Iterative and regenerative DNA sequencing method

• US 6,599,703 B2
• Filed: 02/16/2001
• Issued: 07/29/2003
• Est. Priority Date: 11/01/1996
• Status: Expired due to Fees

• Current Assignee: University of Iowa Research Foundation
• Original Assignee: University of Iowa Research Foundation
• Inventors: Jones, Douglas H.
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/788,038
• Publication Number:
  US 20020072055A1
• Time in Patent Office: 893 Days
• Field of Search: 435/6, 435/91.2
• US Class Current: 435/6.18
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/313 Type II endonucleases, i.e. cutting outside recognition site
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site

Abstract

An iterative and regenerative method for sequencing DNA is described. This method sequences DNA in discrete intervals starting at one end of a double stranded DNA segment. This method overcomes problems inherent in other sequencing methods, including the need for gel resolution of DNA fragments and the generation of artifacts caused by single-stranded DNA secondary structures. A particular advantage of this invention is that it can create offset collections of DNA segments and sequence the segments in parallel to provide continuous sequence information over long intervals. This method is also suitable for automation and multiplex automation to sequence large sets of segments.

Claims

1. A method for automated sequencing of double-strande DNA segments with nested single strand overhang templates, such method compromising the steps of:

i) providing a support array having a plurality of sample holders arrayed in a matrix of positions on the support, and wherein the array is on a stage;

ii) immobilizing a plurality of double-stranded DNA segments at respective sample holders of said array, each DNA segment having an end compromising a single-strand overhang template sequence no longer than about twenty nucleotides in length, wherein said overhang sequence is created by a process including ligation of a strand of a recognition domain to each template and digestion by an enzyme that cuts at a site at least one nucleotide away from the recognition domain, and includes ligation of a DNA sequence that can be used to generate a primer annealing site during DNA amplification in vitro following ligation of the recognition domain and prior to generation of the DNA template;

iii) simultaneously treating all sample holders with one or more reagents which selectively react with at least one nucleotide of said single-strand overhang template to effectively label the material at each holder;

iv) reading said array by automated scan detection to thereby determine at least one nucleotide of said single-strand overhang template; and

v) reducing length of each strand of said DNA segment at each holder by a fixed number n>

1 at said overhang end to produce a homologously ordered array of shorter and nested DNA segments, each with a single-strand overhang template sequence, and further performing steps iii) and iv) to determine at least one nucleotide at each single-strand overhang sequence, wherein the steps of treating, reading and reducing the length of the strands of the DNA segment at each holder by a number of n>

1 nucleotides are iteratively performed as automated process steps to produce nested and progressively shorter DNA segments and to sequence the plurality of DNA segments immobilized at the array of sample holders in situ.

2. The method of claim 1, wherein said array is a chip or a microtiter support array.

3. The method of claim 1, wherein said stage is rotatable for spinning to cause fluid provided at a central position thereof to flow across the array by centrifugal flow, and wherein the step of treating with one or more reagents includes flowing a reagent through said array to alter material immobilized in the sample holders.

4. The method of claim 1, wherein said stage includes heat cycling means for cyclically heating the support array, and the step of treating includes treating at least a portion of material at each sample holder with a primer and operating the heat cycling means to regenerate material at the respective sample holders.

5. The method of claim 1, wherein step i) is preceded by treating each initial DNA segment to produce a set of n DNA segments with respective nested single-strand templates, thereafter reducing the length of each template in intervals of n nucleotides so that the nested sequences from said n templates provides a continuous sequence for said initial DNA segment, thereby increasing the length of continuous DNA sequenced for a given number of steps.

6. The method of claim 1, wherein the step of reducing length to produce a homologously ordered array of DNA segments includes the steps of transferring an aliquot of material from each sample holder to a corresponding sample holder on a separate support array, and enzymatically removing a fixed length >

one nucleotide from each DNA strand.

7. The method claim 5, wherein the step of treating each initial DNA segment to produce a set of n DNA segments with respective nested single-strand templates includes the steps of transferring an aliquot of material from each sample holder to a corresponding sample holder on a separate support array.

8. The method of claim 3, wherein the step of reducing the length of each strand by n nucleotides reduces by n<

60 nucleotides, and said automated process steps are performed by arranging around a circumference on said stage m support arrays A₁, A₂ . . . A_m, each of said m support arrays communicating at a radially inner point with one fluid support channel of a set m fluid supply channels C₁, C₂ . . . C_m, such that all sample holders of an array are treated with a flow of a common reagent.

9. The method of claim 8, wherein m≧

n, and arranging that each array A_i receives reagents along channel C_i to form an overhang at position i with respect to the original DNA segment, whereby each sample is sequenced in steps >

1 and ≦

n nucleotides and the m arrays span the full sequence of nucleotides over a continuous span of each double-stranded DNA segment.

10. The method of claim 8, wherein said m fluid supply channels are provided with reagents effective to label the templates in array A₁, A₂ . . . A_m, and further compromising the step of reading m successive nucleotides by scanning the corresponding sample holders on each of the m support arrays after reducing said length.

11. The method of claim 1, wherein the step of immobilizing a plurality of DNA segments at respective sample holders of an array includes immobilizing a plurality of DNA segments and creating a single strand overhang template on each immobilized DNA segment in situ.

12. The method of claim 1, wherein said enzyme is a class-IIS restriction endonuclease.

13. The method of claim 1, wherein DNA amplification in vitro occurs through PCR.

14. The method of claim 1, further compromising the step of separating an aliquot from each sample holder of the array to a further sample holder and amplifying material of the aliquot by DNA amplification in vitro.

15. The method of claim 14, wherein the step of separating an aliquot include immobilizing the aliquot on a hedgehog comb.

16. The method of claim 13, further compromising the step of retaining an aliquot in each sample holder of the array and amplifying material of the aliquot by the DNA amplification in vitro.

17. The method of claim 1, wherein the DNA templates are relatively inaccessible to primer annealing, resulting in a low magnitude of DNA amplification.

18. The method of claim 17, wherein DNA templates are made relatively inaccessible to primer annealing through immobilization.

19. The method of claim 1, further including the step of methylating sites of the segments outside the ligated recognition domain strand.

20. A method for automated sequencing of double-stranded DNA segments, such method compromising the steps of:

i) providing a support array having a plurality of sample holders arrayed in a matrix of positions on the support, wherein the array is on stage;

ii) immobilizing a plurality of double-stranded DNA segments at respective sample holders of said array, each DNA segment having an end compromising a single-strand overhang template sequence no longer than about twenty nucleotides in length;

iii) simultaneously treating all sample holders with one or more reagents which selectively react with at least one nucleotide of said single-strand overhang template to effectively label the material at each holder;

iv) reading said array by automated scan detection to thereby determine at least one nucleotide of said single-strand overhang template;

v) regenerating material at the respective sample holders by DNA amplification in vitro; and

vi) reducing length of each strand of said DNA segment at each holder by a fixed number n>

1 at said overhang end to produce a homologously ordered array of shorter DNA segments, each with a single-strand overhang template sequence, and further performing step iii) to determine at least one nucleotide at each single-strand overhang sequence, wherein the steps of treating, reading, reducing lengths and product regeneration are iteratively performed as automated process steps to produce progressively shorter DNA segments and to sequence the plurality of DNA segments immobilized at the array of sample holders in situ.

21. The method of claim 20, wherein said stage is rotatable for spinning to cause fluid provided at a central position thereof to flow across the array by centrifugal flow, and wherein the step of treating with one or more reagents includes flowing a reagent through said array to alter material immobilized in the sample holders.

22. The method of claim 20, wherein said stage includes heat cycling means for cyclically heating the support array, and the step of treating includes treating at least a portion of material at each sample holder with a primer and operating the heat cycling means to regenerate material at the respective sample holders.

23. The method of claim 20, wherein n>

1, and step i) is preceded by treating each initial DNA segment to produce a set of n DNA segments with respective nested single-strand templates, and thereafter reducing the length of each template in intervals of n nucleotides so that the nested sequences from said n templates provides a continuous sequence for said initial DNA segment, thereby increasing the length of continuous DNA sequenced for a given number for a given number of steps.

24. The method of claim 20, wherein the steps of reducing length to produce a homologously ordered array of DNA segments includes the steps of transferring an aliquot of material from each sample holder to a corresponding sample holder on a separate support array.

25. The method of claim 23, wherein the step of treating each initial DNA segment to produce a set of n DNA segments with a respective nested single-strand templates includes the steps of transferring an aliquot of material from each sample holder to a corresponding sample holder on a separate support array.

26. The method of claim 21, wherein the step of reducing the length of each strand by nucleotides reduces by n<

60 nucleotides, and said automated process steps are performed by arranging around a circumference on said stage m support arrays A₁, A₂ . . . A_m, each of said m support arrays communicating at a radially inner point with one fluid support channel of a set of m fluid supply channels C₁, C₂ . . . C_m, such that all sample holders of an array are treated with a flow of a common reagent.

27. The method of claim 26, wherein m≧

n, and arranging that each array A_i receives reagents along channel C_i to form an overhang at position i with respect to the original DNA segment, whereby each sample is sequenced in steps of >

1 and ≦

n nucleotides and the m arrays span the full sequence of nucleotides over a continuous span of each double-stranded DNA segment.

28. The method of claim 26, wherein said m fluid supply channels are provided with reagents effective to label the templates in array A₁, A₂ . . . A_m, and further compromising the step of reading m successive nucleotides by scanning the corresponding sample holders on each of the m support arrays after reducing said length.

29. The method of claim 20, wherein the step of immobilizing a plurality of DNA segments at respective sample holders of an array includes immobilizing a plurality of DNA segments and creating a single strand overhang template on each immobilizes DNA segment in situ.

30. The method of claim 29, wherein the single strand overhang sequence is created by a process including ligation of a recognition domain strand to each template and digestion by an enzyme that cuts at a site at least on nucleotide away from the recognition domain.

31. The method of claim 30, wherein said enzyme is a class-IIS restriction endonuclease.

32. The method of claim 31, wherein ligation of a strand of a recognition domain includes ligation of a DNA sequence that can be used to generate a primer annealing site during DNA amplification in vitro following ligation of the recognition domain and prior to generation of the DNA template.

33. The method of claim 32, wherein DNA amplification in vitro occurs through PCR.

34. The method of claim 32, further comprising the step of separating an aliquot from each sample holder of the array to a further sample holder and amplifying material of the aliquot by DNA amplification in vitro.

35. The method of claim 34, wherein the step of separating an aliquot includes immobilizing the aliquot on a hedgehog comb.

36. The method of claim 32, further comprising the step of retaining an aliquot in each sample holder of the array and amplifying material of the aliquot by DNA amplification in vitro.

37. The method of claim 32, wherein the DNA templates are relatively inaccessible to primer annealing, resulting in a low magnitude of DNA amplification.

38. The method of claim 37, wherein DNA templates are made relatively inaccessible to primer annealing through immobilization.

39. The method of claim 32, further including the step of methylating sites of the segments outside the ligated recognition domain strand.