Mismatch repair detection
AssigneesThe Board of Trustees of the Leland Stanford Junior University, The Regents of the University of California
InventorsDavid R. Cox, Malek Faham, Siamak Baharloo
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Abstract
Mismatch Repair Detection (MRD), a novel method for DNA-variation detection, utilizes bacteria to detect mismatches by a change in expression of a marker gene. DNA fragments to be screened for variation are cloned into two MRD plasmids, and bacteria are transformed with heteroduplexes of these constructs. Resulting colonies express the marker gene in the absence of a mismatch, and lack expression in the presence of a mismatch. MRD is capable of detecting a single mismatch within 10 kb of DNA. In addition, MRD can analyze many fragments simultaneously, offering a powerful method for high-throughput genotyping and mutation detection.
Claims26
A method of detecting a mismatch between two substantially complementary DNA sequences of interest, the method comprising:
- annealing a first strand comprising a gene encoding a detectable marker and an origin of replication active in a bacterial host cell, wherein said first strand is characterized by the absence of methyl adenine;
- and a substantially complementary second strand, wherein said gene encoding said detectable marker further comprises an inactivating insertion, deletion or substitution of at least about 5 nt, and characterized by the presence of methyl adenine at GATC sites;
- ligating said first strand to a first DNA sequence of interest of from about 20 to 104 nucleotides in length;
- ligating said substantially complementary second strand to a second DNA sequence of interest substantially complementary to said first DNA sequence, and suspected of having at least one mismatch of from 1 to 4 contiguous nucleotides in length;
- circularizing said ligated first and second strands to provide a circular double stranded DNA molecule;
- transforming a bacterial host having an active methyl mismatch repair system with said circular double stranded DNA molecule;
- detecting the presence of bacterial transformants not expressing said detectable marker;
- wherein the presence of transformants not expressing said detectable marker is indicative of a mismatch between said first DNA sequence of interest and said second DNA sequence of interest.
A method of detecting a mismatch between two substantially complementary DNA sequences of interest, the method comprising:
- annealing in a hybridization reaction:
- a vector comprising a gene encoding a detectable marker, an origin of replication active in a bacterial host cell, and a sequence of interest, wherein said vector lacks methyl adenine;
- and
- a substantially complementary second vector, wherein said gene encoding said detectable marker further comprises an inactivating insertion, deletion or substitution of at least 5 nt, wherein said vector comprises methyl adenine at GATC sites;
- and
- a test sequence substantially complementary to said sequence of interest, and suspected of having at least one mismatch of from 1 to 4 contiguous
- nucleotides in length;
- ligating the product of said annealing step;
- transforming a bacterial host having an active methyl mismatch repair system with the product of said ligating step;
- determining the presence of bacterial transformants expressing or not expressing said detectable marker;
- wherein the presence of transformants not expressing said detectable marker is indicative of a mismatch between said sequence of interest and said test sequence.
A kit for identifying the presence of a mismatch between two substantially complementary DNA sequences, the kit comprising:
- a first DNA vector comprising a gene encoding a detectable marker, an origin of replication active in a bacterial host cell, and a sequence(s) of interest, wherein said vector lacks methyl adenine;
- and
- a substantially complementary second vector, wherein said gene encoding said detectable marker further comprises an inactivating insertion, deletion or substitution of at least 5 nt, wherein said vector comprises methyl adenine at GATC sites
Dependencies are inferred from the claim text.