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Method of detecting genetic mutations

A technology for detecting markers and sequencing primers, which is applied in the field of detecting genetic variants, and can solve the problems of complex samples of small amounts of RNA molecules, difficulty in designing primer sets for pol genes, and inability to use sequences, etc.

Inactive Publication Date: 2009-04-29
DUKE UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

Therefore, all resistance mutations cannot be obtained from a single viral genome, and linkage analysis cannot be performed using sequences obtained by either method
Due to the high level of genetic variability, it is difficult to design primer sets to cover the pol gene that can contain all resistance mutations
In both methods, the preparation of small samples of RNA molecules from patient plasma is complicated (Rogers et al, Nature 437:326-7 (2005))

Method used

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  • Method of detecting genetic mutations
  • Method of detecting genetic mutations
  • Method of detecting genetic mutations

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Experimental program
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Embodiment

[0040] Experiment Details

[0041] Preparation of drug-resistant mutant clones. A portion of the pol gene containing the most drug resistance mutation sites was amplified from an almost full-length HIV-1 clone WEAU.A1. By overlapping PCR method, E44D, M90L and M184V mutations were introduced. The wild type (WEAU.wt) and mutant PCR products (WEAU.E44D, WEAU.M90L and WEAU.M184M) were then cloned directly into the pSTBBlue vector (Novagen, Madison, WI). These drug resistance mutations were confirmed by sequencing. For tracer experiments, mutant and wild-type clones were used in different ratios (higher than 10 4 ) for mixing. The concentration of plasmid DNA was determined by NanoDrop spectrophotometer, and the number of molecules in each DNA sample was calculated based on DNA concentration and plasmid length (bp).

[0042] Slide handling. Teflon-coated glass slides (Eriescientific, Portsmouth, NH) were exposed to UV light in a PCR hood (PCR hood) for 15 minutes, followed...

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Abstract

The present invention relates to a method of detecting genetic variants, including genetic variants associated with drug resistance, cancer and genetic dysfunctional diseases.

Description

[0001] This application claims priority to U.S. Provisional Application No. 60 / 790,535, filed April 10, 2006, and U.S. Provisional Application No. 60 / 878,700, filed January 5, 2007, both of which are incorporated herein in their entirety Incorporated by reference. [0002] This invention was made with government support under ROI GM065057 awarded by the National Institutes of Health. The government has certain rights in this invention. technical field [0003] The present invention relates to a method of detecting genetic variants, including those associated with drug resistance, cancer, genetic dysfunctional diseases. Background of the invention [0004] Combination drug therapy or highly active antiretroviral therapy (HAART) is the mainstay of treatment for human immunodeficiency virus (HIV)-infected individuals. Since HAART can suppress viral replication in the blood of patients to an undetectable level, it has significantly reduced the morbidity and mortality of HIV-in...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12P19/34
Inventor 高峰朱军
Owner DUKE UNIV
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