DNA amplification and sequencing using DNA molecules generated by random fragmentation
The present invention is directed to methods to prepare a DNA molecule or a plurality of DNA molecules by random fragmentation. In some embodiments, the present invention regards preparing a template for DNA sequencing by random fragmentation. In specific embodiments, the random fragmentation comprises chemical fragmentation, mechanical fragmentation, or enzymatic fragmentation. In further specific embodiments, a universal sequence is attached to the 3′ end of the DNA fragments, such as by ligation of an adaptor sequence or by homopolymeric tailing with terminal deoxynucleotidyltransferase. In other embodiments, a library is prepared with methods of the present invention.
1. A method of preparing a DNA molecule, comprising:
obtaining a DNA molecule;
fragmenting the DNA molecule by thermal or chemical fragmentation to produce DNA fragments, wherein the ends of at least some of the DNA fragments comprise an enzymatically non-extendable 3′ end;
repairing non-extendable 3′ ends on DNA fragments having non-extendable 3′ ends and generating a guanine homopolymer extension on 3′ ends of the DNA fragments using terminal deoxynucleotidyltransferase in the presence of a guanine ribonucleotide, guanine deoxyribonucleotide, or both.
2. The method of claim 1 , further comprising concomitantly sequencing a plurality of the fragments.
3. The method of claim 1 , wherein said fragmentation is by acid catalytic hydrolysis, alkaline catalytic hydrolysis, hydrolysis by metal ions, hydroxyl radicals, irradiation, or heating.
4. The method of claim 1 , wherein said fragmentation is by heating.
5. The method of claim 4 , wherein said heating is to a temperature of between about 40° C. and 120° C.
6. The method of claim 4 , wherein said heating is to a temperature of between about 80° C. and 100° C.
7. The method of claim 4 , wherein said heating is to a temperature of between about 90° C. and 100° C.
8. The method of claim 4 , wherein said heating is to a temperature of between about 92° C. and 98° C.
9. The method of claim 4 , said heating is to a temperature of between about 93° C. and 97° C.
10. The method of claim 4 , wherein said heating is to a temperature of between about 94° C. and 96° C.
11. The method of claim 4 , wherein said heating is to a temperature of about 95° C.
12. The method of claim 4 , wherein said heating of the DNA molecule is in a solution having from 0 to about 100 mM concentration of a salt.
13. The method of claim 4 , wherein said heating is in a solution having from about 0 to about 10 mM concentration of salt.
14. The method of claim 4 , wherein said heating is in a solution having from about 0.1 to about 1 mM concentration of salt.
15. The method of claim 4 , wherein said heating is in a solution having from about 0.1 to about 0.5 mM concentration of salt.
16. The method of claim 4 , wherein said heating is in a solution of 10 mM Tris, pH 8.0; 1 mM EDTA.
17. The method of claim 4 , wherein said heating is in a solution of water.
18. The method of claim 1 , further comprising preparing a library of DNA molecules.
19. The method of claim 18 , further comprising concomitantly sequencing a plurality of the fragments.
20. The method of claim 1 , further comprising amplifying a plurality of the fragments.
21. The method of claim 20 , wherein the amplification utilizes:
a second primer complementary to a known sequence in the DNA fragments; and
a third primer complementary to the first primer.
22. The method of claim 21 , further comprising the step of sequencing concomitantly said plurality of DNA fragments using a fourth primer complementary to said known sequence in the DNA fragments.
23. The method of claim 22 , wherein said fourth primer is said second primer.
24. The method of claim 21 , further comprising
sequencing said plurality of DNA fragments using a fourth primer complementary to said known sequence in the DNA fragments.
25. The method of claim 24 , wherein said fourth primer is said second primer.
26. The method of claim 1 , further comprising:
concomitantly sequencing a first region in said plurality of DNA molecules using a primer complementary to a known sequence in said plurality of DNA molecules; and
concomitantly sequencing a second region in said plurality of DNA molecules using a primer complementary to sequence determined from the sequencing of the first region,
wherein the next consecutive sequencing of a region in the overlapping series of nucleic acid sequences is produced by initiating sequencing from the sequence obtained in a preceding overlapping sequencing product.
27. The method of claim 1 , wherein the plurality of DNA fragments comprises DNA fragments having part or all of the sequences which generate the secondary structure.
28. The method of claim 27 , further comprising concomitantly sequencing the plurality of primer-linked fragments.
29. The method of claim 27 , wherein said plurality of DNA fragments further comprises DNA fragments having none of the sequences which generate the secondary structure.
30. The method of claim 27 , wherein said secondary structure is a hairpin, a G quartet, or a triple helix.
31. The method of claim 1 , wherein the obtained DNA molecule comprises genomic DNA, BAC DNA, or plasmid DNA.
32. The method of claim 1 , further defined as comprising
attaching a labeled primer having substantially known sequence to at least one end of a plurality of the DNA fragments to produce labeled primer-linked fragments.
33. The method of claim 32 , wherein said attaching step of a labeled primer comprises ligation of an adaptor molecule to at least one end of the DNA fragment, wherein the adaptor molecule comprises the label.
34. The method of claim 32 , wherein the label comprises a radionuclide, an affinity tag, a hapten, an enzyme, a chromophore, or a fluorophore.
35. The method of claim 1 , wherein the guanine ribonucleotide, guanine deoxyribonucleotide, or both, are labeled and/or wherein the attaching further comprises other deoxyribonucleotides or ribonucleotides that are labeled.
36. The method of claim 35 , wherein the label comprises a radionuclide, an affinity tag, a hapten, an enzyme, a chromophore, or a fluorophore.