METHODS FOR NUCLEIC ACID CAPTURE AND SEQUENCING

• US 20130324419A1
• Filed: 08/25/2011
• Published: 12/05/2013
• Est. Priority Date: 08/27/2010
• Status: Abandoned Application

• Current Assignee: Genentech, Inc. (Roche Holding AG)
• Original Assignee: Genentech, Inc. (Roche Holding AG)
• Inventors: Seshagiri, Somasekar
• Application Number:
  US13/819,657
• Publication Number:
  US 20130324419A1
• Time in Patent Office: Days
• US Class Current: 506/2
• CPC Class Codes: C12N 15/1072 Differential gene expression library synthesis, e.g. subtracted libraries, differential screening
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/173 incorporating a polynucleotide run, e.g. polyAs, polyTs
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides
• CPC Class Codes: C12Q 2565/601 being a microscope, e.g. atomic force microscopy [AFM]

Abstract

Methods of capturing and sequencing target nucleic acid molecules are provided. Methods of determining the methylation status of genomic DNA are also provided.

Claims

1. A method of capturing and sequencing a target nucleic acid molecule comprising:

(a) exposing a solid support to a mixture of nucleic acids comprising the target nucleic acid molecule under hybridizing conditions, wherein the target nucleic acid molecule forms a specific hybridization complex with a primer immobilized on the solid support in a priming-competent configuration;

(b) separating unbound and non-specifically bound nucleic acids from the solid support;

(c) exposing the solid support to a polymerase and nucleotides under polymerization conditions; and

(d) determining a nucleic acid sequence of the target nucleic acid molecule by detecting nucleic acid polymerization from the immobilized primer by the polymerase using the target nucleic acid molecule as a template.

2. The method of claim 1, wherein the target nucleic acid molecule is from a region of genomic DNA.

3. The method of claim 2, wherein the target nucleic acid comprises all or part of an exon.

4. The method of claim 1, wherein the target nucleic acid molecule is RNA, the polymerase is a reverse transcriptase, and the primer comprises a 3′

poly-T sequence.

5. The method of claim 1, wherein the target nucleic acid molecule is DNA, and the polymerase is a DNA polymerase.

6. The method of claim 1, wherein the nucleotides are labeled at their terminal phosphates.

7. The method of claim 6, wherein the polymerase is labeled with a FRET donor, and the nucleotides are labeled with a FRET acceptor.

8. The method of claim 7, wherein the FRET donor is a fluorescent nanoparticle.

9. A method of determining the methylation status of a genomic DNA fragment, the method comprising:

(a) immobilizing a genomic DNA fragment on a solid support;

(b) determining a nucleic acid sequence of the immobilized genomic DNA fragment on the solid support by detecting nucleic acid polymerization by a polymerase using the immobilized genomic DNA fragment as a template;

(c) subjecting the immobilized genomic DNA fragment to bisulfite treatment;

(d) determining a nucleic acid sequence of the immobilized, bisulfite-treated genomic DNA fragment on the solid support by detecting nucleic acid polymerization by a polymerase using the immobilized, bisulfite-treated genomic DNA fragment as a template;

(e) comparing the nucleic acid sequence determined in (b) with the sequence determined in (d), wherein conversion of a cytosine residue in the genomic DNA fragment indicates that the residue was unmethylated in the genomic DNA fragment prior to the bisulfite treatment, and wherein absence of conversion of a cytosine residue in the genomic DNA fragment indicates that the residue was methylated in the genomic DNA fragment prior to the bisulfite treatment.

10. The method of claim 9, wherein the genomic DNA fragment is immobilized to the solid support by an adaptor.

11. The method of claim 10, wherein the adaptor contains a primer binding site, and wherein cytosines in the primer binding site are protected.

12. The method of claim 11, wherein the polymerase of (b) and/or (d) polymerizes a nucleic acid strand from a primer annealed to the primer binding site.

13. The method of claim 9, wherein nucleic acid polymerization in (b) and/or (d) is detected by detecting the incorporation of labeled nucleotides.

14. The method of claim 13, wherein the labeled nucleotides are labeled at their terminal phosphates.

15. The method of claim 14, wherein the polymerase of (b) and/or (d) is labeled with a FRET donor, and the nucleotides are labeled with a FRET acceptor.

16. The method of claim 15, wherein the FRET donor is a fluorescent nanoparticle.