Method of producing a DNA library using positional amplification
First Claim
Patent Images
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;
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.
51 Citations
272 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;
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, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 257, 258, 259, 260)
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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)
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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 (139, 140, 141, 142, 143, 144, 145)
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- 138. The method of claim 138, further comprising the step of subdividing the upstream-adaptor attached genomic DNA molecules into a plurality of 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 at low DNA concentrations;
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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187. A method of sequencing a BAC clone, comprising:
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a) cleaving the BAC clone at a cos site with lambda terminase b) ligating an upstream adaptor to the 5′
overhangs;
c) partially cleaving the BAC clone with a frequently cutting enzyme;
d) recombining the partially cleaved BAC clone of step c);
e) adding a homopolymeric tail to the 3′
end of the recombined product with terminal transferase;
f) ligating an adaptor having a homopolymeric 3′
single-strand overhang and a unique double strand sequence at the end to the homopolymeric tail, wherein the homopolymeric single-strand overhang is complementary to the homopolymeric tail of step e);
g) size separating the product of step f);
h) distributing the separated product into the wells of a microplate. i) amplifying the separated products with primers complementary to adaptor sequences such that products are formed which proceed in either a clockwise or counterclockwise direction around the recombined molecule;
j) ligating the amplified product into a cloning vector; and
k) subsequently sequencing said amplified product.
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201. An adaptor construct, wherein said construct comprises:
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a) a first domain comprising nucleotides that facilitate ligation of said construct to a nucleic acid; and
b) a second domain proximal to said first domain, comprising a site which facilitates the initiation of a nick translation reaction and a site that facilitates recombination. wherein ligation of said adaptor construct to a polynucleotide molecule results in the only free 3′
OH group capable of initiating a nick translation reaction within said second domain. - View Dependent Claims (202, 203, 204, 205, 206, 207, 208, 248)
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209. An adaptor construct comprising:
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a) a first oligonucleotide comprising a phosphate group at the 5′
end and a blocking nucleotide at the 3′
end;
b) a second oligonucleotide comprising a blocked 3′
end, a non-phosphorylated 5′
end, and a nucleotide sequence complementary to the 5′
element of said first oligonucleotide; and
c) a third oligonucleotide comprising a 3′
hydroxyl group, a non-phosphorylated 5′
end, and a nucleotide sequence complementary to the 3′
element of said first oligonucleotide. - View Dependent Claims (210, 211, 212, 213, 214, 215, 249)
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216. An adaptor construct comprising
a) a first oligonucleotide comprising a 5′ - phosphate and a 3′
nucleotide blocked to prevent ligation or extension by a polymerase;
b) a second oligonucleotide comprising a domain which facilitates ligation to the template strand and a nucleotide sequence complementary to the 5′
element of said first oligonucleotide;
c) a third oligonucleotide comprising an initiation site for nick-translation and a nucleotide sequence complementary to a region of said first oligonucleotide; and
d) a plurality of oligonucleotides which may be readily removed to expose a 3′
terminus of the adaptor, wherein each of said plurality of oligonucleotides comprise a nucleotide sequence complementary to a region of said first oligonucleotide. - View Dependent Claims (217, 250)
- phosphate and a 3′
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218. An adaptor construct, wherein said construct comprises:
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a) a first domain comprising nucleotides that facilitate ligation of said construct to a nucleic acid; and
b) a second domain proximal to said first domain, comprising a site which facilitates the initiation of a nick translation reaction, c) a third domain proximal to said first domain, comprising a second site which facilitates the initiation of a nick translation reaction, said second or said third domain further comprising a site that facilitates recombination, wherein ligation of said adaptor construct to a polynucleotide molecule results in the only free 3′
OH groups capable of initiating a nick translation reaction within said second and said third domains. - View Dependent Claims (219, 220, 221, 222, 223, 224, 225, 251)
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226. An adaptor construct comprising:
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a) a first oligonucleotide comprising a phosphate group at the 5′
end and a blocking nucleotide at the 3′
end;
b) a second oligonucleotide comprising a blocked 3′
end, a non-phosphorylated 5′
end, and a nucleotide sequence complementary to the 5′
element of said first oligonucleotide;
c) a third oligonucleotide comprising a 3′
hydroxyl group, a non-phosphorylated 5′
end, and a nucleotide sequence complementary to the 3′
element of said first oligonucleotide; and
d) a fourth oligonucleotide comprising a 3′
hydroxyl group, a non-phosphorylated 5′
end, and a nucleotide sequence complementary to the 3′
element of said first oligonucleotide. - View Dependent Claims (227, 228, 229, 230, 231, 232, 252)
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233. An adaptor construct comprising:
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a) a first oligonucleotide comprising a 5′
region comprising a 5′
phosphate group and homopolymeric tract of about 8-20 bases and a 3′
region comprising an about 12- about 100 base primer binding domain; and
b) a second oligonucleotide complementary to the 3′
region of said first oligonucleotide. - View Dependent Claims (234, 253)
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235. An adaptor construct comprising:
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a) a first oligonucleotide of about 12- about 100 bases, wherein the 5′
end of said oligonucleotide comprises a free phosphate group; and
b) a second oligonucleotide comprising a homopolymeric tract of about 8-20 bases, a 3′
blocking nucleotide and wherein the 5′
region of said second oligonucleotide is complementary to said first oligonucleotide. - View Dependent Claims (236, 254)
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237. An adaptor construct comprising:
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a) a first oligonucleotide comprising a 5′
region comprising an about 12-about 100 base primer binding domain and a 3′
region comprising a homopolymeric tract of about 8- about 20 bases; and
b) a second oligonucleotide comprising a blocked 3′
end and a 3′
region complementary to the 5′
region of said first oligonucleotide. - View Dependent Claims (238, 255)
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239. An adaptor construct comprising:
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a) a first oligonucleotide comprising a 5′
region comprising an about 12-about 100 base primer binding domain; and
b) a second oligonucleotide a homopolymeric tract of about 4- about 12 bases at the 5′
end, a blocking nucleotide at the 3′
end, and a 3′
region complementary to said first oligonucleotide. - View Dependent Claims (240, 256)
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241. 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 (242, 243, 244, 245, 246, 247)
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- 261. A method of recombining DNA molecules comprising recombining ends of adaptor attached template molecules in a dilute solution.
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265. A method of recombining DNA molecules comprising:
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a) hybridizing the ends of adaptor attached template molecules in dilute solution;
b) concentrating the molecules; and
c) ligating the molecules.
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266. A method of recombining DNA molecules comprising:
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a) hybridizing the ends of adaptor attached template molecules; and
b) subjecting said molecule to a nick-translation reaction to form a covalent intramolecular junction.
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267. A method of detecting a specific DNA sequence, comprising:
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a) separating adaptor attached nick translate molecules; and
b) identifying said DNA sequence. - View Dependent Claims (268, 269, 270, 271, 272)
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Specification
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Current AssigneeBoard of Regents of the University of Michigan (University of Michigan)
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Original AssigneeBoard of Regents of the University of Michigan (University of Michigan)
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InventorsMakarov, Vladimir L., Langmore, John P.
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Granted Patent
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Time in Patent OfficeDays
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Field of Search
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US Class Current536/24.3
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CPC Class CodesC12N 15/10 Processes for the isolation...C12N 15/1093 General methods of preparin...C12N 15/1096 cDNA Synthesis; Subtracted ...C12P 19/34 Polynucleotides, e.g. nucle...C12Q 1/6844 Nucleic acid amplification ...C12Q 1/6855 Ligating adaptorsC12Q 2521/101 DNA polymeraseC12Q 2521/319 ExonucleaseC12Q 2525/155 incorporating/generating a ...C12Q 2525/191 incorporating an adaptorC12Q 2531/113 PCRC12Q 2533/101 Primer extensionC40B 40/00 Libraries per se, e.g. arra...
