Methods of nucleic acid sequences analysis using solid supports containing identifier sequences

• US 10,144,966 B2
• Filed: 04/15/2015
• Issued: 12/04/2018
• 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:
  US14/687,790
• Publication Number:
  US 20160040231A1
• Time in Patent Office: 1,329 Days
• Field of Search: None
• 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 sample nucleic acid sequence, which comprises:

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

(i) the nucleic acid molecules on each solid support in the collection comprise a single-stranded solid phase nucleic acid species including a primer sequence, an identifier sequence and a probe sequence;

(ii) at least one nucleic acid molecule of the solid phase nucleic acid species on each of the solid supports in the collection has a unique probe sequence different than a probe sequence of the solid phase nucleic acid species of the other solid supports in the collection;

(iii) the probe sequence of each solid phase nucleic acid species on a solid support in the collection hybridizes to a subsequence of the sample nucleic acid sequence when the probe sequence is complementary to a nucleotide subsequence in the sample nucleic acid sequence;

(iv) the solid phase nucleic acid species on each solid support in the collection share a unique identifier sequence with the other solid phase nucleic acid species on the solid support, wherein the unique identifier sequence is different than the identifier sequences of the solid phase nucleic acid species of the other solid supports in the collection;

b. contacting the mixture with extension reagents under conditions in which solid phase nucleic acid species hybridized to the sample nucleic acid in (iii) are extended by the reagents, whereby extended solid phase nucleic acid species comprising the unique identifier sequence of the solid support in (iii) and subsequences of the sample nucleic acid are obtained;

c. amplifying the extended solid phase nucleic acid species of (b) by linear amplification; and

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

whereby the sample nucleic acid sequence is analyzed.

2. The method of claim 1, wherein the solid phase nucleic acid species of one or more solid supports in the collection do not share a common probe sequence.

3. The method of claim 1, wherein the solid phase nucleic acid species of one or more solid supports in the collection share a common probe sequence.

4. The method of claim 1, wherein the solid support is a bead or particle.

5. The method of claim 4, wherein the bead or particle is a microbead, a nanobead, a microparticle or a nanoparticle.

6. The method of claim 4, wherein the bead or particle comprises a gel.

7. The method of claim 4, wherein the bead or particle comprises a magnetic material.

8. The method of claim 1, wherein determining the sequences of the amplification products in (d) comprises:

hybridizing the amplification products to an oligonucleotide comprising a sequence complementary to an immobilized capture nucleic acid sequence, thereby immobilizing the amplification products; and

sequencing the immobilized amplification products.

9. The method of claim 1, wherein the solid phase nucleic acid species of each solid support in the collection share a common primer sequence.

10. The method of claim 1, wherein the primer sequence is a universal primer sequence.

11. The method of claim 1, wherein the solid phase nucleic acid species of all the solid supports in the collection share a common primer sequence.

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

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

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

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

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

17. A method for preparing sample nucleic acid complements, which comprises:

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

(i) the nucleic acid molecules on each solid support in the collection comprising a single-stranded solid phase nucleic acid species including a primer sequence, an identifier sequence and a probe sequence;

(ii) at least one nucleic acid molecule of the solid phase nucleic acid species on each of the solid supports in the collection has a unique probe sequence different than a probe sequence of the solid phase nucleic acid species of the other solid supports in the collection;

(iii) the probe sequence of each solid phase nucleic acid species on a solid support in the collection hybridizes to a subsequence of the sample nucleic acid sequence when the probe sequence is complementary to a nucleotide subsequence in the sample nucleic acid sequence;

(iv) the solid phase nucleic acid species on each solid support in the collection share a unique identifier sequence with the other solid phase nucleic acid species on the solid support, wherein the unique identifier sequence is different than the identifier sequences of the solid phase nucleic acid species of the other solid supports in the collection; and

b. contacting the mixture with extension reagents under conditions in which solid phase nucleic acid species hybridized to the sample nucleic acid in (iii) are extended by the reagents, whereby sample nucleic acid complements comprising the shared, unique identifier sequence of the solid support in (iii) and subsequences of the sample nucleic acid are prepared

c. amplifying the extended solid phase nucleic acid species of (b) by linear amplification.