Isolation of segments of homologous genes with only one conserved amino acid region via PCR

Open Access

15 January 2001

journal article
research article
Published by Oxford University Press (OUP) in Nucleic Acids Research

Vol. 29 (2) , 8e-8
https://doi.org/10.1093/nar/29.2.e8

Abstract

We present a method which allows the isolation of fragments from genes coding for homologous proteins via PCR when only one block of conserved amino acids is available. Sets of degenerated primers are defined by reverse translation of the conserved amino acids such that each set contains not more than 128 different sequences. The second primer binding site is provided by a special cassette that is designed such that it does not allow binding of the second primer prior to being copied by DNA synthesis. The cassette is ligated to partially-digested chromosomal DNA. The second primer is biotinylated to allow elimination of PCR products carrying degenerated primers on both sides via streptavidin binding. Fragments obtained after amplification and enrichment are cloned and sequenced. The feasibility of this method was demonstrated in a model experiment, where degenerated primers were deduced from six conserved amino acids within the family of homologs to the Escherichia coli Vsr protein.

Keywords

This publication has 11 references indexed in Scilit:

Studies on transformation of Escherichia coli with plasmids
Published by Elsevier ,2006
The msh2 Gene of Schizosaccharomyces pombe Is Involved in Mismatch Repair, Mating-Type Switching, and Meiotic Chromosome Organization
Molecular and Cellular Biology, 1999
Cloning and expression of the NaeI restriction endonuclease-encoding gene and sequence analysis of the NaeI restriction-modification system
Gene, 1995
Identification of the XorII methyltransferase gene and a vsr homolog from Xanthomonas oryzae pv. oryzae
Molecular Genetics and Genomics, 1994
Organization and sequence of the HpaII restriction-modification system and adjacent genes
Gene, 1994
The M·AluI DNA-(cytosineC5)-methyltransferase has an unusually large, partially dispensable, variable region
Nucleic Acids Research, 1993
The vsr gene product of E. coli K-12 is a strand- and sequence-specific DNA mismatch endonuclease
Nature, 1991
Vectorette PCR: a novel approach to genomic walking.
Genome Research, 1991
Neucleotide sequence of thedcmlocus ofEsherichia coliK12
Nucleic Acids Research, 1989
Nudeotide sequence of the BsuRI restriction-modification system
Nucleic Acids Research, 1985