METHOD OF MAKING A PAIRED TAG LIBRARY FOR NUCLEIC ACID SEQUENCING

• US 20170226580A1
• Filed: 04/24/2017
• Published: 08/10/2017
• Est. Priority Date: 01/09/2008
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC Legal Department Intellectual Property
• Inventors: LI, Bin, RANADE, Swati, XI, Lei, WANG, Yangzhou
• Granted Patent: US 10,190,164 B2
• Time in Patent Office: Days
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/325 Single stranded exonuclease
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides

Abstract

The present disclosure relates to methods and compositions for making paired tags and paired tag libraries.

Claims

1. A plurality of circular nucleic acid molecules, the individual circular molecules in the plurality comprising:

(a) an adapter sequence common to the plurality of circular nucleic acid molecules, wherein the adapter sequence is 100 bases or less in length, and wherein the adapter sequence comprises a double-stranded nucleic acid; and

(b) a target sequence region that differs between different molecules of the plurality of circular molecules, wherein each target sequence region comprises a fragment of genomic DNA comprising between 200 bp and 15 kb, wherein each of the circular nucleic molecules comprise a first and second nick in the target sequence, one on each strand of the circular nucleic acid molecule, wherein the first and second nick are each located at different positions within the target sequence region.

2. The molecules of claim 1, wherein the adapter sequence further comprises a binding moiety.

3. The molecules of claim 2, wherein the binding moiety comprises biotin.

4. The molecules of claim 2, wherein the binding moiety comprises streptavidin.

5. The molecules of claim 1, further comprising a nick translating enzyme bound to the nucleic acid molecules.

6. The molecules of claim 5, wherein the nick translating enzyme comprises DNA polymerase I.

7. The molecules of claim 1, wherein the target sequence region is derived from the human genome.

8. The molecules of claim 1, wherein the target sequence region is derived from random fragments generated from a genomic library.

9. The molecules of claim 8, wherein the random fragments are generated from an entire genome.

10. The molecules of claim 1, wherein the first nick is located between 10 and 2000 nucleotides from a first end of the adapter sequence and the second nick is located between 10 and 2000 nucleotides from a second end of the adapter sequence.

11. The molecules of claim 1, wherein the first nick is located between 10 and 500 nucleotides from a first end of the adapter sequence and the second nick is located between 10 and 500 nucleotides from a second end of the adapter sequence.

12. The molecules of claim 1, wherein the first nick is located between 10 and 200 nucleotides from a first end of the adapter sequence and the second nick is located between 10 and 200 nucleotides from a second end of the adapter sequence.

13. The molecules of claim 1, wherein the first nick is located between 10 and 100 nucleotides from a first end of the adapter sequence and the second nick is located between 10 and 100 nucleotides from a second end of the adapter sequence.