Polynucleotide mapping and sequencing
The present invention provides methods of obtaining structural information about a biopolymer sample. The methods include labeling portions of a biopolymer, such as DNA or RNA, linearizing the biopolymer in some cases, and determining the distance between the labels. The user can then compare different samples' between-label distances to qualitatively compare different samples and to assay a given sample for additions or deletions of nucleotides in the regions flanked by the labels. The methods also permit sequencing of biopolymers.
1. A method of obtaining sequence information about a DNA sample, comprising:
nicking a first double-stranded DNA sample with a first sequence-specific nicking endonuclease;
incorporating labeled nucleotides into the first double-stranded DNA sample at two or more nicking sites effected by the nicking endonuclease;
linearizing a portion of the first double-stranded DNA sample that includes at least two labeled nucleotides by passing the portion of the first double-stranded DNA sample through a nanochannel; and
registering the relative positions of two or more labeled nucleotides of the portion of the first double-stranded DNA sample, wherein the portion of the first double-stranded DNA sample is residing within the nanochannel, and wherein at least two of the labeled nucleotides are separated from each other by 30 base pairs to 1000 base pairs.
2. The method of claim 1 , wherein at least one of the two or more of the labeled nucleotides comprises a terminator nucleotide.
3. The method of claim 1 , further comprising determining the relative position of the first double-stranded DNA sample within a principal double-stranded DNA sample from which the first double-stranded DNA sample was derived.
4. The method of claim 3 , wherein the first double-stranded DNA sample was obtained from the principal double-stranded DNA sample by digestion.
5. The method of claim 1 , further comprising nicking a second double-stranded DNA sample with a sequence-specific nicking endonuclease, incorporating one or more labeled nucleotides into the second double-stranded DNA sample at two or more nicking sites effected by the nicking endonuclease, linearizing a portion of the second double-stranded DNA sample that includes at least two labeled nucleotides, and registering the relative positions of two or more labeled nucleotides of the portion of the second double-stranded DNA sample.
6. The method of claim 1 , further comprising comparing the relative positions of the two or more labeled nucleotides to the positions of the same labeled nucleotides on a second double-stranded DNA sample contacted with the same nicking endonuclease.
7. The method of claim 1 , wherein registering the relative positions of the two or more labeled nucleotides comprises determining the distances between the two or more labeled nucleotides residing within the linearized portion of the first double-stranded DNA.
8. The method of claim 1 , wherein incorporating labeled nucleotides at the nicking sites comprises contacting the nicking sites with three or more fluorescent-label-bearing nucleotides selected from a first nucleotide bearing a fluorescent label A, a second nucleotide bearing a fluorescent label B, a third nucleotide bearing a fluorescent label C, and a fourth nucleotide bearing a fluorescent label D.
9. The method of claim 8 , further comprising illuminating the first double-stranded DNA sample so as to determine the presence or relative positions of the one or more fluorescent-label-bearing nucleotides.
10. The method of claim 9 , further comprising determining the sequence of at least a portion of the first double-stranded DNA sample by correlating the order of fluorophores visible under illumination to the nucleotides to which one or more of the fluorophores correspond.
11. The method of claim 1 , further comprising nicking the first double-stranded DNA sample with a second sequence-specific nicking endonuclease to introduce nick sites adjacent to the nucleotide residing at the two or more nick sites effected by the first sequence-specific nicking endonuclease.
12. The method of claim 1 , further comprising obtaining sequence information of the DNA sample based on the registered relative positions of the labeled nucleotides.
13. The method of claim 1 , wherein incorporating labeled nucleotides comprises contacting the nicking sites with two or more labeled nucleotides selected from the group consisting of a first nucleotide bearing a label A, a second nucleotide bearing a label B, a third nucleotide bearing a label C, and a fourth nucleotide bearing a label D.
14. The method of claim 13 , wherein at least two of the label A, label B, label C, and label D are different.
15. The method of claim 13 , wherein the nicking sites are contacted with three or more labeled nucleotides, and wherein at least three of the label A, label B, label C, and label D are different.
16. The method of claim 13 , wherein the nicking sites are contacted with four or more labeled nucleotides. and wherein at least four of the label A, label B, label C, and label D are different.
17. The method of claim 1 , wherein the labeled nucleotide is a dye-labeled nucleotide.