Preparation of pools of nucleic acids based on representation in a sample

• US 6,355,419 B1
• Filed: 04/27/1998
• Issued: 03/12/2002
• Est. Priority Date: 04/27/1998
• Status: Expired due to Fees

• Current Assignee: Callida Genomics, Inc. (SBH Genomics, Inc.)
• Original Assignee: Hyseq, Inc. (Arca Biopharma, Inc.)
• Inventors: Alfenito, Mark R.
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US09/067,317
• Time in Patent Office: 1,415 Days
• Field of Search: 435/6, 435/69.1, 435/183.1, 435/810, 422/50, 422/68.1, 436/501, 935/77, 935/78
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides

Abstract

The invention relates to methods for preparing nucleic acid pools useful in hybridization studies. Such methods allow hybridization conditions, such as time, temperature, ionic strength, etc., to be adjusted to increase the likelihood that hybridization to the nucleic acids within each pool is within the linear range of detection (i.e., detectable but not saturating). The methods rely on pooling nucleic acids derived from a sample, based on the degree of representation within the sample, i.e., nucleic acids having similar degrees of representation within in a sample are combined into a pool. The invention also provides arrays and kits produced from pooled nucleic acids, and an improved method for identifying a nucleic acid and/or its representation in a sample.

Claims

1. A method for preparing a plurality of nucleic acid pools comprising:

(a) providing a sample of nucleic acids;

(b) contacting each nucleic acid with each of a plurality of oligonucleotide probes under conditions for hybridization of oligonucleotide probes to nucleic acids;

(c) identifying nucleic acids that hybridize to the same plurality of oligonucleotide probes thereby clustering the nucleic acids in a plurality of groups, wherein each group comprises nucleic acids having tht same or similar nucleotide sequences;

(d) determining the number of nucleic acids in each group as an indication of the degree of representation in the sample of the nucleic acids within said group;

(e) selecting a single nucleic acid from each group to obtain a series of representative nucleic acids;

(f) combining different representative nucleic acids in a plurality of pools based on degree of representation in the sample, wherein the degree of representation of a representative nucleic acid in each pool is sufficiently similar to other different representative nucleic acids in that pool to allow one set of hybridization conditions to be selected for that pool that is suitable for all nucleic acids in that pool.

2. The method of claim 1 additionally comprising affixing said representative nucleic acids to one or more substrates.

3. The method of claim 2 wherein each pool of representative nucleic acids is arrayed on a substrate to form a plurality of arrayed pools of representative nucleic acids on said on said substrate.

4. A method for preparing a plurality of nucleic acid arrays comprising:

(a) providing a series of different nucleic acids derived from a sample;

(b) subdividing said series of nucleic acids into a plurality of pools based on degree of representation in the sample, wherein the degree of representation of a nucleic acid in each pool is sufficienty similar to other different nucleic acids in that pool to allow one set of hybridization conditions to be selected for that pool that is suitable for all nucleic acids in that pool;

(c) affixing each pool of nucleic acids to a substrate to form a plurality of arrays of nucleic acids.

5. An array of pools of different nucleic acids affixed to a substrate, wherein the nucleic acids in each pool have a degree of representation in the sample that is within a predetermined range allowing selection of one set of hybridization conditions that is suitable for all nucleic acids in that pool.

6. A kit comprising a plurality of arrays of different nucleic acids affixed to one or more substrates, wherein the nucleic acids are derived from a sample, and wherein thc nucleic acids in each array have a degree of representation in the sample that is within a predetermined range, said range being different for each array and said range for each array allowing one set of hybridization conditions to be selected for that array that is suitable for all nucleic acids in that array.

7. An improved method for determining the presence or representation of a nucleic acid sequence in a sample comprising:

(a) providing a series of different nucleic acids derived from a sample, wherein said series has been subdivided in a plurality of pools based on degree of representation in the sample;

(b) contacting the nucleic acids in each pool with one or more target nucleic acids and/or oligonucleotide probes under conditions suitable for hybridization, wherein said suitable hybridization conditions are different for each pool, wherein the degree of representation of a nucleic acid in each pool is sufficiently similar to other different representative nucleic acids in that pool to allow one set of hybridization conditions to be selected for that pool that is suitable for all nucleic acids in that pool; and

(c) detecting hybridization between said nucleic acids and said one or more targets nucleic acids and/or probes as an indication of the presence or representation of one or more nucleic acid sequences in said sample.

8. The method of claim 7 wherein said nucleic acids are affixed to one or more substrates.

9. The method of claim 8 wherein each pool of representative nucleic acids is arrayed on a substrate to form a plurality of arrays of nucleic acids.

10. The method of claim 7 wherein each nucleic acid is labeled and is contacted with one or more target nucleic acids and or oligonucleotide probes in solution.