Methods of determining properties of nucleic acids without use of an internal standard and involving stretch and optical intensity
This disclosure features, inter alia, methods for determining at least one property of a nucleic acid. The methods include: (a) fixing the nucleic acid on a planar surface; (b) digesting the nucleic acid into fragments with at least one enzyme; (c) imaging the nucleic acid; and (d) analyzing the imaged nucleic acid to determine the property of the nucleic acid, wherein no internal nucleic acid standard is added during the method.
1. A method for determining at least one property of a nucleic acid, the method comprising:
(a) fixing the nucleic acid on a planar surface;
(b) digesting the nucleic acid into fragments with at least one enzyme;
(c) imaging the nucleic acid; and
(d) analyzing a relationship between known stretch of a nucleic acid fragment and optical intensity of the fragment to determine the property of the nucleic acid, wherein no internal nucleic acid standard is added during the method.
2. The method of claim 1 , wherein the imaging step comprises fluorescently labeling the nucleic acid.
3. The method of claim 2 , wherein said property is a size of at least one fragment of the nucleic acid.
4. The method of claim 3 , wherein the size of the fragment of the nucleic acid is determined by the following formula: (pixel length of the nucleic acid)/(known stretch−kb/pixel)×(fluorescence of a fragment)/(fluorescence of the nucleic acid).
5. The method of claim 2 , wherein the analyzing step further comprises identifying a backbone fluorescence intensity for the fragments, obtaining an intensity profile, and normalizing the fluorescence intensity among the fragments, thereby correcting for sizing biases of the fragments.
6. The method of claim 5 , wherein the intensity profile is expressed as a graph.
7. The method of claim 1 , wherein said nucleic acid is deoxyribonucleic acid.
8. The method of claim 1 , wherein said nucleic acid is ribonucleic acid.
9. The method of claim 1 , wherein said nucleic acid is a human nucleic acid.
10. A method for constructing an ordered restriction map, the method comprising:
(a) fixing a nucleic acid on a planar surface;
(b) digesting the nucleic acid into fragments with at least one enzyme;
(c) imaging the nucleic acid;
(d) analyzing a relationship between known stretch of a nucleic acid fragment and optical intensity of the fragment; and
(e) constructing an ordered restriction map based upon results of step (d), wherein no internal nucleic acid standard is added during the method.
11. The method of claim 10 , wherein the imaging step comprises fluorescently labeling the nucleic acid.
12. The method of claim 11 , wherein the analyzing step comprises sizing of the fragments of the nucleic acid.
13. The method of claim 12 , wherein the sizing of the fragments of the nucleic acid is determined by the following formula: (pixel length of the nucleic acid)/(known stretch−kb/pixel)×(fluorescence of a fragment)/(fluorescence of the nucleic acid).
14. The method of claim 11 , wherein the analyzing step further comprises identifying a backbone fluorescence intensity for the fragments, obtaining an intensity profile, and normalizing the fluorescence intensity among the fragments, thereby correcting for sizing biases of the fragments.
15. The method of claim 14 , wherein the intensity profile is expressed as a graph.
16. The method of claim 10 , wherein said nucleic acid is deoxyribonucleic acid.
17. The method of claim 10 , wherein said nucleic acid is ribonucleic acid.
18. The method of claim 10 , wherein said nucleic acid is a human nucleic acid.