Single molecule nucleic acid sequence analysis processes and compositions

• US 9,034,580 B2
• Filed: 01/15/2009
• Issued: 05/19/2015
• 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.
• Primary Examiner(s): Kim, Young J
• Application Number:
  US12/354,749
• Publication Number:
  US 20090202984A1
• Time in Patent Office: 2,315 Days
• US Class Current: 435/6.11
• 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 obtaining sequence information of a target nucleic acid from sample nucleic acid comprising fragments of the target nucleic acid, the method comprising:

(a) preparing a mixture comprising the sample nucleic acid and a solid support comprising single-stranded solid phase nucleic acid under conditions in which a single molecule of the sample nucleic acid hybridizes to a single-stranded solid phase nucleic acid molecule, wherein;

(i) the single-stranded solid phase nucleic acid comprises a primer sequence, an identifier sequence and a probe sequence, wherein (1) the sequences are orientated 5′

-(primer sequence)-(identifier sequence)-(probe sequence)-3′ and

(2) the 5′

terminal nucleotide of the solid phase nucleic acid is linked to the solid support;

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

(iii) the solid phase nucleic acid comprises one probe sequence species or the solid phase nucleic acid comprises a plurality of probe sequence species; and

(iv) the solid phase nucleic acid comprises one identifier sequence species;

(b) contacting the mixture of (a) with extension reagents, thereby generating an extension product;

(c) contacting the extension product of (b) with a primer, thereby generating a product for amplification;

(d) amplifying the product of (c) by linear amplification, thereby generating amplification products, wherein each amplification product comprises the identifier sequence or complement thereof;

(e) determining the nucleotide sequences of the amplification products of (d) using a nucleotide sequencing method, thereby generating sequencing reads; and

(f) constructing a larger nucleotide sequence using the reads of (e) by identifying overlapping sequences in the reads and by using the identifier sequence, or complement thereof, thereby obtaining sequence information of the target nucleic acid.

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

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

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

15. The method of claim 1, wherein the mixture comprises deoxynucleotides and a polymerase.

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

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

18. The method of claim 17, wherein the amplification process comprises contacting under amplification conditions the amplification products of (d) with a substrate having pits that contain a single solid support.

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

20. The method of claim 1, wherein the molar ratio of the solid phase nucleic acid to the sample nucleic acid is 10:

1 or greater.