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Mutation detection by cycle sequencing

Item Type:Article
Title:Mutation detection by cycle sequencing
Creators Name:Thierfelder, L.
Abstract:Candidate genes are screened for mutations by a DNA sequencing procedure known as cycle sequencing. First, a segment of the candidate gene is PCR amplified from the genomic DNA of an affected individual. The PCR product is then subjected to multiple rounds of further amplification in a thermal cycler using a heat-stable DNA polymerase in the presence of different dideoxynucleotides and a radiolabeled primer. The resulting 32P-labeled sequence reaction products are fractionated on a denaturing polyacrylamide gel and visualized by autoradiography. DNA segments on the order of 200 bp from 10 to 30 individuals can be screened on each gel. Cycle sequencing eliminates the need to subclone genomic fragments or PCR products, which makes it a much simpler method than conventional sequencing for identifying mutations.
Keywords:Autoradiography, DNA, DNA Mutational Analysis, DNA Primers, Polyacrylamide Gel Electrophoresis, Medical Genetics, Mutation, Phosphorus Radioisotopes, Polymerase Chain Reaction, DNA Sequence Analysis
Source:Current Protocols in Human Genetics
Volume:Chapter 7
Number:Unit 7.7
Date:May 2001
Official Publication:https://doi.org/10.1002/0471142905.hg0707s16
PubMed:View item in PubMed

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