Methods and devices for single-molecule whole genome analysis
Provided are methods and devices for single-molecule genomic analysis. In one embodiment, the methods entail processing a double-stranded nucleic acid and characterizing said nucleic acid. These methods are useful in, e.g. determining structural variations and copy number variations between individuals.
1. A method of processing DNA, comprising:
nicking a first DNA strand of a double-stranded DNA to give rise to multiple nicking sites;
incorporating one or more nucleotides at the nicking sites;
extending the first DNA strand along a corresponding region of a second DNA strand of the double-stranded DNA and contacting the first DNA strand with a polymerase, one or more nucleotides, and a ligase; and
labeling one or more sequence-specific locations on the first DNA strand;
stretching the double-stranded DNA at the extended region of the labeled first DNA strand or a portion thereof, thereby linearizing at least a portion of the double-stranded DNA comprising a portion of the extended region of the first DNA strand; and
detecting the relative positions of the nicking sites on the linearized double-stranded DNA.
2. The method of claim 1 , wherein the nicking is effected at one or more sequence-specific locations.
3. The method of claim 1 , wherein the nicking is effected at one or more non-sequence specific locations.
4. The method of claim 1 , wherein the labeling is accomplished by (a) binding at least one complementary probe to at least a portion of the double-stranded DNA, the probe comprising one or more tags, (b) extending the first DNA strand along the corresponding region of the second DNA strand with one or more nucleotides comprising one or more tags, or (a) and (b).
5. The method of claim 4 , further comprising obtaining sequence information derived from one or more tags.
6. The method of claim 5 , wherein the sequence information is derived from two or more locations on the double-stranded DNA.
7. The method of claim 4 , further comprising detecting one or more signals from one or more tags, wherein the signal comprises at least one member selected from the group consisting of: a fluorescent signal, a chemoluminescent signal, an electromagnetic signal, an electrical signal, a potential difference, and a physical size difference.
8. The method of claim 4 , further comprising measuring the distance between two or more tags.
9. The method of claim 8 , wherein the distance between the two tags comprises at least one member selected from the group consisting of: a region comprising a methylation pattern, a location of a CpG island, an epigenomic pattern, and a physiological characteristic.
10. The method of claim 4 , further comprising measuring the intensity of at least one signal from at least one tag.
11. The method of claim 1 , wherein the labeling comprises attaching a tag comprising at least one member selected from the group consisting of: a fluorophore, a quantum dot, a dendrimer, a nanowire, a bead, a peptide, a protein, a magnetic bead, a methyl group, a methyltransferase, a non-cutting restriction enzyme, a zinc-finger protein, an antibody, a transcription factor, a DNA binding protein, a hairpin polyamide, a triplex-forming oligodeoxynucleotide, and a peptide nucleic acid.
12. The method of claim 1 , wherein the linearizing is effected by confinement of the DNA in a channel.
13. A method of labeling a double-stranded DNA, comprising:
processing the double-stranded DNA comprising a first DNA strand and a second DNA strand to nick the first DNA strand and give rise to one or more nicking sites on the first DNA strand and a corresponding region on the second DNA strand;
extending the first DNA strand at the nicking sites along the corresponding region of the second DNA strand and contacting the first DNA strand with a polymerase, one or more nucleotides, and a ligase; and
labeling a portion of the extended first DNA strand, thereby labeling the double-stranded DNA.
14. The method of claim 13 , wherein the nicking is effected at one or more sequence-specific locations.
15. The method of claim 13 , wherein the labeling is accomplished by (a) binding at least one complementary probe to at least a portion of the double-stranded DNA, the probe comprising one or more tags, (b) extending the first DNA strand along the corresponding region of the second DNA strand with one or more nucleotides comprising one or more tags, or (a) and (b).
16. The method of claim 15 , further comprising detecting one or more signals from one or more tags, wherein the signal comprises at least one member selected from the group consisting of: a fluorescent signal, a chemoluminescent signal, an electromagnetic signal, an electrical signal, a potential difference, and a physical size difference.
17. The method of claim 13 , wherein the labeling comprises attaching a tag comprising at least one member selected from the group consisting of: a fluorophore, a quantum dot, a dendrimer, a nanowire, a bead, a peptide, a protein, a magnetic bead, a methyl group, a methyltransferase, a non-cutting restriction enzyme, a zinc-finger protein, an antibody, a transcription factor, a DNA binding protein, a hairpin polyamide, a triplex-forming oligodeoxynucleotide, and a peptide nucleic acid.
18. A method of processing DNA, comprising:
processing a double-stranded DNA comprising a first DNA strand and a second DNA strand and nicking the first DNA strand to give rise to an unhybridized flap of the first DNA strand and a corresponding region on the second DNA strand, wherein the unhybridized flap comprises about 1 to about 1000 bases;
extending the first DNA strand along the corresponding region of the second DNA strand by incorporating more than one nucleotide; and
labeling at least a portion of the unhybridized flap, a portion of the extended first DNA strand, or both; and
linearizing at least a portion of the double-stranded DNA comprising the unhybridized flap and the extended first DNA strand, wherein the linearizing is accomplished by linearizing the labelled portion of the double-stranded DNA comprising the unhybridized flap and the extended first DNA strand in a nanochannel or by applying a fluid, electrical, or pressure gradient.
19. The method of claim 18 , wherein the nicking is effected at one or more sequence-specific locations.
20. The method of claim 18 , wherein the labeling is accomplished by (a) binding at least one complementary probe to at least a portion of the double-stranded DNA, the probe comprising one or more tags, (b) extending the first DNA strand along the corresponding region of the second DNA strand with one or more nucleotides comprising one or more tags, or (a) and (b).