Compositions and methods for analysis of nucleic acids
View Patent ↗Disclosed are a number of methods that can be used in a variety of embodiments, including, creation of a nucleic acid terminated at one or more selected bases, sequence analysis of nucleic acids, mapping of sequence motifs within a nucleic acid, positional mapping of nucleic acid clones, and analysis of telomeric regions. The methods utilize double-stranded templates, and in most aspects involve a strand replacement reaction initiated at one or more random or specific locations created in a nucleic acid molecule, and in certain aspects utilizing an oligonucleotide primer.
1. A method for preparing a DNA molecule comprising the steps of:
a) obtaining a sample of DNA that includes double-stranded DNA molecules that have been fragmented enzymatically;
b) conditioning DNA fragments of the sample to provide a 3′ hydroxyl group thereon through the use of a mixture comprising DNA polymerase and exonuclease III to provide conditioned DNA fragments.
2. The method of claim 1 , wherein the DNA molecules have been fragmented using an endonuclease.
3. The method of claim 2 , wherein the DNA molecules have been fragmented through the use of a restriction endonuclease.
4. The method of claim 3 , wherein the DNA molecules have been fragmented through the use of a restriction endonuclease having a two base recognition sequence.
5. The method of claim 3 , wherein the DNA molecules have been fragmented through the use of a restriction endonuclease having a four base recognition sequence.
6. The method of claim 3 , wherein the restriction endonuclease has introduced random double strand breaks into DNA molecules.
7. The method of claim 2 , wherein the endonuclease introduced a blunt end.
8. The method of claim 1 , wherein the DNA polymerase possesses a 3′ to 5′ exonuclease activity.
9. The method of claim 1 , further comprising attaching an oligonucleotide adaptor to the conditioned DNA fragments.
10. The method of claim 9 , wherein the oligonucleotide adaptor is a double-stranded oligonucleotide adaptor.
11. The method of claim 10 , wherein the double-stranded oligonucleotide adaptor is attached to the conditioned DNA by only one of its two strands.
12. The method of claim 11 , wherein the double stranded adaptor is attached to the conditioned DNA by means of a 5′ terminus of the adaptor.
13. The method of claim 12 , wherein the double-stranded oligonucleotide adaptor is blocked at at least one of its 3′ termini.
14. The method of claim 13 , wherein the double-stranded oligonucleotide adaptor is blocked at both of its 3′ termini.
15. The method of claim 1 , wherein the conditioned DNA fragments are amplified.
16. The method of claim 15 , wherein DNA fragments are amplified through a PCR reaction.
17. The method of claim 16 , wherein the DNA fragments are amplified through a PCR reaction through the use of double-stranded adaptors that have been attached to the conditioned DNA fragments.
18. The method of claim 1 , further defined as comprising the steps of:
a) obtaining a sample of DNA wherein the sample includes DNA fragments that do not include a 3′ hydroxyl group, wherein the sample has been subjected to enzymatic fragmentation;
b) conditioning DNA fragments of the sample that lack a 3′ hydroxyl by incorporating a 3′ hydroxyl group thereon through the use of a mixture comprising a DNA polymerase and exonuclease III;
c) attaching adaptors to DNA fragments of the sample; and
d) amplifying DNA fragments of the sample through the use of the adaptors.