METHOD OF SEQUENCING AND MAPPING TARGET NUCLEIC ACIDS

• US 20090269771A1
• Filed: 04/24/2009
• Published: 10/29/2009
• Est. Priority Date: 04/24/2008
• Status: Abandoned Application

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: SCHROEDER, Benjamin G.
• Application Number:
  US12/430,005
• Publication Number:
  US 20090269771A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2523/125 Bisulfite(s)
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2537/1376 Displacement by an enzyme

Abstract

The present teachings pertain to methods, compositions, reaction mixtures, and kits for mapping a low complexity sequence to a locus in a genome. In some embodiments, the low complexity sequence can be used to determine the methylation profile of a target nucleic acid. A strand-replacing reaction results in a product containing a first strand and a second strand, which can be connected together with a stem-loop adapter to form a single strand. A sequencing reaction can compare the two strands of the product, allowing the experimentalist to both map the sequence to a locus in a reference genome, as well as ascertain the methylation profile of the original target nucleic acid.

Claims

1. A method of determining the methylation profile of a target nucleic acid comprising;

ligating a first adapter to an extendable 3′

end of a 5′

dephosphorylated target nucleic acid, wherein the target nucleic acid comprises a first native strand and a complementary second strand, and wherein a nick is between a 3′

extendable end of the adapter and the second strand of the target nucleic acid;

extending the extendable 3′

end of the adapter with a strand-replacing polymerase and dATP, dGTP, dTTP, 5-methyl-dCTP, to form a fully methylated second strand, wherein the fully methylated strand is complementary to the first strand;

phosphorylating the first strand to form a phosphorylated 5′

end;

ligating the phosphorylated 5′

end of the first strand to an extendable 3′

end of a second adapter, and ligating an extendable 3′

end of the fully-methylated second strand to a phosphoryated 5′

end of the second adapter, to form a dual-adapter ligation product;

converting non-methylated cytosine in the first native strand of the dual-adapter ligation product to uracil to form a converted native strand in a converted dual-adapter ligation product; and

,

comparing the identity of the cytosine positions in the fully methylated strand with the identity of the cytosine positions in the converted native strand to determine the methylation profile of the target nucleic acid.

2. The method according to claim 1 wherein the first adapter is a stem-loop adapter.

3. The method according to claim 2 wherein the stem-loop adapter comprises a 5′

phosphorylated end and an extendable 3′

end.

4. The method according to claim 1 wherein the comparing comprises performing a sequencing reaction.

5. The method according to claim 4 wherein the sequencing reaction is an enzyme-mediated extension reaction selected from the group consisting of a ligase-mediated extension of ligation probes, a polymerase-mediated extension of reversible terminators, and a polymerase mediated extension di-deoxy nucleotides.

6. The method according to claim 1 wherein the converting comprises treating with bisulfite.

7. A method of determining the methylation profile of a target nucleic acid comprising;

ligating a first adapter to an extendable 3′

end of the target nucleic acid, wherein the first adapter is a stem-loop molecule comprising an extendable 3′

end and a phosphorylated 5′

end, wherein the target nucleic acid comprises a native first strand and a complementary second strand, and wherein a nick is between the 3′

extendable end of the first adapter and the second strand of the target nucleic acid;

extending the 3′

end of the stem-loop adapter with dATP, dGTP, dTTP, 5-methyl-dCTP to form a fully methylated strand, wherein the fully methylated strand is complementary to the first native strand;

providing a second adapter, wherein the second adapter comprises a first strand and a second strand, wherein the first strand comprises a first primer portion, and an extendable 3′

end, and the second strand comprises a second primer portion and a phosphorylated 5′

end;

ligating the fully methylated second strand to the phosphorylated 5′

end of the second adapter and ligating the first native strand of the target nucleic acid to the extendable 3′

end of the second adapter, to form a dual-adapter ligation product;

converting non-methylated cytosine in the first native strand of the dual-adapter ligation product to uracil to form a converted native strand in a converted dual-adapter ligation product;

immobilizing the converted dual-adapter ligation product on a solid support;

hybridizing a primer to the second primer portion of the converted dual-adapter ligation product;

sequencing the converted dual-adapter ligation product; and

,

comparing the identity of the cytosine positions in the fully-methylated second strand with the identity of the cytosine positions in the converted strand to determine the methylation profile of the target nucleic acid.

8. The method according to claim 7 wherein the sequencing reaction is an enzyme-mediated extension reaction.

9. The method according to claim 7 wherein the converting comprises treating with bisulfite.

10. The method according to claim 7 wherein the first strand of the second adapter further comprises an affinity moiety, and the immobilizing comprises interacting the affinity moiety with an affinity moiety binding partner.

11. The method according to claim 7 wherein the immobilizing comprises covalently attaching the converted dual-adapter ligation product to a bead.

12. A reaction mixture comprising;

(a) an adapter ligated to a first strand of a target nucleic acid,

wherein the target nucleic acid comprises the first strand and a second strand,

wherein the adapter is a stem-loop adapter comprising an extendable 3′

end, and,

wherein a nick exists between the extendable 3′

end of the stem-loop adapter and the second strand of the target nucleic;

(b) a strand-replacing polymerase;

(c) 5-methyl-dCTP; and

,

(d) at least one of DATP, dTTP, dGTP.

13. A strand replacement product, wherein the strand replacement product comprises a high complexity fully methylated strand and a low complexity converted native strand.

14. The composition according to claim 13 wherein the high complexity fully methylated strand comprises 5-methyl-dCTP.

15. A kit for determining the methylation profile of a target nucleic acid comprising;

(a) a first adapter, wherein the first adapter is a stem-loop adapter, and wherein the stem-loop adapter comprises a phosphorylated 5′

end and an extendable 3′

end;

(b) a second adapter, wherein the second adapter comprises a phosphorylated 5′

end;

(c) a strand-replacing polymerase;

(d) a converting agent;

(e) a kinase;

(f) 5-methyl-dCTP; and

,

(g) at least one of dATP, dTTP, dGTP.

16. The kit according to claim 15 further comprising;

(h) a distal-cutting restriction enzyme.

17. The kit according according to claim 15 further comprising;

(i) sequencing reagents.

18. The kit according to claim 15 wherein the converting agent is bisulfite.

19. A method of mapping a low complexity sequence to a locus of a genome comprising;

generating a strand replacement product comprising a high complexity strand and a low complexity strand;

sequencing the high complexity strand; and

,

comparing the sequence of the high complexity strand to the genome in order to map the low complexity strand to a locus of the genome.

20. The method according to claim 19 wherein the high complexity strand is a fully methylated strand and the low complexity strand is a converted native strand.

21. The method according to claim 19 wherein the fully-methylated strand comprises cytosines that are methylated, and the strand-replacing reaction comprises 5-methyl-dCTP.

22. The method according to claim 19 wherein the fully methylated strand comprises adenines that are methylated, and the strand replacing reaction comprises methylated adenines.

23. A method of forming a single-stranded dual-adapter ligation product comprising;

forming an adapter-ligated single-stranded target nucleic acid;

hybridizing a primer to the adapter of the adapter-ligated single-stranded target nucleic acid;

extending the primer in the presence of 5-methyl dCTP to form a double-stranded product comprising a fully methylated strand; and

,

ligating a stem-loop adapter to the double-stranded product to form a single-stranded dual adapter ligation product.

24. The method according to claim 23 wherein the single-stranded dual adapter ligation product is treated with a converting reagent and sequenced to determine the methylation status of a target nucleic acid.