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Mismatch repair detection

Inactive Publication Date: 2005-10-27
THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIV +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0018] The host repair system is activated by a mismatch in the sequence of interest, and will then “co-repair” the marker gene, to produce an inactive, double stranded copy. When the two strands of the sequence of interest are a perfect match, the marker gene is not altered, and the transformed bacteria will produce active marker. Where a mismatch i

Problems solved by technology

Since these methods are riot designed to screen many genes simultaneously, their usefulness has been limited to testing a handful of candidate genes.
However, mapping of diseases with a complex mode of inheritance has been less successful.

Method used

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Examples

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example 1

[0093] Two pUC-derived plasmids, the A plasmid (pMF200) and the I plasmid (pMF100), are employed in the MRD procedure. A map of the plasmids is shown in FIG. 3. These plasmids are identical except for a five bp insertion into the Lac Zα gene of pMF100. This insertion results in white colonies when bacteria transformed with the I plasmid are grown on LB plates supplemented with indolyl-β-D-galactoside (Xgal) and isopropyl-β-D-thiogalactoside (IPTG). In contrast, bacteria transformed with the A plasmid result in blue colonies when grown under these conditions.

[0094] The initial step of the MRD procedure consists of cloning one of two DNA fragments to be screened for differences into the A plasmid and cloning of the second DNA fragment into the I plasmid. The A plasmid construct is then transformed into a dam− bacterial strain, resulting in a completely unmethylated plasmid while the I plasmid construct is transformed into a dam+ bacterial strain, resulting in a fully methylated plasm...

example 2

[0098] As an initial test of the sensitivity and specificity of the MRD system, a single nucleotide mismatch was detected in a 550 base pair DNA fragment derived from the promoter of the mouse beta globin gene (Myers et al. (1985) Science 229:242). MRD was used to compare this DNA fragment, which contains a T at position −49 (relative to the functional transcription start site of the gene) with a second DNA fragment identical in sequence except for at C position −49. The mismatch was located about 700 base pairs from the five nucleotide Lac Zα loop in the vector. Comparison of the two DNA molecules by using MRD resulted in 90% white colonies. In contrast, comparison of the same two DNA molecules with no mismatch (−49T / −49T), resulted in only 7% white colonies. The data is shown in Table 1.

TABLE 1Detection of Known Point Mutations using MRDSequenceDistance from% White (Inactive)Variation*Fragment Size{circumflex over ( )}Loop{circumflex over ( )}Colonies@None10.55N / A7G_C10.550.789A...

example 3

[0103] MRD was used to detect unknown mutations in genomic DNA fragments generated by the polymerase chain reaction (PCR). PCR is a practical method for obtaining a particular genomic DNA fragment of interest from many different individuals. Recent advances in PCR technology makes it possible to isolate DNA products greater than 10 kb in length (Barnes (1994) P.N.A.S. 91:2216; Cheng et al. (1994) P.N.A.S. 91:5695). However, the introduction of errors during the PCR reaction severely limits the use of individual cloned PCR products. In an effort to overcome this limitation, an MRD protocol was developed to enrich for molecules that are free of PCR-induced errors. Following this “cleaning” protocol, the cloned PCR products can be compared for DNA sequence differences by using the MRD procedure described above.

[0104] The basic principle underlying the MRD cleaning protocol is the fact that any single PCR-induced mutation will make up a very small fraction of all the molecules generate...

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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.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation-in-part of U.S. patent application Ser. No. 08 / 713,751, filed Sep. 13, 1996; which claims priority to U.S. Provisional Patent Application No. 60 / 004,664, filed Oct. 2, 1995.GOVERNMENT GRANTS [0002] This invention was made with government support under Contract Nos. HD 24610 07-10 and 5T32GM07618 awarded by the National Institutes of Health. The Government has certain rights in this invention.INTRODUCTION [0003] Background [0004] The detection of mutations in genomic DNA plays a critical role in efforts to elucidate the genetic basis of human disease. For many types of genetic screening and analysis, knowledge of the presence of a mutated copy of a gene is essential. Such information may be used in prenatal and other genetic testing, as well as analysis of tumor cells and other somatic mutations. For many genes, there are a number of different mutations that can affect function. [0005] Common diseases s...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74C12Q1/68
CPCC12Q1/6827C12Q2600/156C12Q1/6897
Inventor COX, DAVIDFAHAM, MALEKBAHARLOO, SIAMAK
Owner THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIV
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