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 conducting a DNA sequence analysis by fragmenting DNA to provide DNA probes therefrom, the method comprising:
(a) obtaining a DNA sample comprising primer-linked DNA fragments, the DNA fragments having been prepared by subjecting the DNA sample to thermal fragmentation by heating said sample under conditions to effect fragmentation of DNA molecules in the sample to provide single stranded DNA fragments; and either
i) forming double-stranded DNA fragments from said single-stranded fragments that include a primer or primers having a known sequence at least at one end of a plurality of the double-stranded DNA fragments, to provide double-stranded primer-linked fragments; or
ii) providing the DNA probes from said sample by attaching a primer or primers having a known sequence to at least one end of a plurality of the single-stranded DNA fragments, to provide primer-linked single-stranded fragments; and
(b) conducting a DNA sequence analysis of at least a portion of one or more of said primer-linked DNA fragments or copies thereof.
2. The method of claim 1 , wherein said heating is to a temperature of between about 40° C. and 120° C.
3. The method of claim 2 , wherein said heating is to a temperature of between about 80° C. and 100° C.
4. The method of claim 3 , wherein said heating is to a temperature of between about 90° C. and 100° C.
5. The method of claim 4 , wherein said heating is to a temperature of between about 92° C. and 98° C.
6. The method of claim 5 , said heating is to a temperature of between about 93° C. and 97° C.
7. The method of claim 6 , wherein said heating is to a temperature of between about 94° C. and 96° C.
8. The method of claim 7 , wherein said heating is to a temperature of about 95° C.
9. The method of claim 1 , wherein said heating of the DNA molecule is in a solution having from 0 to about 100 mM concentration of a salt.
10. The method of claim 9 , wherein said heating is in a solution having from about 0 to about 10 mM concentration of salt.
11. The method of claim 10 , wherein said heating is in a solution having from about 0.1 to about 1 mM concentration of salt.
12. The method of claim 11 , wherein said heating is in a solution having from about 0.1 to about 0.5 mM concentration of salt.
13. The method of claim 1 , wherein said heating is in a solution of 10 mM Tris, pH 8.0; 1 mM EDTA.
14. The method of claim 1 , wherein said heating is in a solution of water.
15. The method of claim 1 , wherein double-stranded DNA fragments are formed from said single-stranded fragments by
(a) attaching a first primer sequence to one end of a plurality of said single-stranded fragments; and
(b) forming double-stranded DNA fragments using a second primer complementary to said first primer.
16. The method of claim 15 , wherein copies of said double-stranded DNA fragments are prepared using a third primer complementary to a sequence present in said single-stranded fragments in a DNA polymerase reaction.
17. The method of claim 15 , wherein copies of said double-stranded DNA fragments are prepared by ligating primers having a known sequence to one or both ends of said double-stranded DNA and forming copies thereof fragments using complementary primers in a DNA polymerase reaction.
18. The method of claim 15 , wherein said first primer sequence is a homopolymeric sequence and is attached by a terminal transferase.
19. The method of claim 15 , wherein said first primer sequence is attached to one end by ligating a single-stranded adaptor thereto.
20. The method of claim 1 , wherein the DNA sequence analysis is performed by sequencing at least a portion of the primer-linked single-stranded fragments or copies thereof.
21. The method of claim 1 , wherein said conducting a sequence analysis is further defined as concomitantly sequencing a plurality of primer-linked fragments.
22. The method of claim 1 , wherein said conducting a sequence analysis comprises the steps of:
obtaining a plurality of DNA molecules having overlapping sequences;
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.