Preparation of templates for methylation analysis

Patent metadata

Field	Value
Publication number	US-10954554-B2
Application number	US-201815867283-A
Country	US
Kind code	B2
Filing date	Jan 10, 2018
Priority date	Feb 7, 2007
Publication date	Mar 23, 2021
Grant date	Mar 23, 2021

Abstract

The invention relates to a kit for preparing a library of template polynucleotides suitable for use as templates in solid-phase nucleic acid amplification and sequencing reactions to determine the methylation status of the cytosine bases in the library. In particular, the invention relates to a kit for preparing a library of template polynucleotides suitable for methylation analysis.

First claim

The invention claimed is:

1. A kit for use in preparing a 5′ and 3′ modified library of template polynucleotide molecules comprising known sequences at their 5′ and 3′ ends, the kit comprising methylated adaptor polynucleotides and oligonucleotide primers capable of annealing to the methylated adaptor polynucleotides, wherein said methylated adaptor polynucleotides are forked universal adaptors comprising a region of double stranded nucleic acids comprising 5 or more consecutive base pairs and at least one region of single stranded nucleic acids comprising at least 10 consecutive nucleotides; the forked universal adaptors are phosphorylated at the 5′ end of the region of double stranded nucleic acids; all cytosine bases in the forked universal adaptors are methylated; and the at least one region of single stranded nucleic acids is complementary to an oligonucleotide primer of SEQ ID NO: 6 over at least 15 consecutive nucleotides.

2. The kit according to claim 1 , wherein the oligonucleotide primers are capable of annealing to the at least one region of single stranded nucleic acids of the universal adaptor polynucleotides.

3. The kit according to claim 1 , wherein the kit comprises two or more amplification primers, at least one of the amplification primers comprising a region that hybridizes to the at least one region of single stranded nucleic acids of the forked universal adaptor.

4. The kit according to claim 1 , wherein the kit comprises two or more amplification primers, at least one of the amplification primers comprising a region that extends beyond the 5′ end of the at least one region of single stranded nucleic acids of the forked universal adaptor.

5. The kit according to claim 1 , wherein the kit comprises two or more amplification primers, at least one of the amplification primers comprising a region that hybridizes to, and extends beyond, the 5′ end of the at least one region of single stranded nucleic acids of the forked universal adaptor.

6. The kit according to claim 1 , wherein the kit comprises first oligonucleotide primers capable of annealing to one of the strands of the forked universal adaptor and second oligonucleotide primers capable of annealing to a region of extended strands produced by extension of the first oligonucleotide primers.

7. The kit according to claim 6 , wherein the first and second oligonucleotide primers have different nucleotide sequences.

8. The kit according to claim 7 , wherein the first and second oligonucleotide primers are capable of annealing to one of the strands in the region of the double stranded nucleic acids of the forked universal adaptor.

9. The kit according to claim 7 , wherein the first oligonucleotide primers and the second oligonucleotide primers comprise one or more of SEQ ID NO: 3 and SEQ ID NO: 4.

10. The kit according to claim 1 , wherein the forked universal adaptors are formed by annealing partially complementary first methylated and second methylated polynucleotide strands, wherein a sequence of 5 or more consecutive nucleotides at 3′ end of the first strand is complementary to a sequence of 5 or more consecutive nucleotides at the 5′ end of the second strand, wherein a duplex region of 5 or more consecutive base pairs is formed by annealing the first and second strands and wherein a sequence of at least 10 consecutive nucleotides at the 5′ end of the first strand and a sequence of at least 10 consecutive nucleotides at the 3′ end of the second strand are not complementary such that a mismatched single stranded region of at least 10 consecutive nucleotides on each strand remains single stranded when the duplex region is annealed.

11. The kit according to claim 10 , wherein the duplex region formed when the two strands are annealed is 5 to 20 consecutive base pairs in length.

12. The kit according to claim 10 , wherein the mismatched single stranded region comprises 10 to 50 consecutive unpaired nucleotides on each strand.

13. The kit according to claim 1 , wherein the forked universal adaptors comprise one or more of SEQ ID NO: 1 and SEQ ID NO: 2.

14. The kit according to claim 1 , wherein the methylated adaptor polynucleotides and/or oligonucleotide primers are provided in a lyophilized or dried form.

15. The kit according to claim 14 , further comprising a diluent suitable for reconstitution of the methylated adaptor polynucleotides and/or oligonucleotide primers.

16. The kit according to claim 1 , further comprising an amplification reagent selected from the group consisting of a buffer, an enzyme, dNTPs, and a combination thereof.

17. The kit according to claim 16 , wherein the enzyme is a polymerase.

Assignees

• Illumina Cambridge Ltd
• Massachusetts Inst Technology

Inventors

• Gormley Niall
• Gnirke Andreas
• Jaffe David
• Nusbaum Harris

Classifications

• C12Q1/6855 Primary

  Ligating adaptors · CPC title

• C12Q1/6827

  for detection of mutation or polymorphism · CPC title

• C12Q1/6809

  Methods for determination or identification of nucleic acids involving differential detection · CPC title

• C12Q2523/125

  Bisulfite(s) · CPC title

• C12Q2565/501

  being an array of oligonucleotides · CPC title