SINGLE MOLECULE NUCLEIC ACID SEQUENCE ANALYSIS PROCESSES AND COMPOSITIONS

• US 20090202984A1
• Filed: 01/15/2009
• Published: 08/13/2009
• Est. Priority Date: 01/17/2008
• Status: Active Grant

• Current Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Original Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Cantor, Charles R.
• Granted Patent: US 9,034,580 B2
• Time in Patent Office: Days
• US Class Current: 435/5
• 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 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2531/101 Linear amplification, i.e. non exponential
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer

Abstract

Improved solid supports and methods for analyzing target nucleotide sequences are provided herein. Certain improvements are directed to efficiently preparing nucleic acids that comprise nucleotide sequences identical to or substantially identical to one or more target nucleotide sequences, or complement thereof. The prepared nucleic acids include a reference sequence that facilitates sequence analysis. The solid supports and methods provided herein minimize the number of steps required by published sequence analysis methodologies, and thereby offer improved sequence analysis efficiency.

Claims

1. A method for analyzing a target nucleic acid sequence, which comprises:

a. preparing a mixture comprising sample nucleic acid a solid support under conditions in which a single molecule of the sample nucleic acid hybridizes to a solid support molecule, wherein;

the solid support comprises single-stranded solid phase nucleic acid comprising a primer sequence, an identifier sequence and a probe sequence;

the probe sequence hybridizes to sample nucleic acid when the probe sequence is complementary to a nucleotide sequence in the sample nucleic acid;

the solid phase nucleic acid shares a common probe sequence or the solid phase nucleic acid does not share a common probe sequence; and

the solid phase nucleic acid shares a common identifier sequence;

b. contacting the mixture with extension reagents under conditions in which solid phase nucleic acid hybridized to sample nucleic acid is extended by the reagents;

c. amplifying the extended solid phase nucleic acid of (b); and

d. analyzing the sequences of the amplification products of (c);

whereby the target nucleic acid sequence is analyzed.

2. The method of claim 1, wherein a single molecule of sample nucleic acid hybridizes to a solid support molecule in and emulsion.

3. The method of claim 1, wherein the sample nucleic acid comprises DNA.

4. The method of claim 3, wherein the sample nucleic acid consists of DNA.

5. The method of claim 4, wherein the sample nucleic acid is fragmented DNA.

6. The method of claim 1, wherein the sample nucleic acid comprises RNA.

7. The method of claim 6, wherein the sample nucleic acid consists of RNA.

8. The method of claim 1, wherein the sample nucleic acid is from a single sample.

9. The method of claim 1, wherein the sample nucleic acid is from a mixture or pool of samples.

10. The method of claim 1, wherein the sample nucleic acid is from a viral mixture.

11. The method of claim 10, wherein the viral mixture is a hepatitis viral mixture.

12. The method of claim 1, wherein the sample nucleic acid is from a mixed viral population.

13. The method of claim 1, wherein the sample nucleic acid is single-stranded nucleic acid.

14. The method of claim 1, wherein the sample nucleic acid is from one chromosome.

15. The method of claim 1, wherein the sample nucleic acid is from one chromosome and comprises nucleic acid fragments.

16. The method of claim 1, wherein the reagents include deoxynucleotides and a polymerase.

17. The method of claim 1, with the proviso that the sample nucleic acid is not ligated to one or more heterologous nucleic acids.

18. The method of claim 1, wherein the amplification products of (d) are further amplified and sequences of further amplified products are analyzed.

19. The method of claim 18, wherein the amplification process comprises contacting the amplification products with a substrate having pits that contain a single solid support.

20. The method of claim 1, wherein the sequences of the amplification products are analyzed directly.

21. The method of claim 1, wherein the ratio of the concentration of the solid support to the concentration of the sample nucleic acid is ten or greater.