Re-sequencing pathogen microarray

Patent metadata

Field	Value
Publication number	US-9430610-B2
Application number	US-10051908-A
Country	US
Kind code	B2
Filing date	Apr 10, 2008
Priority date	Jul 2, 2004
Publication date	Aug 30, 2016
Grant date	Aug 30, 2016

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.

First claim

What we claim is:

1. A method comprising:
providing a resequencing DNA microarray;
contacting an unknown sample comprising genetic material to the resequencing DNA microarray;
wherein the resequencing DNA microarray comprises multiple groups of oligonucleotide probes of a length ranging from 13 to 70 nucleotides immobilized to a solid phase support;
wherein each group of oligonucleotide probes is selected to span a region of a reference sequence that is a single gene or subsequence that represents a genus, species, or subspecies of organisms;
wherein the reference sequence is a consensus sequence that is not identical to any sequence in the genus, species, or subspecies of organisms;
wherein each group of oligonucleotide probes occupies a tiled region of the microarray;
wherein each group of oligonucleotide probes comprises at least four probes arranged in a parallel fashion within the tiled region of the array, wherein the four probes are selected as follows:
i) a first probe that is exactly complementary to the reference sequence; and
ii) three additional probes, each of which is identical to the first probe but for the nucleotide at a central position, which is different in each of the three additional probes such that all four nucleotide bases are present on the microarray;

hybridizing a nucleic acid in the unknown sample to the resequencing DNA microarray;
generating a sequence of base calls based on a hybridization response between the nucleic acid in the unknown sample and the probes on the resequencing DNA microarray; and
determining the sequence of a full-length gene or genomic fragment corresponding to the nucleic acid in the unknown sample by comparing the sequence of base calls to a sequence database containing sequences from the genus, species, or subspecies of organisms.

2. The method of claim 1 , wherein the method further comprises:
determining the identity of the drug-resistance marker or the particular class of organism species or subspecies by sequence comparison between a DNA sequence identified by the method and known sequences.

3. The method of claim 1 , wherein the unknown sample is a biological sample, a nasal wash specimen, a nasal aspirate, a throat swab, a blood sample, a sputum sample, blood cells, a tissue sample, a fine needle biopsy sample, a urine specimen, a peritoneal fluid sample, a visceral fluid sample, a pleural fluid sample, a soil sample, an air sample, or a water sample.

4. The method of claim 1 , wherein prior to the hybridizing, the unknown sample is subjected to at least one of process selected from the group consisting of:
(i) isolation of the genetic material within the sample,
(ii) enrichment for target sequences of interest within the sample,
(iii) amplification of the genetic material contained within the sample,
(iv) labeling the genetic material within the sample, and
(v) subtractive hybridization.

5. The method of claim 1 , wherein prior to the hybridizing one or more target nucleic acids of interest in the unknown sample, the unknown sample is subjected to at least one method selected from the group consisting of specific reverse transcription, PCR, multiplex PCR, random PCR, random primed amplification, isothermal Klenow polymerase-based amplification, Φ29DNA polymerase-based amplification, tandem amplification, multiplex PCR amplification, and total amplification.

6. The method of claim 1 , wherein the genetic material or one or more target nucleic acids of interest present in the unknown sample are enriched by subtraction of the background nucleic acids from the sample, reverse-transcriptase subtractive hybridization, or selective removal of the target nucleic acids from a mixture of nucleic acids presenting the unknown sample.

7. The method of claim 1 , wherein the length of the probes is 13-35 nucleotides.

8. The method of claim 1 , wherein the length of the probes is 25 nucleotides.

9. The method of claim 1 , wherein the region of the reference sequence that is spanned by the first probe moves by one nucleotide across the reference sequence for each adjacent tiled region across the microarray surface.

10. The method of claim 1 , wherein the reference sequences represent genotypes of pathogen families, a family or group of adenoviruses, or a family or group of influenza viruses.

11. The method of claim 1 , wherein the reference sequences encode a drug-resistance marker.

12. The method according to claim 1 , wherein the base calls are made by determining the absolute intensity of the hybridization signals on the resequencing DNA microarray.

13. The method according to claim 1 , further comprising:
determining the percentage of base calls, both as a percentage of the total tile region size and as a percentage of base calls within a selected subsequence satisfying a sliding window algorithm.

Assignees

• Agan Brian K
• Hanson Eric H
• Kruzelock Russell P
• Lin Baochuan
• Rowley Robb K
• Seto Donald
• Stenger David A
• Johnson Jennifer
• Tibbetts Clark J
• Thach Dzung C
• Vora Gary J
• Walter Elizabeth A
• Wang Zheng
• Us Navy

Inventors

• Agan Brian K
• Hanson Eric H
• Kruzelock Russell P
• Lin Baochuan
• Rowley Robb K
• Seto Donald
• Stenger David A
• Johnson Jennifer
• Tibbetts Clark J
• Thach Dzung C
• Vora Gary J
• Walter Elizabeth A
• Wang Zheng

Classifications

• G06F19/22 Primary

  Physics · mapped topic

• C12Q1/689

  for bacteria · CPC title

• C12Q1/6874

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12Q1/701

  Specific hybridization probes · CPC title

• C12Q1/6893

  for protozoa · CPC title