Re-sequencing pathogen microarray

a pathogen and microarray technology, applied in the field of resequencing pathogen microarrays, can solve the problems of pathogen detection, introduction of a new set of problems, and conventional methods for amplification that do not scale well

Inactive Publication Date: 2006-09-21
THE UNITED STATES OF AMERICA AS REPRESENTED BY THE SECRETARY OF THE NAVY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

One technical challenge for pathogen detection with microarrays arises due to the difficulty in obtaining samples with a sufficient quantity of pathogen nucleic acid.
Unfortunately, conventional methods for this amplification do not scale well in comparison to the number of probes that can be placed on a microarray chip.
Although PCR-b...

Method used

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  • Re-sequencing pathogen microarray
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  • Re-sequencing pathogen microarray

Examples

Experimental program
Comparison scheme
Effect test

preparation example 1

RPM Version 1 Chip Design

[0433] DNA sequences were provided to Affymetrix for creation of the resequencing microarray chip (RPM Version 1 chip) utilized in the following examples. Submission of the DNA sequence and instruction files to Affymetrix were in accordance with the manufacturer instructions CustomSeq™ Array Protocol and product literature. Probe lengths were nominally 25-nucleotides long and contained a variable (interrogation point) central nucleotide for each of four possible variants (A, C, T or G) in both the sense and antisense directions.

[0434] The target genes selected for the RPMV1 pathogens listed above are described in the version 1 layout shown in Table 8 and the Sequence Listing along with the respective PCR primers used for amplification of the same. The sequences submitted for tiling and chip fabrication were based on the Affymetrix instruction file summarized in Table 7, which corresponds to the sequences appearing as SEQ ID NOs: 1-58. The corresponding “in...

preparation example 2

PCR Primer Design and Amplification Protocols

Degenerate PCR Primers Design

[0442] The objective of primer selection to support conserved (degenerate) multiplex PCR is to design primers that target the conserved regions flanking species-specific variable regions of E1A, fiber, and hexon genes. In general, this method may be applied to any organism, as conserved sequences within a species are a ubiquitous in nature. These target genes were selected based on their function and location within the linear adenoviral genome. E1A is located at the 5′ end of the adenoviruses genome and encodes a trans-acting transcriptional regulatory factor that is necessary for transcriptional activation of early genes. The hexon and fiber genes, which are located in the middle and 3′ end of the adenovirus genome, encode antigenic determinants ε and γ respectively, which determine the viral serotype. Thus, detection and serotyping of ARD-causing adenoviruses can be effectuated by targeting the nucleic a...

preparation example 3

REPI Software

[0447] Raw sequence data from the resequencing microarray chips is provided by the Genetic Data Analysis Software version 2.0 (GDAS) packaged with the microarray reader from Affymetrix. GDAS base calling is based on a previously described base-calling algorithm (Cutler et al., 2001). Each of the FASTA output files containing the base calls obtained from the GDAS software was analyzed using specialized software (REPI) that the present inventors developed.

[0448] In the case of the present invention, the sequence output of GDAS is most often a scattered mixture of contiguous sequence calls (A, T, C or G) that are interspersed with varying amounts of no-calls (n's) where the GDAS software does not make a base call due to lack of amplification, weak hybridization signal on the chip and / or high background hybridization caused by non-specific binding (Cutler et al., 2001). An example output of the GDAS output for the Adenovirus 4 prototype sample for the Ad4FIBER tile region...

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Abstract

The present invention relates to pathogen detection and identification by use of DNA resequencing microarrays. The present invention also provides resequencing microarray chips for differential diagnosis and serotyping of pathogens present in a biological sample. The present invention further provides methods of detecting the presence and identity of pathogens present in a biological sample.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] The present application claims priority to U.S. provisional Application Ser. No. 60 / 590,931, filed on Jul. 2, 2004, U.S. provisional Application Ser. No. 60 / 609,918 filed on Sep. 15, 2004, U.S. provisional Application Ser. No. 60 / 631,437 filed on Nov. 29, 2004, U.S. provisional Application Ser. No. 60 / 631,460 filed on Nov. 29, 2004 and U.S. provisional Application Ser. No. 60 / 691,768 filed on Jun. 16, 2005. This application is also related to U.S. non-provisional application Ser. No. ______, titled “Computer-Implemented Biological Sequence Identifier System and Method,” filed along with this application on Jul. 2, 2005. The entire contents of these applications are incorporated herein by reference. STATEMENT REGARDING FEDERALLY FUNDED PROJECT [0002] The United States Government owns rights in the present invention pursuant to funding from the Defense Threat Reduction Agency (DTRA; Interagency Cost Reimbursement Order (IACRO #02-4118), M...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12M1/34G16B30/10
CPCC12Q1/6874C12Q1/6888C12Q1/689C12Q1/6893C12Q1/701G06F19/22G16B30/00G16B30/10
Inventor AGAN, BRIAN K.HANSON, ERIC H.KRUZELOCK, RUSSELL P.LIN, BAOCHUANROWLEY, ROBB K.SETO, DONALDSTENGER, DAVID A.JOHNSON, JENNIFERTIBBETTS, CLARK J.THACH, DZUNG C.VORA, GARY J.WALTER, ELIZABETH A.WANG, ZHENG
Owner THE UNITED STATES OF AMERICA AS REPRESENTED BY THE SECRETARY OF THE NAVY
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