Nucleic acid sample preparation
First Claim
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1. A method for preparing an amplified mixture of nucleic acid molecules wherein each amplified molecule in the mixture has a known region at each end, the method comprising:
- a) providing a sample containing a population of nucleic acid molecules,b) treating the population to produce first single stranded oligonucleotides of different sequence from each of the nucleic acid molecules in the population,c) joining a hairpin oligonucleotide sequence to each of the first single stranded oligonucleotides of different sequence using template independent nucleic acid polymerase, wherein the hairpin oligonucleotide sequence comprises a single stranded region comprising a 5′
-triphosphate, a region of self-complementary double stranded sequence, a 3′
-overhang which hybridises to the first single stranded oligonucleotide, and a blocking moiety at the 3′
end,d) removing the 3′
blocking moiety,e) producing a full length copy of each of the first single stranded oligonucleotides by extending the deblocked 3′
hydroxyl of the hairpin oligonucleotide sequence to create a blunt end,f) attaching a further oligonucleotide sequence to each full length copy of the first single stranded oligonucleotides,g) cleaving the hairpin oligonucleotide sequence, thereby producing a mixture of double stranded nucleic acid molecules comprising the first single stranded oligonucleotides of different sequence and full length copies thereof, wherein each full length copy thereof has a known region at each end, andh) amplifying the double stranded nucleic acid molecules of step g), thereby generating an amplified mixture of nucleic acid molecules wherein each amplified molecule in the mixture has a known region at each end.
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Abstract
This invention relates to the preparation of nucleic acid samples for analysis. The invention may be particularly useful for single stranded samples. Embodiments of the invention involve the attachment of double stranded or hairpin oligonucleotides using template independent polymerase enzymes in the preparation of nucleic acid sequencing libraries.
39 Citations
14 Claims
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1. A method for preparing an amplified mixture of nucleic acid molecules wherein each amplified molecule in the mixture has a known region at each end, the method comprising:
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a) providing a sample containing a population of nucleic acid molecules, b) treating the population to produce first single stranded oligonucleotides of different sequence from each of the nucleic acid molecules in the population, c) joining a hairpin oligonucleotide sequence to each of the first single stranded oligonucleotides of different sequence using template independent nucleic acid polymerase, wherein the hairpin oligonucleotide sequence comprises a single stranded region comprising a 5′
-triphosphate, a region of self-complementary double stranded sequence, a 3′
-overhang which hybridises to the first single stranded oligonucleotide, and a blocking moiety at the 3′
end,d) removing the 3′
blocking moiety,e) producing a full length copy of each of the first single stranded oligonucleotides by extending the deblocked 3′
hydroxyl of the hairpin oligonucleotide sequence to create a blunt end,f) attaching a further oligonucleotide sequence to each full length copy of the first single stranded oligonucleotides, g) cleaving the hairpin oligonucleotide sequence, thereby producing a mixture of double stranded nucleic acid molecules comprising the first single stranded oligonucleotides of different sequence and full length copies thereof, wherein each full length copy thereof has a known region at each end, and h) amplifying the double stranded nucleic acid molecules of step g), thereby generating an amplified mixture of nucleic acid molecules wherein each amplified molecule in the mixture has a known region at each end. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
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Specification
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Current AssigneeBiomodal Limited
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Original AssigneeCambridge Epigenetix Limited
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InventorsOst, Tobias William Barr, Bell, Neil Matthew
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Primary Examiner(s)DUNSTON, JENNIFER ANN
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Application NumberUS15/054,227Publication NumberTime in Patent Office634 DaysField of SearchUS Class CurrentCPC Class CodesC12Q 1/6806 Preparing nucleic acids for...C12Q 1/6855 Ligating adaptorsC12Q 1/6869 Methods for sequencingC12Q 1/6874 involving nucleic acid arra...C12Q 2521/131 Terminal transferaseC12Q 2523/107 Chemical cleaving agentsC12Q 2523/125 Bisulfite(s)C12Q 2525/121 incorporating both deoxyrib...C12Q 2525/186 incorporating a non-extenda...C12Q 2525/191 incorporating an adaptorC12Q 2525/301 Hairpin oligonucleotidesC12Q 2535/122 Massive parallel sequencingC12Q 2543/101 in situ amplificationC12Q 2565/1015 labels being on the same ol...
