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Mono-allelic mutation analysis for identifying germline mutations

a technology of monoallelic mutations and germline mutations, applied in the field of monoallelic mutation analysis for identifying germline mutations, can solve the problems of presenting a continuing challenge in the sensitive and specific dissection of germline mutations

Inactive Publication Date: 2003-07-01
THE JOHN HOPKINS UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

These and other objects of the invention are provided by one or more of the embodiments described below. In one embodiment a method of detecting mutations in a gene of interest on a chromosome of a human is provided. The method comprises the steps of: obtaining cells of the human; fusing said cells to rodent cell recipients to form a human-rodent cell hybrid; testing said human-rodent cell hybrid to confirm the presence of said chromosome of the human in said hybrid; testing said hybrid which contains said chromosome to detect a protein product of said gene, absence of said protein product or diminished amounts of said protein product indicating the presence of a mutation in the gene of interest of the human.
The present invention thus provides the art with a useful diagnostic tool in the evaluation of inherited diseases.

Problems solved by technology

Dissection of germline mutations in a sensitive and specific manner presents a continuing challenge.

Method used

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  • Mono-allelic mutation analysis for identifying germline mutations
  • Mono-allelic mutation analysis for identifying germline mutations
  • Mono-allelic mutation analysis for identifying germline mutations

Examples

Experimental program
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example 1

This example demonstrates the development of a simple scheme for producing somatic cell hybrids from routinely available clinical samples such as peripheral blood lymphocytes (PBL).

Cells. The Urd-A cell line.sup.16 was grown in F-12 supplemented with 10%(v / v) dialyzed fetal calf serum and 30 mM uridine at 37.degree. C. The UCW56 cell line.sup.17 was grown in Dulbeco's Modified Eagles medium (DMEM) supplemented with 10%(v / v) fetal calf serum (FCS) at 32.degree. C. Lymphoblastoid lines were grown in RPMI 1640 supplemented with 10%(v / v) FCS. Peripheral blood lymphocytes (PBL) were isolated with Histopaque 1077 (Sigma) according to the manufacturer's protocol. The cells were used immediately for fusions, or resuspended in RPMI 1640 / 10% FCS, with or without 10 mg / ml PHA and 50 U / ml IL2, and incubated at 37.degree. C. overnight. Alternatively, Histopaque isolated PBL were frozen at -80.degree. C. in RPMI 1640 / 10% FCS containing 10% DMSO.

Isolation of somatic cell hybrids. The following pro...

example 2

This example demonstrates that single alleles were isolated in the somatic cell hybrid clones.

DNA analysis. Confluent cells in 48 well plates were washed with Hank's balanced salt solution, and 100 ml of proteinase K (100 mg / ml) in TE 10 (10 mM Tris [pH 8.0], 1 mM EDTA) were added to each well. The plates were incubated for 2 hr at 58.degree. C. The liquid was transferred to 1.5 ml tubes and boiled for 5 minutes. Cells growing in suspension were collected by centrifugation, and DNA was prepared similarly, except that the 58.degree. C. incubation was performed in 1.5 ml tubes. Two ml of each DNA sample were used for each PCR reaction. The microsatellite markers used for chromosome 2 were YH5 and CA7.sup.4,18, and for chromosome 5 were D5S82.sup.19 and LSCA.sup.20. The PCR reactions were carried out in 96 well plates with end-labelled primers in 10 ml reactions in PCR Master Mix (Boehringer Mannheim), by heating to 95.degree. C. for 30 seconds, 50.degree. C for 1 minute, 70.degree. C....

example 3

This example demonstrates that mutations that result in greatly reduced expression of the full length gene product are detectable by Western blotting of hybrid proteins using appropriate antibodies.

Western blot analysis. Cells were resuspended in buffer composed of 0.0625M Tris (pH 6.8), 5% beta-mercaptoethanol, 2% SDS, 10% glycerol, / 0.025% bromophenol blue, and quantitated according to previously published methods.sup.22. Western blot analysis for APC was carried out according to ref. 23. Briefly, proteins were separated by electrophoresis in 3% low melting point agarose gels and transferred to polyvinyldifluoride membranes (Immobilon; Millipore) by capillary action in TBS (100 mM Tris, 150 mM NaCl) containing 0.05% SDS. These membranes were treated as described below. For hMSH2, proteins were separated on 10% polyacrylamide / SDS gels, and transferred to polyvinyldifluoride membranes for 1 hr in 40 mM glycine, 48 mM Tris, 0.0375% SDS buffer using a semi-dry electroblotter (ISS). Th...

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Abstract

A diagnostic strategy for detection of inherited diseases caused by germline mutations is based on somatic cell hybridization. Each allele of a human gene involved in the inherited disease is isolated in a somatic cell hybrid. The products of the isolated human allele are then observed in the absence of the other allele of the human.

Description

BACKGROUND OF THE INVENTIONDissection of germline mutations in a sensitive and specific manner presents a continuing challenge. In dominantly inherited diseases, mutations occur in only one allele and are often masked by the normal allele. For example, it is estimated that 20-40% of both APC and hMSH2 mutations are difficult or impossible to detect with standard techniques based on PCR analysis of genomic DNA or RNA transcripts.sup.6-10. Thus there is a need in the art for a technique which is relatively simple to perform and which will detect a broad spectrum of mutations in genes of clinical interest.SUMMARY OF THE INVENTIONIt is an object of the invention to provide a method for detecting mutations in the germline.It is another object of the invention to provide a method for detecting mutations which are not detected by standard methods.It is yet another object of the invention to provide a method for detecting mutations which lead to diminished or loss of expression of a gene pr...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): C12Q1/68G01N33/68G01N33/50
CPCC12Q1/6827G01N33/5005G01N33/68C12Q2561/107
Inventor VOGELSTEIN, BERTKINZLER, KENNETH W.PAPADOPOULOS, NICKOLAS
Owner THE JOHN HOPKINS UNIV SCHOOL OF MEDICINE
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