Molecular encoding of nucleic acid templates for PCR and other forms of sequence analysis

• US 20070020640A1
• Filed: 07/21/2005
• Published: 01/25/2007
• Est. Priority Date: 07/21/2005
• Status: Abandoned Application

• Current Assignee: University of Washington
• Original Assignee: University of Washington
• Inventors: Burden, Alice, McCloskey, Megan, Stoger, Reinhard, Miner, Brooks, Laird, Charles, Hansen, R.
• Application Number:
  US11/186,636
• Publication Number:
  US 20070020640A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid

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.

Claims

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.

2. The method of claim 1, wherein the 5′

end of the bar-coded oligonucleotide comprises a tethering sequence that is complementary to a sequence 5′

to the first sequence.

3. The method of claim 1, wherein the bar-coded oligonucleotide further comprises a fourth sequence providing experimental identification information.

4. The method of claim 1, wherein the target nucleic acid molecule is a DNA molecule.

5. A method for authenticating a DNA amplification product 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 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.

6. The method of claim 5, wherein the bar-coded oligonucleotide further comprises a fourth sequence providing experimental identification information and wherein step (d) further comprises authenticating the amplification product by detecting the presence of the fourth sequence in the amplification product.

7. The method of claim 5, wherein the target nucleic acid molecule is a DNA molecule.

8. A method for authenticating a DNA amplification product comprising the steps of:

(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.

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.

10. The composition of claim 9, wherein fourth sequence provides experimental identification information.