Perry D J, Carrell R W
Department of Haematology, University of Cambridge, MRC Centre.
J Clin Pathol. 1992 Feb;45(2):158-60. doi: 10.1136/jcp.45.2.158.
To develop a simple and rapid technique for detecting DNA mutations based on the polymerase chain reaction, followed by electrophoresis, in a novel polymer--Hydrolink D5000--specifically designed to separate double stranded DNA fragments.
Eleven subjects with previously characterised mutations within the antithrombin gene (including single base pair mutations and insertions) and three normal controls were studied. DNA was amplified and one sixth of the PCR product electrophoresed in a 20 cm x 20 cm x 1 mm 100% Hydrolink D5000 gel for two to six hours, followed by staining in ethidium bromide for 20 minutes. The gel was then visualised under ultraviolet light.
After amplification and electrophoresis a single additional band was observed in five out of nine variants in which the mutations involved a single base pair substitution, while two additional bands were seen in four out of nine mutants which arose as a result of a single base pair insertion. No abnormality was detected in two known variants.
This method provides a simple and rapid approach to the screening and detection of mutations at the DNA level which does not involve the use of either toxic reagents or radioisotopes. It may also provide evidence about the type of mutation.
开发一种基于聚合酶链反应并随后进行电泳的简单快速技术，用于检测DNA突变，该技术采用一种专门设计用于分离双链DNA片段的新型聚合物——Hydrolink D5000。
对11名抗凝血酶基因内存在先前已鉴定突变（包括单碱基对突变和插入突变）的受试者以及3名正常对照进行研究。扩增DNA，将六分之一的PCR产物在20 cm×20 cm×1 mm的100% Hydrolink D5000凝胶中电泳两至六小时，然后用溴化乙锭染色20分钟。随后在紫外光下观察凝胶。
扩增和电泳后，在9个涉及单碱基对替换突变的变体中有5个观察到一条额外条带，而在9个由单碱基对插入导致的突变体中有4个观察到两条额外条带。在2个已知变体中未检测到异常。
该方法为DNA水平的突变筛查和检测提供了一种简单快速的方法，既不涉及使用有毒试剂也不涉及使用放射性同位素。它还可能提供有关突变类型的证据。
