Quantitative analysis of hybridization patterns and intensities in oligonucleotide arrays

• US 6,489,096 B1
• Filed: 10/15/1998
• Issued: 12/03/2002
• Est. Priority Date: 10/15/1998
• Status: Expired due to Term

• Current Assignee: Princeton University
• Original Assignee: Princeton University
• Inventors: Levine, Arnold J., Alon, Uri
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US09/174,364
• Time in Patent Office: 1,510 Days
• Field of Search: 422/50, 422/68.1, 422/69, 422/82.05, 435/6, 435/283.1, 435/287.1, 435/287.2, 435/287.9, 435/288.7, 435/289.1, 364/178, 364/550, 364/551.01, 364/825, 702/20
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2545/114 involving a quantitation step
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression

Abstract

Systems and methods for enhanced quantitative analysis of hybridization intensity measurements obtained from oligonucleotide probes and other probes exposed to target samples are provided by virtue of the present invention. One embodiment ameliorates the effects of high frequency noise superimposed on a hybridization intensity measurement signal measured over successive probe alignments to a target sample sequence. Detection of expressed genes and ESTs and quantitative measurement of expression level may be improved. Mutation detection and base calling may be improved.

Claims

1. A method for analyzing a sample nucleic acid sequence comprising:

providing a plurality of hybridization intensities of probes including exposing said probes to said sample nucleic acid sequence, said probes including pairs of perfect match and mismatch probes, each pair including a perfect match probe perfectly complementary to a subsequence of said nucleic acid sequence and a mismatch probe having at least one base mismatch with said particular subsequence;

applying a non-linear low-pass filter to said plurality of hybridization intensities, wherein applying the non-linear filter includes averaging hybridization intensities of perfect match probes over subsequences aligned to a particular base position and subsequences aligned to surrounding base positions, excluding outlying hybridization intensities; and

comparing pairs of relative hybridization intensities, wherein comparing pairs of said relative hybridization intensities determines an expression of a gene specified by said sample nucleic acid sequence.

2. The method of claim 1 wherein applying a non-linear filter comprises:

for a particular base position, averaging hybridization intensities of mismatch probes over subsequences aligned to said particular base position and subsequences aligned to surrounding base positions, excluding outlying hybridization intensities.

3. A method for analyzing a sample nucleic acid sequence comprising:

providing a plurality of hybridization intensities of probes including exposing said probes to said sample nucleic acid sequence, said probes including pairs of perfect match and mismatch probes, each pair including a perfect match probe perfectly complementary to a subsequence of said nucleic acid sequence and a mismatch probe having at least one base mismatch with said particular subsequence;

applying a non-linear low-pass filter to said plurality of hybridization intensities;

comparing pairs of relative hybridization intensities, wherein comparing pairs of said relative hybridization intensities determines an expression of a gene specified by said sample nucleic acid sequence;

evaluating relative hybridization intensities for a plurality of subsequences by comparison of hybridization intensities between perfect match and mismatch probes in individual ones of said pairs, wherein applying the non-linear filter comprises for a particular base position, averaging relative hybridization intensities over subsequences aligned to said particular base position and subsequences aligned to surrounding base positions, excluding subsequences having outlying relative hybridization intensities.

4. A method for analyzing a sample nucleic acid sequence comprising:

providing a plurality of hybridization intensities of probes including exposing said probes to said sample nucleic acid sequence, said probes including pairs of perfect match and mismatch probes, each pair including a perfect match probe perfectly complementary to a subsequence of said nucleic acid sequence and a mismatch probe having at least one base mismatch with said particular subsequence;

applying a non-linear low-pass filter to said plurality of hybridization intensities, wherein applying the non-linear filter includes obtaining a median of hybridization intensities of perfect match probes of subsequences aligned to a particular base position and subsequences aligned to surrounding base positions, excluding outlying hybridization intensities; and

comparing pairs of relative hybridization intensities, wherein comparing pairs of said relative hybridization intensities determines an expression of a gene specified by said sample nucleic acid sequence.

5. A method for analyzing a sample nucleic acid sequence comprising:

providing a plurality of hybridization intensities of probes including exposing said probes to said sample nucleic acid sequence, said probes including pairs of perfect match and mismatch probes, each pair including a perfect match probe perfectly complementary to a subsequence of said nucleic acid sequence and a mismatch probe having at least one base mismatch with said particular subsequence;

applying a non-linear low-pass filter to said plurality of hybridization intensities, wherein applying the non-linear filter includes obtaining a median of hybridization intensities mismatch probes of subsequences aligned to a particular base position and subsequences aligned to surrounding base positions, excluding outlying hybridization intensities; and

comparing pairs of relative hybridization intensities, wherein comparing pairs of said relative hybridization intensities determines an expression of a gene specified by said sample nucleic acid sequence.

6. A method for analyzing a sample nucleic acid sequence comprising;

providing a plurality of hybridization intensities of probes including exposing said probes to said sample nucleic acid sequence, said probes including pairs of perfect match and mismatch probes, each pair including a perfect match probe perfectly complementary to a subsequence of said nucleic acid sequence and a mismatch probe having at least one base mismatch with said particular subsequence;

applying a non-linear low-pass filter to said plurality of hybridization intensities;

comparing pairs of relative hybridization intensities, wherein comparing pairs of said relative hybridization intensities determines an expression of a gene specified by said sample nucleic acid sequence;

evaluating relative hybridization intensities for a plurality of subsequences by comparison of hybridization intensities between perfect match and mismatch probes in individual ones of said pairs, wherein applying the non-linear filter comprises for a particular base position, obtaining a median of relative hybridization intensities over subsequences aligned to said particular base position and subsequences aligned to surrounding base positions, excluding subsequences having outlying relative hybridization intensities.