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 population of DNA fragments, comprising:
(a) fragmenting a DNA molecule to produce DNA fragments;
(b) producing a homopolymer extension on 3′ ends of the DNA fragments; and
(c) amplifying a plurality of the fragments with a population of primers complementary to a known sequence in the DNA fragments and a population of primers comprising a region complementary to the homopolymer extension, wherein a portion of each primer in the population of primers comprising a region complementary to the homopolymer extension comprises a random sequence.
2. The method of claim 1 , wherein the fragmenting is mechanical, thermal, or chemical fragmentation.
3. The method of claim 2 , wherein the fragmentation is by hydrodynamic shearing, sonication, acid catalytic hydrolysis, alkaline catalytic hydrolysis, hydrolysis by metal ions, hydroxyl radicals, irradiation, or heating.
4. The method of claim 1 , wherein the homopolymer extensions are generated by terminal deoxynucleotidyltransferase.
5. The method of claim 1 , further comprising sequencing a plurality of the amplified fragments.
6. The method of claim 1 , wherein the portion of each primer in the population of primers comprising a region complementary to the homopolymer extension that comprises a random sequence is about 50% of the sequence of each primer.
7. The method of claim 1 , wherein the portion of each primer in the population of primers comprising a region complementary to the homopolymer extension that comprises a random sequence is about 40% of the sequence of each primer.
8. The method of claim 1 , wherein the portion of each primer in the population of primers comprising a region complementary to the homopolymer extension that comprises a random sequence is about 30% of the sequence of each primer.
9. The method of claim 1 , wherein the portion of each primer in the population of primers comprising a region complementary to the homopolymer extension that comprises a random sequence is about 20% of the sequence of each primer.
10. The method of claim 1 , wherein the portion of each primer in the population of primers comprising a region complementary to the homopolymer extension that comprises a random sequence is about 10% of the sequence of each primer.
11. The method of claim 1 , wherein the portion of each primer in the population of primers comprising a region complementary to the homopolymer extension that comprises a random sequence is about 5% of the sequence of each primer.
12. The method according to claim 1 , wherein the method comprises preparing a plurality of populations of DNA fragments from a plurality of DNA molecules.
13. The method according to claim 1 , wherein the DNA molecule is a cDNA molecule.
14. The method according to claim 13 , wherein the method further comprises reverse transcribing the cDNA molecule from an mRNA template.