Multiplexed polymerase chain reaction for genetic sequence analysis

A chain reaction and polymerase technology, which is applied in the field of multiplex polymerase chain reaction for genetic sequence analysis, can solve the problem of insensitivity to sequence diversity, difficulty in distinguishing, and inability to distinguish closely related strains of the same organism And other issues

Inactive Publication Date: 2008-09-24
THE UNITED STATES OF AMERICA AS REPRESENTED BY THE SECRETARY OF THE NAVY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

A major limitation of these systems is the inability to distinguish closely related strains of the same organism, since the detected hybridization events may not be sensitive to local sequence diversity
For example, spotted microarray probes can non-specifically cross-hy...

Method used

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  • Multiplexed polymerase chain reaction for genetic sequence analysis
  • Multiplexed polymerase chain reaction for genetic sequence analysis
  • Multiplexed polymerase chain reaction for genetic sequence analysis

Examples

Experimental program
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Embodiment 1

[0022] RPM v.1 Chip Design - The RPM v.1 (Respiratory Pathogen Microarray) chip design includes 57 tiles that allow resequencing of 29.7kb sequences from 27 respiratory pathogens and biological warfare agents and was detailed in previous studies Described (Lin et al., "Broad-spectrum respiratory tract pathogen identification using resequencing DNA microarrays" Genome Res., 16(4), 527-535 (2006)). Briefly, RPM arrays consist of 25-mer perfectly matched probes representing sequences at each base within (and centered around) sequences selected from the genome of a target organism. In addition, for each perfectly matched probe, three mismatched probes representing the three possible single nucleotide polymorphisms (SNPs) at the central location were also tiled on the array. Thus, hybridization to a series of perfectly matched probes provides redundant presence / absence information, while hybridization to mismatched probes reveals strain-specific SNP data. On the chip, two pathogen...

Embodiment 2

[0028] Clinical samples-archived throat swabs were collected from patients with symptoms of ARI at multiple Army recruit training centers, the US-Mexico border zone, and on board Navy ships deployed in 1999-2005. These throat swabs were immediately placed in chilled 2 mL vials containing 1.5 mL of viral transport medium (VTM), frozen and stored at -80°C or below to maintain virions during transport. Samples were then shipped to the Naval Health Research Center (NHRC, San Diego, CA), thawed and aliquoted, and tested for HAdV and influenza virus using a CAP-certified diagnostic RT-PCR / PCR and culture assay. to test. Frozen aliquots were subsequently submitted for microarray-based detection in blinded fashion.

Embodiment 3

[0030] Nucleic Acid Extraction - Using MasterPure TM DNA purification kit (Epicentre Technologies, Madison, WI), omit RNA digestion, or use MagNA Pure Compact Nucleic Acid Isolation Kit I (Roche Nucleic acids were extracted from clinical samples by Roche Applied Science, Indianapolis, IN, following the manufacturer's recommended protocol.

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Abstract

A PCR method involving: providing a biological sample suspected of containing one or more pathogen nucleic acids; adding a plurality of PCR primers corresponding to genes found in the pathogens; and performing a polymerase chain reaction on the sample to amplify a subset of the nucleic acids that correspond to the genes. The primers include at least one primer pair for each pathogen, and the primers contain a tail sequence that is not homologous any pathogen DNA or to any background DNA in the sample. The concentration of at least one primer in the polymerase chain reaction is no more than about 100 nM.

Description

[0001] This application is a reference to U.S. Provisional Patent Application No. 60 / 691,768 filed June 16, 2005; U.S. Provisional Patent Application No. 60 / 735,876 filed November 14, 2005; U.S. Provisional Patent Application No. filed November 14, 2005 60 / 735,824 and U.S. Provisional Patent Application No. 60 / 743,639, filed March 22, 2006, and U.S. Patent Application No. 11 / 422,425, filed June 06, 2006, and U.S. Patent Application No. 11 / 422,425, filed June 06, 2006 11 / 422,431 claims priority. This application is a continuation-in-part of US Patent Application Nos. 11 / 177,647, filed July 02, 2005; 11 / 177,646, filed July 02, 2005; and 11 / 268,373, filed November 07, 2005. These non-provisional applications are complementary to U.S. Provisional Patent Application No. 60 / 590,931, filed July 02, 2004; U.S. Provisional Patent Application No. 60 / 609,918, filed September 15, 2004; U.S. Provisional Patent Application No. 60 / 609,918, filed November 05, 2004 Application No. 60 / 626,500; U...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12P19/34
Inventor 林宝川凯特·M.·布莱尼安东尼·P.·马拉诺斯基乔尔·M.·施努尔大卫·A.·斯滕格
Owner THE UNITED STATES OF AMERICA AS REPRESENTED BY THE SECRETARY OF THE NAVY
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