, 10) which introduce a Hind III site into the amplicons when the template genomic DNA that is used contains the factor V gene with A at nt 1691 which causes the Arginine-506 to Glutamine mutation. No such Hind III site is generated for normal DNA containing G at nt 1691. 30 cycles of PCR amplification (1 min at 94” C, 2 min at 6o” C, 3 min at 72 C) were performed in a 50~ 1 reaction containing 1 pg genomic DNA and 50 pmol of each primer, 200 PM of each dNTP, 1.5 mM MgCl,, 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, and 1 unit of Taq polymerase (Promega, Madison, WI). 10~ 1 of the reaction mixture were subjected to Hind III (5 units) digestion and the products were resolved on an agarose gel (Fig. 1).