Method of producing a DNA library using positional amplification based on the use of adaptors and nick translation
First Claim
1. A method of preparing a DNA molecule having an amplifiable region comprising:
- a) obtaining a DNA sample comprising DNA molecules having regions to be amplified;
b) attaching upstream adaptor molecules to ends of DNA molecules of the sample to provide a nick translation initiation site;
c) subjecting the DNA molecules to nick translation comprising DNA polymerization and 5′
-3′
exonuclease activity to produce nick translate molecules; and
d) attaching downstream adaptor molecules to the nick translate molecules to produce adaptor attached nick translate molecules.
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Abstract
The disclosed invention relates to general and specific methods to use the Primer Extension/Nick Translation (PENT) reaction to create an amplifiable DNA strand, called a PENTAmer. A PENTAmers can be made for the purpose of amplifying a controlled length of DNA located at a controlled position within a DNA molecule, a process referred to as Positional Amplification by Nick Translation (PANT). In contrast to PCR, which amplifies DNA between two specific sequences, PANT can amplify DNA between two specific positions. PENTAmers can be created to amplify very large regions of DNA (up to 500,000 bp) as random mixtures (unordered positional libraries), or as molecules sorted according to position (ordered positional libraries). PANT is fast and economical, because PENTAmer preparation can be multiplexed. A single PENTAmer preparation can include very complex mixtures of DNA such as hundreds of large-insert clones, complete genomes, or cDNA libraries. Subsequent PCR amplification of the preparation using a single specific primer can positionally amplify contiguous regions along a specific clone, along a specific genomic region, or along a specific expressed sequence.
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Citations
206 Claims
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1. A method of preparing a DNA molecule having an amplifiable region comprising:
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a) obtaining a DNA sample comprising DNA molecules having regions to be amplified;
b) attaching upstream adaptor molecules to ends of DNA molecules of the sample to provide a nick translation initiation site;
c) subjecting the DNA molecules to nick translation comprising DNA polymerization and 5′
-3′
exonuclease activity to produce nick translate molecules; and
d) attaching downstream adaptor molecules to the nick translate molecules to produce adaptor attached nick translate molecules. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 182, 183, 186, 187, 188, 196, 197, 198, 199, 206)
a) digesting the DNA molecule with a first sequence-specific endonuclease;
b) ligating both strands of an adaptor molecule to the sequence-specific termini of the template molecules;
c) digesting the DNA molecules with a second sequence-specific endonuclease;
d) incubating the DNA molecules under conditions to favor intramolecular circularization and reduce undesirable intermolecular ligation;
e) concentrating the DNA molecules;
f) initiating a nick-translation reaction for a controlled time; and
g) attaching a down-stream adaptor.
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45. The method of claim 39, wherein said recombination comprises:
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a) methylating the DNA molecules;
b) ligating a first and second adaptor to the ends of the DNA molecule to form a recognition sequence, a single nick-translation initiation site, and a single Eco RI restriction recognition sequence within the recombination domain;
c) activating the adaptors by incubation with a restriction enzyme or nuclease;
d) incubating the DNA molecules under conditions to favor intramolecular circularization and reduce undesirable intermolecular ligation;
e) concentrating the DNA molecules;
f) initiating a nick-translation reaction for a controlled time; and
g) attaching a down-stream adaptor.
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46. The method of claim 1, wherein the adaptor attached nick translate molecules are between 0.5 and 500 kB in length.
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47. The method of claim 1, wherein the DNA sample is cDNA.
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48. The method of claim 1, wherein the DNA sample is genomic DNA.
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49. The method of claim 1, wherein the DNA sample is cloned DNA.
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50. The method of claim 1, wherein the DNA sample is a BAC.
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51. The method of claim 1, wherein the DNA sample is a YAC.
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52. The method of claim 1, wherein the DNA sample is a cosmid.
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53. The method of claim 1, wherein the DNA sample is insert clone comprising up to 500 kB.
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54. A method of shotgun sequencing of DNA comprising the steps of:
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a) preparing a DNA library in accordance with claim 30;
b) sequencing the library using primers specific for known loci to derive the sequence of adjacent unknown regions.
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55. The method of claim 1, further comprising:
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a) recombining the DNA molecules after adaptor attachment;
b) size separating the DNA molecules;
c) amplifying the DNA.
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56. The method of claim 55, wherein the size separated DNA is distributed into the wells of a multi-well plate.
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57. The method of claim 55, wherein the amplified DNA is sequenced.
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58. The method of claim 55, wherein the amplified DNA is subsequently cloned into a vector.
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59. The method of claim 1, further comprising:
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a) amplifying the DNA molecules after adaptor attachment;
b) hybridizing the amplified DNA to a microarray;
c) analyzing the hybridization patterns.
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60. The method of claim 1, wherein the DNA sample is modified.
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61. The method of claim 60, wherein the DNA sample is methylated.
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62. The method of claim 1, further comprising:
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a) initiating a second nick translation reaction at the upstream adaptor comprising subjecting the DNA molecules to nick translation using a DNA polymerase having 5′
-3′
exonuclease activity;
b) attaching second downstream adaptor molecules to the 5′
end of the molecules to produce adaptor attached nick translate molecules.
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63. The method of claim 1, further comprising:
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a) denaturing the adaptor attached nick translation product and separating the single stranded DNA;
b) replicating the second strand of the adaptor attached molecule to form a double stranded product;
c) subjecting the DNA molecules to nick translation using a DNA polymerase having 5′
-3′
exonuclease activity, to produce nick translate molecules;
d) attaching additional downstream adaptor molecules to the nick translation initiation site of the nick translate molecules to produce adaptor attached nick translate molecule molecules.
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64. The method of claim 1, wherein an affinity adaptor is ligated to said DNA molecules.
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65. The method of claim 64, wherein said affinity adaptor is used to separate DNA molecules.
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66. The method of claim 3, wherein said restriction digestion is carried out with a frequent cutter.
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67. The method of claim 3, wherein said restriction digestion is carried out with an infrequent cutter.
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68. The method of claim 3, wherein said restriction digestion results in partial cleavage.
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69. The method of claim 1, further comprising attaching the upstream adaptor molecule to both the proximal and distal ends of said DNA molecules to create a circular product.
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70. The method of claim 69, wherein the initiation of nick translation occurs in the direction of the distal end of the nick translate molecule subjected to circularization.
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71. The method of claim 69, wherein different internal regions of the nick translate molecules are exposed as distal ends.
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72. The method of claim 19, wherein nick translation is carried out on a DNA sample with a plurality of upstream adaptors in a single tube.
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73. The method of claim 22, wherein nick translation is carried out on a DNA sample with a plurality of downstream adaptors, in a single tube.
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74. The method of claim 1, wherein the nick translation reaction proceeds through a known sequence on the DNA molecule.
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75. The method of claim 74, wherein PCR primers are constructed to recognize regions within said known sequence.
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76. The method of claim 75, wherein PCR amplification of nick translate products occurs using a primer specific to said known sequence and a primer specific to an attached adaptor.
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77. The method of claim 1, further comprising circularizing the adaptor attached, nick translate product by:
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a) incubating said adaptor attached, nick translate product with a linker oligonucleotide to form a nick site; and
b) ligating the ends of said adaptor attached, nick translate product with a DNA ligase.
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78. The method of claim 77, wherein said linker oligonucleotide is 20-200 bp. long.
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79. The method of claim 77, wherein said linker oligonucleotide has a region complementary to the upstream adaptor and a region complementary to the downstream adaptor.
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80. The method of claim 1, wherein:
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a) the DNA molecules of the DNA sample are restricted with one or more restriction enzymes;
b) upstream adaptor molecules are attached at both ends of the restricted DNA molecules;
c) nick translation is carried out from both upstream adaptors; and
d) the ends of the DNA molecules are recombined.
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81. The method of claim 80, further comprising separating the recombined molecules according to size.
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82. The method of claim 80, wherein said restriction enzyme is a frequent cutter.
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83. The method of claim 82, wherein said restriction digestion is a partial digest.
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84. The method of claim 80, wherein each end of the DNA molecule is created with a different restriction enzyme.
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85. The method of claim 1, wherein:
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a) the DNA molecules of the DNA sample are restricted with an infrequent cutting restriction enzyme;
b) upstream adaptor molecules are attached at ends of the restricted DNA molecules;
c) nick translation is carried out from the upstream adaptors;
d) the nick translate molecules are partially restricted with a frequent cutter;
e) internal adaptor molecules are attached at ends of the restricted DNA molecules;
f) nick translation is carried out from the internal adaptors; and
g) the ends of the DNA molecules are recombined.
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86. The method of claim 1, wherein nucleotides integrated by nick translation are modified.
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87. The method of claim 86, wherein the modified nucleotides are exonuclease resistant.
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88. The method of claim 87, wherein said modified nucleotides facilitate the differentiation of the nick translate product from the template strand.
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182. The method of claim 39, wherein said upstream and downstream adaptors further comprise 3′
- tails up to 200 bases in length.
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183. The method of claim 39, wherein said upstream and downstream adaptors comprise a nick site that facilitates nick translation through an intermolecular junction.
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186. The method of claim 1, wherein said DNA sample comprises template molecules of 1-20 kb.
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187. The method of claim 1, wherein said adaptor attached nick translate molecules are distributed as an ordered microarray.
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188. The method of claim 187, wherein said microarray is probed with complementary nucleic acid.
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196. The method of claim 1, wherein said adaptor attached nick translate molecules are assembled as a microarray, and wherein said nick translate molecules are amplified prior to said assembly.
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197. The method of claim 196, wherein said microarray is assembled on a DNA chip.
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198. The method of claim 197, wherein said DNA chip comprises an array of adaptor attached nick translate molecules that facilitate analysis of a patient sample to determine chromosomal mutations.
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199. The method of claim 197, wherein said DNA chip comprises an array of adaptor attached nick translate molecules that facilitate diagnostic mutation analysis.
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206. The method of claim 1, wherein said nick translate molecules are denatured.
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89. A method of preparing a DNA molecule having an amplifiable region comprising:
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a) obtaining a DNA sample comprising DNA molecules having regions to be amplified;
b) attaching upstream adaptor molecules to the proximal end of DNA molecules of the sample to provide a nick translation initiation site;
c) subjecting the DNA molecules to nick translation comprising DNA polymerization and 5′
-3′
exonuclease activity, for a specific time T;
d) attaching downstream adaptor molecules to the 5′
end of the degraded template strand to produce adaptor attached nick translate molecules.- View Dependent Claims (90, 91)
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92. A method of preparing a DNA molecule having an amplifiable region comprising:
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a) obtaining a DNA sample comprising DNA molecules having regions to be amplified;
b) attaching upstream adaptor molecules to the proximal end of DNA molecules of the sample to provide a nick translation initiation site;
c) subjecting the DNA molecules to a first nick translation comprising DNA polymerization and 5′
-3′
exonuclease activity, for a specific time T;
d) attaching first downstream adaptor molecules to the 3′
end of the nick translate product to produce adaptor attached nick translate molecules;
e) subjecting the DNA molecules to a second nick translation initiated from the upstream adaptor comprising DNA polymerization and 5′
-3′
exonuclease activity, for a specific time T; and
f) attaching second downstream adaptor molecules to the 5′
end of the degraded nick translate product.- View Dependent Claims (93, 94, 95)
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96. A method of preparing a DNA molecule having an amplifiable region comprising:
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a) obtaining a DNA sample comprising DNA molecules having regions to be amplified;
b) attaching upstream adaptor molecules to the proximal end of DNA molecules of the sample to provide a nick translation initiation site;
c) subjecting the DNA molecules to a first nick translation comprising DNA polymerization and 5′
-3′
exonuclease activity, for a specific time T;
d) attaching a first downstream adaptor molecules to the 3′
end of the nick translate product;
e) separating the nick translate product from the template molecule;
f) replicating the nick translate product via primer extension;
g) subjecting the product of step f) to a second nick translation comprising DNA polymerization and 5′
-3′
exonuclease activity, for a specific time T; and
h) attaching a second downstream adaptor molecules to the 3′
end of the product of step g).- View Dependent Claims (97, 98, 99)
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100. A method of preparing a DNA molecule having an amplifiable region comprising:
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a) obtaining a DNA sample comprising DNA molecules having regions to be amplified;
b) ligating an affinity adaptor to the proximal ends of said DNA molecules;
c) subjecting the affinity adaptor attached molecules to partial cleavage;
d) separating the affinity adaptor attached molecules;
e) attaching upstream adaptor molecules to ends of the affinity adaptor attached molecules to provide a nick translation initiation site;
f) subjecting the affinity adaptor attached molecules to nick translation comprising DNA polymerization and 5′
-3′
exonuclease activity; and
g) attaching downstream adaptor molecules to the nick translate molecules to produce adaptor attached nick translate molecules. - View Dependent Claims (101, 102, 103, 104)
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105. A method of preparing a DNA molecule having an amplifiable region comprising:
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a) obtaining a DNA sample comprising DNA molecules having regions to be amplified;
b) attaching the first end of a recombination adaptor to one end of said DNA molecules;
c) attaching the second end of said recombination adaptor to the opposite end of said DNA molecules;
d) subjecting the adaptor attached molecules to nick translation comprising DNA polymerization and 5′
-3′
exonuclease activity; and
e) attaching downstream adaptor molecules to the nick translate molecules to produce adaptor attached nick translate molecules. - View Dependent Claims (106, 107)
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108. A method of preparing a DNA molecule having an amplifiable region comprising:
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a) obtaining a DNA sample comprising DNA molecules having regions to be amplified;
b) attaching the first end of a recombination adaptor to the proximal end of said DNA molecules;
c) partially cleaving said DNA molecules to produce cleavage products having a plurality of lengths;
d) attaching the second end of said recombination adaptor to distal ends produced by said partial cleavage;
e) subjecting the adaptor attached molecules to nick translation comprising DNA polymerization and 5′
-3′
exonuclease activity;
f) attaching downstream adaptor molecules to the nick translate molecules to produce adaptor attached nick translate molecules; and
g) separating said adaptor attached nick translate molecules. - View Dependent Claims (109, 110, 111, 112, 113)
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114. A method of preparing DNA molecules having an amplifiable region comprising:
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a) obtaining a first DNA template;
b) attaching a first upstream adaptor molecules to said DNA template to provide a nick translation initiation site;
c) obtaining a second DNA template;
d) attaching a second upstream adaptor molecules to said DNA template to provide a nick translation initiation site;
e) mixing said first and said second templates;
f) subjecting the adaptor attached template molecules to nick translation initiated from the upstream adaptor comprising DNA polymerization and 5′
-3′
exonuclease activity, for a specific time T; and
g) attaching a downstream adaptor molecules to the nick translate molecules to produce adaptor attached nick translate molecules. - View Dependent Claims (115, 116)
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117. A method of preparing DNA molecules having an amplifiable region comprising:
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a) obtaining a plurality of DNA templates;
b) attaching a plurality of different first upstream adaptor molecules to said DNA templates to provide a nick translation initiation site;
c) mixing said plurality of templates;
d) subjecting the adaptor attached template molecules to nick translation initiated from the upstream adaptor comprising DNA polymerization and 5′
-3′
exonuclease activity, for a specific time T; and
e) attaching a downstream adaptor molecules to the nick translate molecules to produce adaptor attached nick translate molecules. - View Dependent Claims (118, 119)
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120. A method of constructing a genomic library, comprising:
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a) obtaining genomic DNA;
b) fragmenting the genome to a desired size;
c) attaching upstream adaptor molecules to ends of the fragmented genomic DNA molecules of the sample to provide a nick translation initiation site;
d) subjecting the DNA molecules to nick translation comprising DNA polymerization and 5′
-3′
exonuclease activity; and
e) attaching downstream adaptor molecules to the nick translate molecules to produce adaptor attached nick translate molecules. - View Dependent Claims (121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136)
a) incubating said linking oligonucleotide with said adaptor attached nick translate molecule to form a nick; and
b) ligating the adaptor attached nick translate molecule with a DNA ligase.
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135. The method of claim 134, wherein said ligase is thermostable.
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136. The method of claim 134, wherein said recombination is performed under conditions to favor intramolecular circularization and reduce undesirable intermolecular ligation.
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137. A method of constructing a genomic library, comprising:
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a) obtaining a genomic DNA;
b) fragmenting the genomic DNA;
c) attaching upstream adaptor molecules to ends of the fragmented genomic DNA molecules of the sample to provide a nick translation initiation site;
d) subjecting the DNA molecules to nick translation comprising DNA polymerization and 5′
-3′
exonuclease activity, for a specific time T; and
e) attaching downstream adaptor molecules to the nick translate molecules to produce adaptor attached nick translate molecules. - View Dependent Claims (138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155)
a) incubating said linking oligonucleotide with said adaptor attached nick translate molecule to form a nick; and
b) ligating the adaptor attached nick translate molecule to the linking oligonucleotide with a DNA ligase.
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153. The method of claim 152, wherein said ligase is thermostable.
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154. The method of claim 152, wherein said recombination is performed under conditions to favor intramolecular circularization and reduce undesirable intermolicular ligation.
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155. The method of claim 138, wherein the specific time T varies for different reaction vessels.
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156. A method of preparing an unordered DNA library comprising:
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a) obtaining a DNA sample comprising DNA molecules;
b) cleaving said DNA molecules;
c) attaching recombination adaptors to termini of the cleaved DNA molecules;
d) subjecting the DNA molecules to nick translation comprising DNA polymerization and 5′
-3′
exonuclease activity, to produce nick translate molecules wherein said nick translation is initiated from both ends of the cleaved DNA molecules; and
e) recombining the ends of the nick translate molecules produced by step d). - View Dependent Claims (157, 158, 159, 160)
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161. A method of producing an ordered DNA library comprising:
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a) obtaining a DNA sample comprising DNA molecules;
b) cleaving said DNA molecules;
c) partially cleaving the cleaved DNA molecules;
d) attaching adaptors to termini of the DNA molecules;
e) subjecting the DNA molecules to nick translation comprising DNA polymerization and 5′
-3′
exonuclease activity, to produce nick translate molecules wherein said nick translation is initiated from both ends of the DNA molecules;
f) separating the nick translate molecules; and
g) subjecting the separated nick translate molecules to recombination. - View Dependent Claims (163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175)
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162. A method of producing an ordered library comprising:
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a) obtaining a DNA sample comprising DNA molecules;
b) cleaving said DNA molecules;
c) attaching recombination adaptors to termini of the DNA molecules;
d) subjecting the DNA molecules to nick translation comprising DNA polymerization and 5′
-3′
exonuclease activity, to produce nick translate molecules wherein said nick translation is initiated from both ends of the DNA molecules;
e) recombining the ends of the DNA molecules produced by step d);
f) separating the nick translate molecules according to size.
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176. A method of creating a DNA library, comprising:
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a) obtaining a DNA sample comprising DNA molecules;
b) cleaving said DNA molecules with an infrequently-cutting restriction enzyme;
c) attaching upstream adaptor molecules to ends of said cleaved DNA molecules of the sample to provide a nick translation initiation site;
d) subjecting the DNA molecules to nick translation comprising DNA polymerization and 5′
-3′
exonuclease activity;
e) attaching downstream adaptor molecules to the nick translate molecules to produce adaptor attached nick translate molecules. f) partially cleaving the adaptor attached nick translate molecules with a frequently cutting restriction enzyme;
g) attaching upstream adaptor molecules to the ends of the adaptor attached nick translate molecules produced by said partial digestion;
h) subjecting the DNA molecules to nick translation comprising DNA polymerization and 5′
-3′
exonuclease activity; and
i) attaching downstream adaptor molecules to the nick translate molecules to produce adaptor attached nick translate molecules;
j) subjecting the product of step i) to recombination. - View Dependent Claims (177, 178, 179, 180, 181)
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184. A method of preparing a DNA molecule having an amplifiable region comprising:
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a) obtaining a DNA sample comprising DNA molecules having regions to be amplified;
b) ligating at least a first upstream adaptor and at least a second upstream adaptor to said DNA molecules;
c) subjecting the DNA molecules to recombination under conditions to favor intramolecular circularization and reduce undesirable intermolecular ligation;
d) subjecting the recombined DNA molecules to nick translation comprising DNA polymerization and 5′
-3′
exonuclease activity; and
e) attaching downstream adaptor molecules to the nick translate molecules to produce adaptor attached nick translate molecules. - View Dependent Claims (185)
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189. A method of preparing a DNA molecule having an amplifiable region comprising:
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a) obtaining a DNA sample comprising DNA molecules having regions to be amplified;
b) attaching upstream adaptor molecules to ends of DNA molecules of the sample to provide a nick translation initiation site;
c) subjecting the DNA molecules to nick translation comprising DNA polymerization, to produce nick translate molecules; and
d) attaching downstream adaptor molecules to the nick translate molecules to produce adaptor attached nick translate molecules. - View Dependent Claims (190, 191, 192, 193, 194, 195)
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200. A method of detecting a specific DNA sequence, comprising:
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a) providing adaptor attached nick translate molecules, wherein said molecules are generated by;
i) attaching upstream adaptor molecules to ends of DNA molecules, thereby providing a nick translation initiation site;
ii) subjecting the DNA molecules to nick translation to produce nick translate molecules;
iii) attaching downstream adaptor molecules to the nick translate molecules;
b) separating the adaptor attached nick translate molecules;
c) amplifying the adaptor attached nick translate molecules by means of sequence on the adaptor; and
d) identifying said DNA sequence. - View Dependent Claims (201, 202, 203, 204, 205)
a) hybridizing said adaptor attached nick translate molecules to a DNA microarray; and
b) detecting said hybridization.
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202. The method of claim 200, wherein a plurality of specific DNA sequences are detected.
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203. The method of claim 200, wherein the adaptor attached nick translate molecules are from a human individual.
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204. The method of claim 200, wherein the adaptor attached nick translate molecules are from a plurality of human individuals.
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205. The method of claim 200, wherein the adaptor attached nick translate molecules are from a plurality of microorganisms.
Specification