A genotypic mutation system measuring mutations in restriction recognition sequences.

Details

Ressource 1Download: BIB_7B162C1AAF5E.P001.pdf (1528.40 [Ko])
State: Public
Version: Final published version
Serval ID
serval:BIB_7B162C1AAF5E
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
A genotypic mutation system measuring mutations in restriction recognition sequences.
Journal
Nucleic Acids Research
Author(s)
Felley-Bosco E., Pourzand C., Zijlstra J., Amstad P., Cerutti P.
ISSN
0305-1048 (Print)
ISSN-L
0305-1048
Publication state
Published
Issued date
1991
Peer-reviewed
Oui
Volume
19
Number
11
Pages
2913-2919
Language
english
Notes
Publication types: Journal Article ; Research Support, Non-U.S. Gov't Publication Status: ppublish
Abstract
The RFLP/PCR approach (restriction fragment length polymorphism/polymerase chain reaction) to genotypic mutation analysis described here measures mutations in restriction recognition sequences. Wild-type DNA is restricted before the resistant, mutated sequences are amplified by PCR and cloned. We tested the capacity of this experimental design to isolate a few copies of a mutated sequence of the human c-Ha-ras1 gene from a large excess of wild-type DNA. For this purpose we constructed a 272 bp fragment with 2 mutations in the PvuII recognition sequence 1727-1732 and studied the rescue by RFLP/PCR of a few copies of this 'PvuII mutant standard'. Following amplification with Taq-polymerase and cloning into lambda gt10, plaques containing wild-type sequence, PvuII mutant standard or Taq-polymerase induced bp changes were quantitated by hybridization with specific oligonucleotide probes. Our results indicate that 10 PvuII mutant standard copies can be rescued from 10(8) to 10(9) wild-type sequences. Taq polymerase errors originating from unrestricted, residual wild-type DNA were sequence dependent and consisted mostly of transversions originating at G.C bp. In contrast to a doubly mutated 'standard' the capacity to rescue single bp mutations by RFLP/PCR is limited by Taq-polymerase errors. Therefore, we assessed the capacity of our protocol to isolate a G to T transversion mutation at base pair 1698 of the MspI-site 1695-1698 of the c-Ha-ras1 gene from excess wild-type ras1 DNA. We found that 100 copies of the mutated ras1 fragment could be readily rescued from 10(8) copies of wild-type DNA.
Keywords
Base Sequence, DNA-Directed DNA Polymerase, Genotype, Molecular Sequence Data, Mutation, Nucleic Acid Hybridization, Polymerase Chain Reaction, Polymorphism, Restriction Fragment Length, Taq Polymerase
Pubmed
Web of science
Create date
14/01/2016 18:57
Last modification date
20/08/2019 14:37
Usage data