Labeling and Sequencing of Nucleic Acids

• US 20090068645A1
• Filed: 10/11/2005
• Published: 03/12/2009
• Est. Priority Date: 10/11/2004
• Status: Abandoned Application

• Current Assignee: Interaseq Genetics Limited
• Original Assignee: Interaseq Genetics Limited
• Inventors: Sibson, Ross
• Application Number:
  US11/577,024
• Publication Number:
  US 20090068645A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2521/313 Type II endonucleases, i.e. cutting outside recognition site
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid

Abstract

The invention provides for methods of end labelling a ds DNA molecule with a bar-code as well as adaptor mediated methodology for creating single stranded overhanging ends, lengthening single stranded overhanging ends, controlled size reduction of labelled ds DNA molecules, and adaptor mediated sequencing. Also disclosed are methods for parallel sequencing of multiple ds DNA molecules in a mixture by visual means.

Claims

1. A method of differentially labelling one end of a double stranded (ds) DNA molecule on the basis of its nucleic acid sequence, comprising:

(a) providing said ds DNA molecule in linear form with at least one single stranded overhanging end;

(b) incubating, under conditions suitable to allow for DNA ligation, said ds DNA molecule having at least one single stranded overhanging end with a pool of different indexing molecules, the different indexing molecules of the pool having complementary single stranded ends for annealing to the at least one overhanging end of the ds DNA molecule, said different indexing molecules being labelled and distinguishable from one another, to produce a linear ligation product having indexing molecules at each end thereof;

(c) circularising the linear ligation product of step (b) by incubating under conditions suitable to allow for DNA ligation;

(d) linearising the circular product of step (c) by cleavage with a restriction enzyme having a cleavage site that is physically displaced from its recognition site, said recognition site being present in a portion of the circular product which is not derived from the original ds DNA molecule to be labelled, and said cleavage site being located within a portion of said circular product which is derived from the original ds DNA molecule to be labelled, such that a linear DNA molecule is produced having single stranded overhanging ends with nucleotide sequences characteristic of the ds DNA molecule; and

, optionally

(e) repeating steps (b) to (d) one or more times

2. A method as claimed in claim 1 wherein the ds DNA molecule is one of a plurality of DNA molecules in a mixture, said plurality of ds DNA molecules of the mixture having different nucleic acid sequences, wherein two or more of the plurality of ds DNA molecules are differentially labelled.

3. A method as claimed in claim 1 or claim 2 wherein both ends of the ds DNA molecule of step (a) are provided with single stranded overhanging ends.

4. A method as claimed in claim 1 or claim 2 wherein one end of the ds DNA molecule of step (a) is provided with a single stranded overhanging end and the opposite end is blunt ended.

5. A method of any preceding claim wherein the pool of indexing molecules comprises indexing molecule having all possible complementary single stranded ends of a predetermined length for annealing and ligating to all possible single stranded overhanging ends of the ds DNA molecule.

6. A method of any preceding claim wherein the restriction enzyme having a cleavage site that is physically displaced from its recognition site is a Type IIs restriction enzyme or an interrupted palindrome restriction enzyme.

7. A method for controlled size reduction of a ds DNA molecule labelled according to the methods of any preceding claim, comprising:

(a) incubating, under conditions suitable to allow for DNA ligation, said labelled ds DNA molecule with a pool of different adaptor molecules, the different adaptor molecules of the pool having;

(i) complementary single stranded ends for annealing to the overhanging end of the ds DNA molecule distal to the indexing molecule derived label thereof; and

(ii) a recognition site for a restriction enzyme having a cleavage site that is physically displaced from its recognition site

in order to provide a labelled ds DNA molecule/adaptor ligation product wherein the cleavage site for the restriction enzyme of step (a)(ii) is located within a portion of the labelled ds DNA molecule/adaptor ligation product derived from the labelled ds DNA molecule;

(b) cleaving said labelled ds DNA molecule/adaptor ligation product with said restriction enzyme of step (a)(ii) in order to remove the adaptor molecule and to reduce the length of the labelled ds DNA molecule by a controlled number of nucleic acid bases; and

, optionally

(c) repeating steps (a) and (b) one or more times to achieve further controlled size reductions.

8. A method of claim 7 wherein said labelled ds DNA molecule is one of a plurality of labelled DNA molecules in a mixture, said plurality of labelled ds DNA molecules of the mixture having different nucleic acid sequences, and wherein the length of two or more of the plurality of labelled ds DNA molecules is reduced by a controlled number of bases.

9. A method of claim 7 or claim 8 wherein the pool of different adaptor molecules comprises adaptor molecules having all possible complementary single stranded ends of a predetermined length for annealing and ligating to all possible single stranded overhanging ends of the ds DNA molecule.

10. A method of any of claims 7 to 9 wherein the restriction enzyme having a cleavage site that is physically displaced from its recognition site is a Type IIs restriction enzyme or an interrupted palindrome restriction enzyme.

11. A method of determining one or more bases of a ds DNA molecule labelled according to a method of any of claims 1 to 6, comprising:

(a) incubating, under conditions suitable to allow for DNA ligation, said labelled ds DNA molecule with a pool of different sequencing adaptor molecules, the different sequencing adaptor molecules of the pool having complementary single stranded ends for annealing to the overhanging end of the ds DNA molecule distal to the indexing molecule derived label thereof, each of the different sequencing adaptor molecules being differentially labelled, in order to provide a labelled ds DNA molecule/sequencing adaptor ligation product; and

(b) detecting the sequencing adaptor ligated to the labelled ds DNA molecule in step (a) and determining one or more bases of the ds DNA molecule on the basis of the sequencing adaptor detected.

12. A method of claim 11 wherein the size of the labelled ds DNA molecule has been reduced in a controlled manner according to a method of any of claims 7 to 10.

13. A method of claim 11 or claim 12 wherein said labelled ds DNA molecule is one of a plurality of labelled DNA molecules in a mixture, said plurality of labelled ds DNA molecules of the mixture having different nucleic acid sequences, and wherein one or more nucleic acid bases are determined for two or more of said plurality of labelled ds DNA molecules.

14. A method for determining at least a partial nucleic acid sequence of two or more ds DNA molecules in a mixed population of ds DNA molecules having different nucleotide sequences, said method comprising:

(a) differentially labelling one end of said two or more ds DNA molecules in said mixed population according to a method as claimed in any of claims 1 to 6;

(b) conducting controlled size reduction of the differentially labelled ds DNA molecules in the mixed population according to a method of any of claims 7 to 10; and

(c) determining by adaptor mediated sequencing methods one or more nucleic acid bases for two or more of the labelled ds DNA molecules in a sample having undergone controlled size reduction.

15. A method as claimed in claim 14 wherein one or more nucleic acid bases is determined for said two or more labelled ds DNA molecules in at least two samples, each sample having undergone controlled size reduction of the labelled ds DNA molecules therein to a different extent.

16. A method of claim 15 wherein multiple rounds of controlled size reduction are carried out and said determining is carried out for each round of controlled size reduction.

17. A method of claim 15 wherein the mixed population of differentially labelled ds DNA molecules resulting from step (a) is separated into individual pools for controlled size reduction, each pool being subjected to controlled size reduction to a different extent.

18. A method of claim 15 wherein multiple rounds of controlled size reduction are carried out on a single pool, and that pool is sampled after at least one of the rounds of controlled size reduction for the determination of one or more nucleic acid bases for two or more of the labelled ds DNA molecules therein.

19. A method for determining at least a partial nucleic acid sequence of two or more ds DNA molecules in a mixed population of ds DNA molecules having different nucleotide sequences, said method comprising:

(a) differentially labelling one end of said two or more ds DNA molecules in said mixed population according to a method of any of claims 1 to 6;

(b) separating the product of step (a) into two or more separate pools for controlled size reduction of the labelled ds DNA molecules therein;

(c) conducting controlled size reduction on each of the separate pools created in step (b) according to a method of any of claims 7 to 10, wherein each separate pool is subjected to a different number of rounds of controlled size reduction; and

(d) for each separate pool of size reduced ds DNA molecules produced in step (c), determining one or more nucleic acid bases for two or more of the labelled ds DNA molecules using adaptor mediated sequencing.

32. A method of any of claims 1 to 19 wherein at least one single stranded overhanging end for annealing and ligation to indexing molecules, adaptors or sequencing adaptors is created by a method of any of claims 20 to 24 or lengthened according to a method of any of claims 25 to 31.

38. A method as claimed in any preceding claim wherein samples from multiple sources are analysed together in a multiplexed assay e.g., wherein an indexer that identifies the individual of origin is ligated to individual samples before pooling.

20. A method of providing a single stranded overhanging end on a double stranded (ds) DNA molecule comprising engineering a nick in one strand of said ds DNA molecule, and dissociating away from said ds DNA molecule a single stranded fragment from said nicked strand, said fragment extending from the first end to the site of the nick.

21. A method of claim 20 wherein said engineering of the nick comprises ligating a double stranded adaptor molecule to the ds DNA molecule.

22. A method of claim 21 wherein said adaptor molecule is modified such that upon ligation to the ds DNA molecule only one strand of the adaptor molecule is covalently linked to the ds DNA molecule, thereby creating a nick in the other strand.

23. A method of claim 21 wherein said adaptor molecule comprises a recognition site for a nicking endonuclease and wherein following ligation, the nicking site for said nicking endonuclease is located in a portion of the ligation product derived from the ds DNA molecule, the method further comprising the step of incubating the ligation product with said nicking endonuclease to create a nick in one strand.

24. A method of claim 20 wherein the nick is engineered to be within 18 bases of the end of the ds DNA molecule such that an overhang of up to 18 bases results from the step of dissociation of the single stranded fragment.

25. A process for lengthening a single stranded overhanging end on a double stranded (ds) DNA molecule comprising:

(a) ligating to said single stranded overhanging end a lengthening adaptor molecule, said lengthening adaptor molecule having a recognition site for a nicking endonuclease, or being designed to create a recognition site for a nicking endonuclease on ligation to said ds DNA molecule, wherein the nick site for said nicking endonuclease following ligation to the ds DNA molecule is located within a portion of the ligation product derived from the ds DNA molecule;

(b) incubating the ligation product of step (a) with a nicking endonuclease that recognises said nicking endonuclease recognition site in order to nick one strand of the ds DNA molecule; and

(c) cleaving the lengthening adaptor in a predetermined position so that the ds DNA molecule remains ligated to a nucleotide sequence derived from the lengthening adaptor on the second strand, and so that the nucleotide sequence between the point of cleavage and the nick site on the first strand disassociates to leave a lengthened single stranded overhang.

26. A method of claim 25 wherein cleavage of the lengthening adaptor occurs before incubation with the nicking endonuclease.

27. A method of claim 25 wherein cleavage of the lengthening adaptor occurs after incubation with the nicking endonuclease.

28. A method of claim 25 wherein the lengthening adaptor is designed to include a recognition site for a restriction endonuclease to allow for cleavage by that enzyme.

29. A method of claim 25 wherein cleavage of the lengthening adaptor is brought about by the action of a restriction endonuclease.

30. A method of claim 25 wherein the lengthening adaptor contains dUTP at the predetermined cleavage site, and wherein cleavage is brought about by the action of uracil-DNA glycosylase.

31. A process for lengthening a single stranded overhanging end on a double stranded (ds) DNA molecule, comprising:

(a) ligating to said single stranded overhang a lengthening adaptor molecule, wherein said lengthening adaptor molecule;

(i) comprises a recognition site for a type II restriction endonuclease; and

(ii) comprises a recognition site for a nicking endonuclease between the type II endonuclease recognition site and the end of the lengthening adaptor to be ligated to the ds DNA molecule;

or

(iii) is designed to create, on ligation to the ds DNA molecule, a recognition site for a nicking endonuclease between the type II endonuclease recognition site and the ds DNA molecule

and wherein the nick site for said nicking endonuclease, following ligation, is located within a portion of the ligation product derived from the ds DNA molecule;

(b) incubating the ligation product of step (a) with a nicking endonuclease that recognises said nicking endonuclease recognition site in order to nick one strand of the ds DNA molecule;

(c) incubating the nicked ligation product of step (b) with a type II restriction endonuclease that recognises said type II restriction endonuclease recognition site;

optionally wherein the order of steps (b) and (c) are reversed.

33. A method of determining nucleic acid sequence of a DNA molecule comprising:

(a) random cleavage of the DNA molecule into fragments, e.g., by shearing or digestion with a restriction nuclease;

(b) filling in any cohesive ends of fragments produced in step (a) to create blunt ends;

(c) ligating a lengthening adapter to the ends of the fragments, said adapter having restriction sites being suitable for creating a lengthened cohesive end for indexing;

(d) cleaving the adapted fragments with a restriction endonuclease to provide a constant end shared by the adaptored fragments;

(e) ligating an adapter to said constant end, e.g. to provide a PCR primer binding site;

(f) cleaving the adapted fragments with restriction enzymes whose recognition sites are found in the lengthening adapter, so as to provide a lengthened cohesive for indexing distal to the adaptored constant end;

(g) indexing said lengthened cohesive end by ligation to a pool of sequencing adapters, said pool of sequencing adapters comprising adapters having cohesive ends complementary to possible cohesive ends exposed in the DNA molecule by the process of end lengthening, each of the different sequencing adapters having means for detectable differentiation from the other adapters in the pool; and

(h) separating the fragments by electrophoresis and determining the particular sequencing adapter ligated to fragments of different length.

34. A method as claimed in claim 33 wherein said means for detectable differentiation are distinguishable labels.

35. A method of claim 34 wherein said distinguishable labels are fluorescent labels.

36. A method of claims 33 to 35 wherein said means for detectable differentiation comprise a PCR primer binding site.

37. A method of claim 36 wherein determination of the sequencing adapter ligated to fragments of different lengths is by PCR analysis.