A simple method for detection of factor V R506Q (Leiden) mutation in dried blood spots.
A simple method for detection of factor V R506Q (Leiden) mutation in dried blood spots.
复制标题
一种检测干血斑中 V 因子 R506Q (Leiden) 突变的简单方法。
DOI：
10.1016/s0009-8981(99)00024-8
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Ganev,VS
中科院分区：
文献类型：
--
作者：
Russeva,MG;Janakiev,PJ;Kirov,SA;Paskaleva,ID;Kremensky,IM;Penner,JA;Hassouna,HS;Ganev,VS
2. Materials and methodsDNA-samples from whole venous blood samples were extracted [5] and genotyped by MnlI RFLP-analysis. The PCR mix (20 μl) contains: 100 ng DNA, 10 pmol each primer (F-5′ CAGGCAGGAACAACACCATG3′; and R-5′ CTTGAAGGAAATGCCCCATTA3′), 100 μmol/l each dNTPs, 0.25 U Taq polymerase in buffer XL3, Boehringer (50 mmol/l Tris–HCl, pH 9.2, 16 mmol/l (NH 4) 2 S0 4, 2.25 mmol/l MgCl 2, 2%(v/v) DMSO, 0.1% Tween® 20). PCR-amplification was performed (Robocycler Gradient 40, Stratagene) at 1 denaturation step (94 C, 2.30 min), followed by 35 cycles of: 95 C, 25 s; 55 C, 50 s; 72 C, 30 s. The PCR-products separated by Sephacryl S-300 (17 μl) were digested in a final volume of 20 μl with 2.5 U MnlI (1 h, 37 C), buffer 1 (New England Biolabs) 10 mmol/l Bis Tris Propane–HCl, 10 mmol/l MgCl 2, 1 mmol/l DTT, pH 7.0. Ten microliters of each restriction reaction products were separated on 7× 10 cm PAG (100 g/l; 19: 1) in TBE, 15 V/lane, 70 min. The gels were stained by the silver technique and vacuum dried.
