Molecular encoding of nucleic acid templates for PCR and other forms of sequence analysis
First Claim
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1. A method for bar-coding a nucleic acid molecule comprising the steps of:
- (a) contacting a target nucleic acid molecule in a sample with a bar-coded oligonucleotide under suitable conditions to anneal the bar-coded oligonucleotide to the target nucleic acid molecule, wherein the bar-coded oligonucleotide comprises a first sequence complementary to the target nucleic acid molecule, a second sequence providing a random barcode, and a third sequence that is not complementary to any sequence in the sample; and
(b) extending the annealed bar-coded oligonucleotide using the target nucleic acid molecule as a template to produce a bar-coded target nucleic acid molecule.
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Abstract
In a first aspect, the present invention provides methods for authenticating a nucleic acid molecule and its sequence with a molecular barcode and batch-stamp. In another aspect, the present invention provides methods for authenticating a nucleic acid amplification product. In a further aspect, the present invention provides compositions for encoding both single-stranded and double-stranded target nucleic acids with coded oligonucleotides. The compositions are useful in the practice of the methods of the invention.
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Citations
10 Claims
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1. A method for bar-coding a nucleic acid molecule comprising the steps of:
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(a) contacting a target nucleic acid molecule in a sample with a bar-coded oligonucleotide under suitable conditions to anneal the bar-coded oligonucleotide to the target nucleic acid molecule, wherein the bar-coded oligonucleotide comprises a first sequence complementary to the target nucleic acid molecule, a second sequence providing a random barcode, and a third sequence that is not complementary to any sequence in the sample; and
(b) extending the annealed bar-coded oligonucleotide using the target nucleic acid molecule as a template to produce a bar-coded target nucleic acid molecule. - View Dependent Claims (2, 3, 4)
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5. A method for authenticating a DNA amplification product comprising the steps of:
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(a) contacting a target nucleic acid molecule in a sample with a bar-coded oligonucleotide under suitable conditions to anneal the bar-coded oligonucleotide to the target nucleic acid molecule, wherein the bar-coded oligonucleotide comprises a first sequence complementary to the target nucleic acid molecule, a second sequence providing a random barcode, and a third sequence that is not complementary to any sequence in the sample, and wherein the 5′
end of the bar-coded oligonucleotide comprises a tethering sequence that is complementary to a sequence 5′
to the first sequence;
(b) extending the annealed bar-coded oligonucleotide using the target nucleic acid molecule as a template to produce a bar-coded target nucleic acid molecule;
(c) amplifying the bar-coded target nucleic acid molecule using a primer that binds to the third sequence to produce an amplification product; and
(d) authenticating the amplification product by detecting the presence of the second sequence in the amplification product. - View Dependent Claims (6, 7)
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8. A method for authenticating a DNA amplification product comprising the steps of:
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(a) ligating a hairpin linker to a double-stranded target DNA molecule to produce a ligated target DNA molecule, wherein the hairpin linker comprises a first sequence providing experimental identification information, a second sequence providing a random barcode comprising nucleotides selected from the group consisting of adenosines, guanidines, and thymidines, and a third sequence complementary to the first sequence;
(b) treating the ligated target DNA molecule of step (a) under suitable conditions to convert cytosines in the ligated target DNA molecule to uracils;
(c) amplifying the treated ligated target DNA molecule of step (b) to produce an amplification product; and
(d) authenticating the amplification product of step (c) by detecting the presence of the first and second sequences in the amplification product.
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9. A composition comprising a target nucleic acid molecule and a bar-coded oligonucleotide, wherein the bar-coded oligonucleotide comprises a first sequence complementary to the target nucleic acid molecule, a second sequence providing a random barcode, and a third sequence that is not complementary to any sequence in the sample, and wherein the 5′
- end of the bar-coded oligonucleotide comprises a tethering sequence that is complementary to a sequence 5′
to the first sequence. - View Dependent Claims (10)
- end of the bar-coded oligonucleotide comprises a tethering sequence that is complementary to a sequence 5′
Specification