Nucleic acid analysis techniques

• US 6,344,316 B1
• Filed: 06/25/1997
• Issued: 02/05/2002
• Est. Priority Date: 01/23/1996
• Status: Expired due to Term

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Gunderson, Kevin, Lee, Danny, Chaoqiang, Lai, Tran, Huu M., Matsuzaki, Hajime, Lockhart, David J., Cronin, Maureen T., Chee, Mark, Wodicka, Lisa
• Primary Examiner(s): JONES, WILLIAM G
• Application Number:
  US08/882,649
• Time in Patent Office: 1,686 Days
• Field of Search: 435/6, 536/24.3
• US Class Current: 506/9
• CPC Class Codes: C07H 19/052 Imidazole radicals
• CPC Class Codes: C07H 19/12 Triazine radicals
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2561/125 Ligase Detection Reaction [LDR]
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C40B 40/00 Libraries per se, e.g. arrays, mixtures
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
• CPC Class Codes: G16B 25/20 Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/10 Sequence alignment; Homology search

Abstract

The present invention provides a simplified method for identifying differences in nucleic acid abundances (e.g., expression levels) between two or more samples. The methods involve providing an array containing a large number (e.g. greater than 1,000) of arbitrarily selected different oligonucleotide probes where the sequence and location of each different probe is known. Nucleic acid samples (e.g. mRNA) from two or more samples are hybridized to the probe arrays and the pattern of hybridization is detected. Differences in the hybridization patterns between the samples indicates differences in expression of various genes between those samples. This invention also provides a method of end-labeling a nucleic acid. In one embodiment, the method involves providing a nucleic acid, providing a labeled oligonucleotide and then enzymatically ligating the oligonucleotide to the nucleic acid. Thus, for example, where the nucleic acid is an RNA, a labeled oligoribonucleotide can be ligated using an RNA ligase. In another embodiment, the end labeling can be accomplished by providing a nucleic acid, providing labeled nucleoside triphosphates, and attaching the nucleoside triphosphates to the nucleic acid using a terminal transferase.

Claims

1. A method of identifying differences in nucleic acid levels between two or more nucleic acid samples, each sample comprising a population of mRNA or nucleic acids derived therefrom;

said method comprising the steps of;

(a) providing one or more nucleic acid probe arrays each comprising probes not chosen to hybridize to nucleic acids derived from particular preselected genes or mRNAs;

(b) hybridizing said nucleic acid samples to said one or more arrays to form hybrid duplexes between nucleic acids in said nucleic acid samples and probes in said one or more arrays that are complementary to said nucleic acids or subsequences thereof; and

(c) determining a first value for a difference in hybridization between two probes differing from each other in at least one position to a first sample, and a second value for a difference in hybridization between the two probes to a second sample, a difference in hybridization values between said nucleic acid samples indicating a difference in a level of a nucleic acid between the first and second samples.

3. The method of claim 1, wherein said nucleotide sequence or nucleotide subsequences are all possible oligonucleotides of a preselected length selected from the group consisting of:

all possible 6 mers, all possible 7 mers, all possible 8 mers, all possible 9 mers, all possible 10 mers, all possible 11 mers, and all possible 12 mers.

4. The method of claim 1 or 2, wherein the one or more nucleic acid probe arrays comprising probes wherein said probes comprise a constant region and a variable region.

6. The method of claim 1, wherein the determining comprises determining a plurality of first values for differences in hybridization between a plurality of paired probes, a pair of probes differing from each other in at least one position, to a first sample, and a plurality of second values for differences in hybridization between the probe pairs to the second sample, differences between corresponding values between said nucleic acid samples indicating differences in a plurality of levels of nucleic acids between said first and second samples.

7. The method of claim 1, wherein the one or more nucleic acid probe arrays each comprising probes wherein said probes comprise a nucleotide sequence or subsequences selected according to a process selected from the group consisting of a random selection, a haphazard selection, and a nucleotide composition biased selection, and all possible probes of a preselected length.

8. The method of claim 1, wherein the first and second samples each comprises an mRNA population or a nucleic acid population derived therefrom.

17. The method of claim 1 or 2, wherein the probes are at least 20 nucleotides long.

18. The method of claim 4, wherein the constant region is double stranded, and the variable region is single stranded, and one of more of the probes binds to a target nucleic acid through the variable region of the probe, and the method further comprises ligating the one or more hybridized nucleic acids to the constant region of the respective probes to which the one or more target nucleic acids are hybridized.

19. The method of claim 1 or 2, wherein one or more target nucleic acids hybridize to one or more probes, and the method further comprises ligating one or more oligonucleotides to the one or more probes whereby the ligated oligonucleotides hybridizes to the one or more target nucleic acids.

20. The method of claim 19, wherein the one or more oligonucleotides are labelled.

21. The method of claim 18, further comprising ligating one or more oligonucleotides to the variable regions of the one or more probes whereby the ligated oligonucleotides hybridize to the one or more target nucleic acids.

2. A method of identifying differences in nucleic acid levels between two or more nucleic acid samples, each sample comprising a population of mRNA or nucleic acids derived therefrom;

said method comprising the steps of;

(a) providing one or more oligonucleotide arrays each comprising probe oligonucleotides wherein said probe oligonucleotides comprise a nucleotide sequence selected according to a process selected from the group consisting of a random selection, a haphazard selection, and a nucleotide composition biased selection;

(b) hybridizing said nucleic acid samples to said one or more arrays to form hybrid duplexes between nucleic acids in said nucleic acid samples and probe oligonucleotides in said one or more arrays that are complementary to said nucleic acids or subsequences thereof; and

(c) determining differences in hybridization between said nucleic acid samples wherein said differences in hybridization indicate differences in said nucleic acid levels.

9. The method of claim 2, wherein the nucleic acid samples are mRNA populations or nucleic acids derived therefrom.

10. The method of claim 2, wherein probes are designed to encode preferred codons from a species from which the samples were obtained.

11. The method of claim 2, wherein at least some codons have an inosine residue in the 5′

position.

12. The method of claim 2, wherein the selection comprises excluding probes having palindromic sequences from an initial probe set.

13. The method of claim 2, further comprising comparing the sequence of a probe showing differential hybridization to the two samples to sequences in a database to identify a gene showing complementarity to the probe.

14. The method of claim 2, further comprising using the sequence of a probe showing differential hybridization to the two samples to synthesize a primer, and using the synthesized primer to generate an amplification product from a genomic or cDNA library.

15. The method of claim 2, further comprising using the sequence of a probe showing differential hybridization to the two samples to synthesize a further probe, and using the further probe as to isolate a complementary nucleic acid.

16. The method of claim 2, further comprising determining the identity of genes that are differentially expressed between the samples, and synthesizing a further nucleic acid probe array comprising probes complementary to the genes.

5. A method of identifying differences in nucleic acid levels between two or more nucleic acid samples each comprising a population of nucleic acids, said method comprising the steps of:

(a) providing one or more nucleic acid probe arrays each comprising probes comprising a nucleotide sequence selected according to a process selected from the group consisting of a random selection, a haphazard selection, and a nucleotide composition biased selection;

(b) providing software describing the location and sequence of probes on said array;

(c) hybridizing said nucleic acid samples to said one or more arrays to form hybrid duplexes between nucleic acids in said nucleic acid samples and probes in said one or more arrays that are complementary to said nucleic acids or subsequences thereof;

(d) operating said software such that said hybridizing indicates differences in said nucleic acid levels.

22. A method of analyzing a nucleic acid population comprising:

converting the nucleic acid population into nucleic acid fragments;

ligating the nucleic acid fragments to a labelled nucleic acid molecule to form labelled nucleic acid fragments;

hybridizing the labelled nucleic acid fragments to an array of nucleic acid probes, and

determining hybridizations signals of the probes as an indication of levels of the nucleic acids in the nucleic acid population.

23. The method of claim 22, wherein the nucleic acid population is an mRNA population or nucleic acids derived therefrom.

24. The method of claim 23, wherein the nucleic acid population is contacted with the array without prior amplification.

25. A method of analyzing a nucleic acid population, comprising:

hybridizing a population of nucleic acids targets to an array of nucleic acid probes, whereby one or more of the nucleic acids hybridizes to probes in the array;

contacting the array with a labelled nucleotide and terminal deoxytransferase, whereby the labelled nucleotide is added to hybridized target molecules;

detecting label hybridized to probes as an indication of which nucleic acids are present in the nucleic acid population.

26. The method of claim 25, wherein the population of nucleic acids is an mRNA population or nucleic acids derived therefrom.

27. A method of analyzing a nucleic acid population, comprising:

hybridizing a population of nucleic acid targets to an array of nucleic acid probes, whereby a plurality of nucleic acid targets hybridize to complementary probes in the array;

forming a common tail on the hybridized nucleic acid targets or their complementary probes;

contacting the array with a labelled moiety having specific affinity for the tail, whereby the moiety binds to the tails;

detecting label hybridized to probes as an indication of the levels of the nucleic acids in the nucleic acid population.

28. The method of claim 27, wherein the population of nucleic acids is an mRNA population or nucleic acids derived therefrom.