Iterative and regenerative DNA sequencing method

• US 20030175780A1
• Filed: 02/24/2003
• Published: 09/18/2003
• Est. Priority Date: 11/01/1996
• Status: Abandoned Application

• Current Assignee: University of Iowa Research Foundation
• Original Assignee: University of Iowa Research Foundation
• Inventors: Jones, Douglas H.
• Application Number:
  US10/372,696
• Publication Number:
  US 20030175780A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• 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 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• 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
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2537/149 Sequential reactions

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 identifying a first nucleotide n and a second nucleotide n+x in a double stranded nucleic acid segment, comprising:

a) digesting said double stranded nucleic acid segment with a restriction enzyme to produce a double stranded molecule having a single stranded overhang sequence corresponding to an enzyme cut site;

b) providing an adaptor having a cycle identification tag, a restriction enzyme recognition domain, a sequence identification region, and a detectable label;

c) hybridizing said adaptor to said double stranded nucleic acid having said single-stranded overhang sequence to form a ligated molecule;

d) identifying said nucleotide n by identifying said ligated molecule;

e) amplifying said ligated molecule from step (d) with a primer specific for said cycle identification tag of said adaptor; and

f) repeating steps (a) through (d) on said amplified molecule from step (e) to yield the identity of said nucleotide n+x,

wherein x is less than or equal to the number of nucleotides between a recognition domain for a restriction enzyme and an enzyme cut site.

2. The method of claim 1, wherein said enzyme cut site is the cut site located the farthest away from said recognition domain.

3. The method of claim 1, wherein said restriction enzyme of step (a) is a class-IIS restriction endonuclease.

4. The method of claim 3, wherein said class-IIS restriction endonuclease is selected from the group consisting of AccBSI, AceIII, AciI, AclWI, AlwI, Alw26I, AlwXI, Asp26HI, Asp27HI, Asp35HI, Asp36HI, Asp40HI, Asp50HI, AsuHPI, BaeI, BbsI, BbvI, BbvII, Bbv16II, Bce83I, BcefI, BcgI, Bco5I, Bco116I BcoKI, BinI, Bli736I, BpiI, BpmI, Bpu10I, BpuAI, BsaI, BsaMI, Bsc91I, BscAI, BscCI, BseII, Bse3DI, BseNI, BseRI, BseZI, BsgI, BsiI, BsmI, BsmAI, BsmBI, BsmFI, Bsp24I, Bsp423I, BspBS31I, BspIS4I, BspKT5I, BspLU11III, BspMI, BspPI, BspST5I, BspTS514I, BsrI, BsrBI, BsrDI, BsrSI, BssSI, Bst11I, Bst71I, Bst2BI, BstBS32I, BstD1021, BstF51, BstTS51, Bsu6l, CjeI, CjePI, Eam1104I, EarI, Eco31I, Eco57I, EcoA41, EcoO44I, Esp3I, FauI, FokI, GdiII, GsuI, HgaI, HphI, Ksp632I, MboII, MlyI, MmeI, Mn1I, Mva1269I, PhaI, PieI, RleAI, SapI, SfaNI, SimI, StsI, TaqII, TspII, TspRI, Tth11II, and VpaK32I.

5. The method of claim 1, wherein a nucleic acid ligase is used to attach at least one strand of said restriction enzyme recognition domain of step (b) to said nucleic acid segment.

6. The method of claim 1, wherein said method further comprises blocking an enzyme recognition domain lying outside said enzyme recognition domain of step (b).

7. The method of claim 6, wherein said blocking occurs through an in vitro primer extension.

8. The method of claim 7, wherein said in vitro primer extension is DNA amplification in vitro.

9. The method of claim 8, wherein said DNA amplification in vitro occurs during said amplification in step (e).

10. The method of claim 7, wherein said in vitro primer extension occurs following said amplification in step (e).

11. The method of claim 7, wherein said method further comprises hemi-methylating an enzyme recognition domain lying outside said enzyme recognition domain of step (b).

12. The method of claim 11, wherein said hemi-methylation occurs through an in vitro primer extension using a primer having a portion of said enzyme recognition domain that blocks enzyme recognition if it is hemi-methylated.

13. The method of claim 12, wherein said primer extension occurs with a methylated nucleotide.

14. The method of claim 7, wherein said restriction endonuclease recognizes a hemi-methylated recognition domain, and the primer contains at least one methylated nucleotide in a methylated portion of said recognition domain.

15. The method of claim 1, wherein said nucleic acid segment is a genomic DNA.

16. The method of claim 1, wherein said nucleic acid segment is a cDNA.

17. The method of claim 1, wherein said nucleic acid segment is a product of an in vitro DNA amplification.

18. The method of claim 1, wherein said nucleic acid segment is a PCR product.

19. The method of claim 1, wherein said nucleic acid segment is a product of a strand displacement amplification.

20. The method of claim 1, wherein said nucleic acid segment is a vector insert.

21. The method of claim 1, wherein said detectable label is selected from the group consisting of one or more fluorescent, near infra-red, radionucleotide and chemilluminescent labels.

22. The method of claim 1, wherein said nucleic acid segment is attached to a solid matrix.

23. The method of claim 22, wherein said solid matrix is a magnetic streptavidin.

24. The method of claim 22, wherein said solid matrix is a magnetic glass particle.

25. The method of claim 1, wherein said adaptor of step (b) is attached to a solid matrix.

26. The method of claim 25, wherein said solid matrix is a magnetic streptavidin.

27. The method of claim 25, wherein said solid matrix is a magnetic glass particle.

28. A method for sequencing an interval within a double stranded nucleic acid segment by identifying a first nucleotide n and a second nucleotide n+x in a plurality of staggered double stranded molecules produced from said double stranded nucleic acid segment, comprising:

a) attaching an enzyme recognition domain to different positions along said double stranded nucleic acid segment within an interval no greater than the distance between a recognition domain for a restriction enzyme and an enzyme cut site, such attachment occurring at one end of said double. stranded nucleic acid segment;

b) digesting said double stranded nucleic acid segment with a restriction enzyme to produce a plurality of staggered double stranded molecules each having a single stranded overhang sequence corresponding to said cut site;

c) providing an adaptor having a restriction enzyme recognition domain, a sequence identification region, and a detectable label;

d) hybridizing said adaptor to said double stranded nucleic acid having said single-stranded overhang sequence to form a ligated molecule;

e) identifying a nucleotide n within a staggered double stranded molecule by identifying said ligated molecule;

f) repeating steps (b) through (e) to yield the identity of said nucleotide n+x in each of said staggered double stranded molecules having said single strand overhang sequence thereby sequencing an interval within said double stranded nucleic acid segment,

wherein x is greater than one and no greater than the number of nucleotides between a recognition domain for a restriction enzyme and an enzyme cut site.

29. The method of claim 28, wherein said enzyme cut site is the cut site located the farthest away from said recognition domain.

30. The method of claim 28, wherein said restriction enzyme of step (b) is a class-IIS restriction endonuclease.

31. The method of claim 30, wherein said class-IIS restriction endonuclease is selected from the group consisting of AceBSI, AceIII, AciI, AclWI, AlwI, Alw26I, AlwXI, Asp26HI, Asp27HI, Asp35HI, Asp36HI, Asp40Hl, Asp50HI, AsuHPI, BaeI, BbsI, BbvI, BbvII, Bbv116I, Bce83I, BcefI, BcgI, Bco5I, Bco116I BcoKI, BinI, Bli736I, BpiI, BpmI, Bpu10I, BpuAI, BsaI, BsaMI, Bsc91I, BscAI, BscCI, BseII, Bse3DI, BseNI, BseRI, BseZI, BsgI, BsiI, BsmI, BsmAI, BsmBI, BsmFI, Bsp24I, Bsp423I, BspBS31I, BspIS4I, BspKT5I, BspLU11III, BspMI, BspPI, BspST5I, BspTS514I, BsrI, BsrBI, BsrDI, BsrSI, BssSI, BstI11I, Bst71I, Bst2BI, BstBS32I, BstD102I, BstF5I, BstTS5I, Bsu6I, CjeI, CjePI, Eam1104I, EarI, Eco31I, Eco57I, EcoA4I, EcoO44I, Esp3I, FauI, FokI, GdiII, GsuI, HgaI, HphI, Ksp632I, MboII, MlyI, MmeI, Mn1I, Mva1269I, PhaI, PieI, RleAI, SapI, SfaNI, SimI, StsI, TaqII, TspII, TspRI, Tth111II, and VpaK32I.

32. The method of claim 28, wherein a nucleic acid ligase is used to attach at least one strand of said restriction enzyme recognition domain of step (c) to said nucleic acid segment.

33. The method of claim 28, wherein said method further comprises blocking an enzyme recognition domain lying outside said enzyme recognition domain of step (c).

34. The method of claim 33, wherein said method further comprises methylating an enzyme recognition domain lying outside said enzyme recognition domain of step (c).

35. The method of claim 34, wherein said methylation occurs through in vitro reaction with a methylase that recognizes the enzyme recognition domain of step (c).

36. The method of claim 35, wherein said methylase is a FokI methylase.

37. The method of claim 33, wherein said blocking occurs through an in vitro primer extension.

38. The method of claim 37, wherein said in vitro primer extension is DNA amplification in vitro.

39. The method of claim 37, wherein said method further comprises hemi-mythylating an enzyme recognition domain lying outside said enzyme recognition domain of step (c).

40. The method of claim 39, wherein said hemi-methylation occurs through an in vitro primer extension using a primer having a portion of said enzyme recognition domain that blocks enzyme recognition if it is hemi-methylated.

41. The method of claim 40, wherein said primer extension occurs with a methylated nucleotide.

42. The method of claim 37, wherein said restriction endonuclease recognizes a hemi-methylated recognition domain, and the primer contains at least one methylated nucleotide in a methylated portion of said recognition domain.

43. The method of claim 28, wherein said nucleic acid segment is a genomic DNA.

44. The method of claim 28, wherein said nucleic acid segment is a cDNA.

45. The method of claim 28, wherein said nucleic acid segment is a product of an in vitro DNA amplification.

46. The method of claim 28, wherein said nucleic acid segment is a PCR product.

47. The method of claim 28, wherein said nucleic acid segment is a product of a strand displacement amplification.

48. The method of claim 28, wherein said nucleic acid segment is a vector insert.

49. The method of claim 28, wherein said detectable label is selected from the group consisting of one or more fluorescent, near infra-red, radionucleotide and chemilluminescent labels.

50. The method of claim 28, wherein said nucleic acid segment is attached to a solid matrix.

51. The method of claim 50, wherein said solid matrix is a magnetic streptavidin.

52. The method of claim 50, wherein said solid matrix is a magnetic glass particle.

53. The method of claim 28, wherein said adaptor of step (c) is attached to a solid matrix.

54. The method of claim 53, wherein said solid matrix is a magnetic streptavidin.

55. The method of claim 53, wherein said solid matrix is a magnetic glass particle.

56. A method for identifying a first nucleotide n and a second nucleotide n+x in a double stranded nucleic acid segment, comprising:

a) digesting said double stranded nucleic acid segment with a restriction enzyme to produce a double stranded molecule having a 5′

single stranded overhang sequence corresponding to an enzyme cut site;

b) identifying said nucleotide n by template-directed polymerization with a labeled nucleotide or nucleotide terminator;

c) providing an adaptor having a cycle identification tag and a restriction enzyme recognition domain;

d) ligating said adaptor to said double stranded nucleic acid to form a ligated molecule;

e) amplifying said ligated molecule from step (d) with a primer specific for said cycle identification tag of said adaptor; and

f) repeating steps (a) through (b) on said amplified molecule from step (e) to yield the identity of said nucleotide n+x,

wherein x is less than or equal to the number of nucleotides between a recognition domain for a restriction enzyme and an enzyme cut site.

57. The method of claim 56, wherein said enzyme cut site is the cut site located the farthest away from said recognition domain.

58. The method of claim 56, wherein said restriction enzyme of step (a) is a class-IIS restriction endonuclease.

59. The method of claim 58, wherein said class-IIS restriction endonuclease is selected from the group consisting of AccBSI, AceIII, AciI, AclWI, AlwI, Alw26I, AlwXI, Asp26HI, Asp27HI, Asp35HI, Asp36HI, Asp40Hl, Asp50HI, AsuHPI, BaeI, BbsI, BbvI, BbvII, Bbv16II, Bce83I, BcefI, BcgI, Bco5I, Bco116I BcoKI, BinI, Bli736I, BpiI, BpmI, Bpu10I, BpuAI, BsaI, BsaMI, Bse9II, BscAI, BscCI, BseII, Bse3DI, BseNI, BseRI, BseZI, BsgI, BsiI, BsmI, BsmAI, BsmBI, BsmFI, Bsp24I, Bsp423I, BspBS31I, BspIS4I, BspKT5I, BspLU11III, BspMI, BspPI, BspST5I, BspTS514I, BsrI, BsrBI, BsrDI, BsrSI, BssSI, Bst11I, Bst71I, Bst2BI, BstBS321, BstD102I, BstF5I, BstTS5I, Bsu6I, CjeI, CjePI, Eam1104I, EarI, Eco31I, Eco57I, EcoA4I, EcoO44I, Esp3I, FauI, FokI, GdiII, GsuI, HgaI, HphI, Ksp632I, MboII, MlyI, MmeI, Mn1I, Mva1269I, PhaI, PieI, RleAI, SapI, SfaNI, SimI, StsI, TaqII, TspII, TspRI, Tth111II, and VpaK32I.

60. The method of claim 56, wherein a nucleic acid ligase is used to attach at least one strand of said restriction enzyme recognition domain of step (c) to said nucleic acid segment.

61. The method of claim 56, wherein said method further comprises blocking an enzyme recognition domain lying outside said enzyme recognition domain of step (c).

62. The method of claim 61, wherein said blocking occurs through an in vitro primer extension.

63. The method of claim 62, wherein said in vitro primer extension is DNA amplification in vitro.

64. The method of claim 63, wherein said DNA amplification in vitro occurs during said amplification in step (e).

65. The method of claim 62, wherein said in vitro primer extension occurs following said amplification in step (e).

66. The method of claim 62, wherein said method further comprises hemi-methylating an enzyme recognition domain lying outside said enzyme recognition domain of step (c).

67. The method of claim 66, wherein said hemi-methylation occurs through an in vitro primer extension using a primer having a portion of said enzyme recognition domain that blocks enzyme recognition if it is hemi-methylated.

68. The method of claim 67, wherein said primer extension occurs with a methylated nucleotide.

69. The method of claim 62 wherein said restriction endonuclease recognizes a hemi-methylated recognition domain, and the primer contains at least one methylated nucleotide in a methylated portion of said recognition domain.

70. The method of claim 56, wherein said nucleic acid segment is a genomic DNA.

71. The method of claim 56, wherein said nucleic acid segment is a cDNA.

72. The method of claim 56, wherein said nucleic acid segment is a product of an in vitro DNA amplification.

73. The method of claim 56, wherein said nucleic acid segment is a PCR product.

74. The method of claim 56, wherein said nucleic acid segment is a product of a strand displacement amplification.

75. The method of claim 56, wherein said nucleic acid segment is a vector insert.

76. The method of claim 56, wherein said label is selected from the group consisting of one or more fluorescent, near infra-red, radionucleotide and chemilluminescent labels.

77. The method of claim 56, wherein said nucleic acid segment is attached to a solid matrix.

78. The method of claim 77, wherein said solid matrix is a magnetic streptavidin.

79. The method of claim 77, wherein said solid matrix is a magnetic glass particle.

80. The method of claim 56, wherein said adaptor of step (c) is attached to a solid matrix.

81. The method of claim 80, wherein said solid matrix is a magnetic streptavidin.

82. The method of claim 80, wherein said solid matrix is a magnetic glass particle.

83. The method of claim 56, wherein said step (a) is modified to generate a blunt end in said nucleic acid segment.

84. The method of claim 83, wherein said step (b) is modified to identify a nucleotide in said blunt end of said nucleic acid segment by using a 3′

exonuclease activity of a DNA polymerase to generate a single nucleotide long single-stranded nucleic acid template.

85. The method of claim 84, said method further comprising sequencing said nucleotide by a template-directed polymerization with a labeled nucleotide or nucleotide terminator.

86. The method of claim 85, wherein said template-directed polymerization is followed by identification of an incorporated label.

87. A method for sequencing an interval within a double stranded nucleic acid segment by identifying a first nucleotide n and a second nucleotide n+x in a plurality of staggered double stranded molecules produced from said double stranded nucleic acid segment, comprising:

a) attaching an enzyme recognition domain to different positions along said double stranded nucleic acid segment within an interval no greater than the distance between a recognition domain for a restriction enzyme and an enzyme cut site, such attachment occurring at one end of said double stranded nucleic acid segment;

b) digesting said double stranded nucleic acid segment with a restriction enzyme to produce a plurality of staggered double stranded molecules each having a 5′

single stranded overhang sequence corresponding to said cut site;

c) identifying a nucleotide n within a staggered double stranded molecule by template-directed polymerization with a labeled nucleotide or nucleotide terminator;

d) providing an adaptor having a restriction enzyme recognition domain;

e) ligating said adaptor to said double stranded nucleic acid to form a ligated molecule;

f) repeating steps (b) through (c) to yield the identity of said nucleotide n+x in each of said staggered double stranded molecules having said single strand overhang sequence thereby sequencing an interval within said double stranded nucleic acid segment,

wherein x is greater than one and no greater than the number of nucleotides between a recognition domain for a restriction enzyme and an enzyme cut site.

88. The method of claim 87, wherein said enzyme cut site is the cut site located the farthest away from said recognition domain.

89. The method of claim 87, wherein said restriction enzyme of step (b) is a class-IIS restriction endonuclease.

90. The method of claim 89, wherein said class-IIS restriction endonuclease is selected from the group consisting of AccBSI, AceIII, AciI, AclWI, AlwI, Alw26I, AlwXI, Asp26HI, Asp27HI, Asp35HI, Asp36HI, Asp40HI, Asp50HI, AsuHPI, BaeI, BbsI, BbvI, BbvII, Bbv16II, Bce83I, BcefI, BcgI, Bco5I, Bco116I BcoKI, BinI, Bli736I, BpiI, BpmI, Bpu10I, BpuAI, BsaI, BsaMI, Bsc91I, BscAI, BscCI, BseII, Bse3DI, BseNI, BseRI, BseZI, BsgI, BsiI, BsmI, BsmAI, BsmBI, BsmFI, Bsp24I, Bsp423I, BspBS31I, BspIS4I, BspKT5I, BspLU11III, BspMI, BspPI, BspST5I, BspTS514I, BsrI, BsrBI, BsrDI, BsrSI, BssSI, Bst11I, Bst71I, Bst2BI, BstBS32I, BstD102I, BstF5, BstTS5I, Bsu6I, CjeI, CjePI, Eam1104I, EarI, Eco31I, Eco57I, EcoA4I, EcoO44I, Esp3I, FauI, FokI, GdiII, GsuI, HgaI, HphI, Ksp632I, MboII, MlyI, MmeI, Mn1I, Mva1269I, PhaI, PieI, RleAI, SapI, SfaNI, SimI, StsI, TaqII, TspII, TspRI, Tth111II, and VpaK32I.

91. The method of claim 87, wherein a nucleic acid ligase is used to attach at least one strand of said restriction enzyme recognition domain of step (d) to said nucleic acid segment.

92. The method of claim 87, wherein said method further comprises blocking an enzyme recognition domain lying outside said enzyme recognition domain of step (d).

93. The method of claim 92, wherein said method further comprises methylating an enzyme recognition domain lying outside said enzyme recognition domain of step (d).

94. The method of claim 93, wherein said methylation occurs through in vitro reaction with a methylase that recognizes the enzyme recognition domain of step (d).

95. The method of claim 94, wherein said methylase is a FokI methylase.

96. The method of claim 92, wherein said blocking occurs through an in vitro primer extension.

97. The method of claim 96, wherein said in vitro primer extension is DNA amplification in vitro.

98. The method of claim 96, wherein said method further comprises hemi-mythylating an enzyme recognition domain lying outside said enzyme recognition domain of step (d).

99. The method of claim 98, wherein said hemi-methylation occurs through an in vitro primer extension using a primer having a portion of said enzyme recognition domain that blocks enzyme recognition if it is hemi-methylate.

100. The method of claim 99, wherein said primer extension occurs with a methylated nucleotide.

101. The method of claim 96, wherein said restriction endonuclease recognizes a hemi-methylated recognition domain, and the primer contains at least one methylated nucleotide in a methylated portion of said recognition domain.

102. The method of claim 87, wherein said nucleic acid segment is a genomic DNA.

103. The method of claim 87, wherein said nucleic acid segment is a cDNA.

104. The method of claim 87, wherein said nucleic acid segment is a product of an in vitro DNA amplification.

105. The method of claim 87, wherein said nucleic acid segment is a PCR product.

106. The method of claim 87, wherein said nucleic acid segment is a product of a strand displacement amplification.

107. The method of claim 87, wherein said nucleic acid segment is a vector insert.

108. The method of claim 87, wherein said detectable label is selected from the group consisting of one or more fluorescent, near infra-red, radionucleotide and chemilluminescent labels.

109. The method of claim 87, wherein said nucleic acid segment is attached to a solid matrix.

110. The method of claim 109, wherein said solid matrix is a magnetic streptavidin.

111. The method of claim 109, wherein said solid matrix is a magnetic glass particle.

112. The method of claim 87, wherein said adaptor of step (d) is attached to a solid matrix.

113. The method of claim 112, wherein said solid matrix is a magnetic streptavidin.

114. The method of claim 112, wherein said solid matrix is a magnetic glass particle.

115. The method of claim 87, wherein said step (b) is modified to generate a blunt end in said nucleic acid segment.

116. The method of claim 115, wherein said step (c) is modified to identify a nucleotide in said blunt end of said nucleic acid segment by using a 3′

exonuclease activity of a DNA polymerase to generate a single nucleotide long single-stranded nucleic acid template.

117. The method of claim 116, said method further comprising sequencing said nucleotide by a template-directed polymerization with a labeled nucleotide or nucleotide terminator.

118. The method of claim 117, wherein said template-directed polymerization is followed by identification of an incorporated label.

119. A method for removing all or a part of a primer sequence from a primer extended product, comprising:

a) providing a primer sequence encoding a methylated portion of a restriction endonuclease recognition domain, wherein recognition of said domain by a restriction endonuclease requires at least one methylated nucleotide;

b) polymerizing by a template-directed primer extension using said primer and a nucleic acid segment to generate a primer extended product; and

c) digesting said primer extended product with a restriction endonuclease that recognizes the resulting double-stranded restriction endonuclease recognition domain encoded by said primer sequence in said primer extended product.

120. The method of claim 119, wherein a sequence complimentary to said primer sequence is also removed by said restriction endonuclease digestion in said step (c).

121. The method of claim 119, wherein said restriction endonuclease of step (c) is a class-IIS restriction endonuclease.

122. The method of claim 121, wherein said digestion with said class IIS restriction endonuclease of step (c) generates a single-strand extension no longer than 10 nucleotides in length that is not encoded by said primer encoding at least part of said restriction endonuclease recognition domain.

123. The method of claim 119, wherein said template-directed primer extension in said step (b) occurs during nucleic acid amplification in vitro.

124. The method of claim 123, wherein said nucleic acid amplification in vitro is linear.

125. The method of claim 123, wherein said nucleic acid amplification in vitro is exponential.

126. The method of claim 125, wherein said nucleic acid amplification in vitro is PCR.

127. The method of claim 125, wherein said nucleic acid amplification in vitro is strand displacement amplification.

128. A method for blocking a restriction endonuclease recognition domain in a primer extended product, comprising:

a) providing a primer with at least one modified nucleotide, wherein said modified nucleotide blocks an enzyme recognition domain, and at least a portion of said enzyme recognition domain sequence is encoded in said primer.

b) polymerizing by a template-directed primer extension using said primer and a nucleic acid segment to generate a primer extended product; and

c) digesting said primer extended product with an enzyme that recognizes a double-stranded enzyme recognition domain in said primer extended product.

129. The method of claim 128, wherein said modified nucleotide is a methylated nucleotide.

130. The method of claim 128, wherein said template directed primer extension in said step (b) occurs during nucleic acid amplification in vitro.

131. The method of claim 130, wherein said amplification in vitro is linear.

132. The method of claim 130, wherein said amplification in vitro is exponential.

133. The method of claim 132, wherein said amplification in vitro is PCR.

134. The method of claim 132, wherein said amplification in vitro is strand displacement amplification.

135. The method of claim 128, wherein said nucleic acid template is part of a construct consisting of an insert in a vector.

136. A method for automated sequencing of double-stranded DNA segments with nested single strand overhang templates, such method comprising the steps of

i) providing a support array having a plurality of sample holders arrayed in a matrix of positions on the support

ii) immobilizing a plurality of double-stranded DNA segments at respective sample holders of said array, each DNA segment having an end comprising a single-strand overhang template sequence no long 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, 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.

137. The method of claim 136, wherein said array is a chip or a microtiter support array.

138. The method of claim 136, wherein the array is on a stage.

139. The method of claim 138, 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.

140. The method of claim 138, 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.

141. The method of claim 136, 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, 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 of steps.

142. The method of claim 136, 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 of >

one nucleotide from each DNA strand.

143. The method of claim 141, 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.

144. The method of claim 139, wherein the step of reducing the length of each stand 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, A_{2 . . . Am}, 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.

145. The method of claim 144, 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.

146. The method of claim 144, wherein said m fluid supply channels are provided with reagents effective to label the templates in array A₁, A₂ . . . A_m, and the step of reading m successive nucleotides by scanning the corresponding sample holders on each of the m support arrays after reducing said length.

147. The method of claim 136, 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.

148. The method of claim 147, wherein the single strand 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.

149. The method of claim 148, wherein said enzyme is a class-IIS restriction endonuclease.

150. The method of claim 149, wherein ligation of a recognition domain strand 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.

151. The method of claim 150, wherein DNA amplification in vitro occurs through PCR.

152. The method of claim 150, 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.

153. The method of claim 152, wherein the step of separating an aliquot includes immobilizing the aliquot on a hedgehog comb.

154. The method of claim 151, 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.

155. The method of claim 150, wherein the method of DNA amplification is of low magnitude by making the DNA templates relatively inaccessible to primer annealing.

156. The method of claim 155, wherein DNA templates are made relatively inaccessible to primer annealing through immobilization.

157. The method of claim 150, further including the step of methylating sites of the segments outside the ligated recognition domain strand.

158. A method for automated sequencing of double stranded DNA segments, such method being characterized by steps of

attaching a recognition domain to each segment to form a set of DNA segments having the recognition domain nested at an interval no greater than the distance between the recognition domain and its cut site for a given enzyme that recognizes said recognition domain

treating the DNA segments with an enzyme that recognizes said attached recognition domain, and cuts each strand of each DNA segment to create an overhang template at a distance of >

1 nucleotide along the DNA segment from said recognition domain, and thereby generating a set of nested overhang templates.

determining at least one nucleotide of each of said nested overhang templates, and thereafter

reducing length of each strand at the end of the DNA segment with the overhang template by >

1 nucleotide to produce a corresponding set of shorter DNA segments each with an overhang template, said step of reducing being performed by removing a block of nucleotides, whereby each shorter DNA segment with an overhang template is a known subinterval of a previous DNA segment with overhang.

159. A method for automated sequencing of double-stranded DNA segments, such method comprising the steps of

i) providing a support array having a plurality of sample holders arrayed in a matrix of positions on the support

ii) immobilizing a plurality of double-stranded DNA segments at respective sample holders of said array, each DNA segment having an end comprising a single-strand overhang template sequence no long 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

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 trimmed 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 trimmed DNA segments and to sequence the plurality of DNA segments immobilized at the array of sample holders in situ.

160. The method of claim 159, wherein said array is a chip or a microtiter support array.

161. The method of claim 159, wherein the array is on a stage.

162. The method of claim 161, 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.

163. The method of claim 161, 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.

164. The method of claim 159, 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 of steps.

165. The method of claim 159, 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.

166. The method of claim 164, 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.

167. The method of claim 162, wherein the step of reducing the length of each stand 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 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.

168. The method of claim 167, 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.

169. The method of claim 167, wherein said m fluid supply channels are provided with reagents effective to label the templates in array A₁, A₂ . . . A_m, and the step of reading m successive nucleotides by scanning the corresponding sample holders on each of the m support arrays after reducing said length.

170. The method of claim 159, 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.

171. The method of claim 170, 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 one nucleotide away from the recognition domain.

172. The method of claim 171, wherein said enzyme is a class-IIS restriction endonuclease.

173. The method of claim 172, 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.

174. The method of claim 173, wherein DNA amplification in vitro occurs through PCR.

175. The method of claim 173, 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.

176. The method of claim 175, wherein the step of separating an aliquot includes immobilizing the aliquot on a hedgehog comb.

177. The method of claim 173, 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.

178. The method of claim 173, wherein the method of DNA amplification is of low magnitude by making the DNA templates relatively inaccessible to primer annealing.

179. The method of claim 178, wherein DNA templates are made relatively inaccessible to primer annealing through immobilization.

180. The method of claim 173, further including the step of methylating sites of the segments outside the ligated recognition domain strand.

181. A method for automated sequencing of double stranded DNA segments, such method being characterized by steps of

attaching a recognition domain to each segment to form DNA segments having the recognition domain.

regenerating the template precursor by DNA amplification in vitro

treating the DNA segments with an enzyme that recognizes said attached recognition domain, and cuts each strand of each DNA segment to create an overhang template at a distance of ≧

1 nucleotide along the DNA segment from said recognition domain

determining at least one nucleotide of said overhang template, and thereafter

reducing length of each strand at the end of the DNA segment with the overhang template by ≧

1 nucleotide to produce a corresponding set of trimmed DNA segments each with an overhang template, said step of reducing being performed by removing a block of nucleotides, whereby each trimmed DNA segment with an overhang template is a known subinterval of a previous DNA segment with overhang.

182. A method for identifying a first nucleotide n and a second nucleotide n+x in a double stranded nucleic acid segment, comprising:

a) digesting said double stranded nucleic acid segment with a restriction enzyme to produce a double stranded molecule having a single stranded overhang sequence corresponding to an enzyme cut site;

b) providing an adaptor having a cycle identification tag, a restriction enzyme recognition domain and a sequence identification region;

c) hybridizing said adaptor to said double stranded nucleic acid having said single-stranded overhang sequence to form a ligated molecule;

d) amplifying said ligated molecule from step (c) with a labeled primer specific for said cycle identification tag, restriction enzyme recognition domain, and a portion of said sequence identification region of said adaptor;

e) identifying said nucleotide n by identifying said primer incorporated into the amplification product; and

f) repeating steps (a) through (e) on said amplified molecule from step (e) to yield the identity of said nucleotide n+x, wherein x is less than or equal to the number of nucleotides between a recognition domain for a restriction enzyme and an enzyme cut site.

183. A method for identifying a first nucleotide n and a second nucleotide n+x in a double stranded nucleic acid segment, comprising:

a) digesting said double stranded nucleic acid segment with a restriction enzyme, resulting in a trimmed end in said double stranded molecule;

b) providing an adaptor having a cycle identification tag and a restriction enzyme recognition domain;

c) ligating said adaptor to the trimmed end of said double stranded nucleic acid to form a ligated molecule;

d) amplifying said ligated molecule from step (c) with a labeled primer specific for said cycle identification tag and said restriction enzyme recognition domain of the adaptor, and for a nucleotide in said trimmed end in said double stranded molecule;

e) identifying said nucleotide n by identifying said primer incorporated into the amplification product; and

f) repeating steps (a) through (e) on said amplified molecule from step (e) to yield the identity of said nucleotide n+x, wherein x is less than or equal to the number of nucleotides between a recognition domain for a restriction enzyme and an enzyme cut site.

184. A method for removing all or part of a primer sequence from a primer extended product comprising:

a) providing a primer sequence comprising at least a portion of a restriction endonuclease recognition domain;

b) polymerizing by a template-directed primer extension using said primer, a methylated nucleotide, and a nucleic acid segment to generate a primer extended product during nucleic acid amplification in vitro, wherein the non-methylated nucleotide corresponding to the methylated nucleotide is contained within said portion of the recognition domain sequence in said primer sequence; and

c) digesting said primer extended product with a restriction endonuclease that recognizes the resulting hemi-methylated double-stranded restriction endonuclease recognition domain of said primer sequence in said primer extended product, and does not recognize the double-methylated products resulting from said nucleic acid amplification in vitro.

185. The method of claim 184, wherein a sequence complementary to said primer sequence is also removed by said restriction endonuclease digestion in said step (c).

186. The method of claim 184, wherein said restriction endonuclease of step (c) is a class-IIS restriction endonuclease.

187. The method of claim 186, wherein said digestion with said class IIS restriction endonuclease generates a single-strand extension no longer than 10 nucleotides in length that is not encoded by said primer encoding at least part of said restriction endonuclease recognition domain.

188. The method of claim 184, wherein said nucleic acid amplification in vitro is linear.

189. The method of claim 184, wherein said nucleic acid amplification in vitro is exponential.

190. The method of claim 189, wherein said nucleic acid amplification in vitro is by PCR.

191. The method of claim 189, wherein said nucleic acid amplification in vitro is strand displacement amplification.