Polynucleotide mapping and sequencing
View Patent ↗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 nucleic acid biopolymer, comprising:
binding a first fluorescently labeled sequence specific probe having a first binding sequence to a single-stranded nucleic acid biopolymer;
contacting the single-stranded nucleic acid biopolymer with a first terminator nucleotide bearing a fluorescent label A, with a second terminator nucleotide bearing a fluorescent label B, with a third terminator nucleotide bearing a fluorescent label C, and with a fourth terminator nucleotide bearing a fluorescent label D;
linearizing the nucleic acid biopolymer; and
illuminating the linearized nucleic acid biopolymer so as to determine the presence of, the relative positions of, or both, of the first terminator nucleotide, the second terminator nucleotide, the third terminator nucleotide, the fourth terminator nucleotide, or any combination thereof, adjacent to the first labeled sequence-specific probe.
2. The method of claim 1 , wherein the first binding sequence comprises from 4 to 6 nucleotides.
3. The method of claim 1 , wherein at least two of fluorescent label A, fluorescent label B, fluorescent label C, and fluorescent label D have different excitation wavelengths.
4. The method of claim 3 , wherein at least one of fluorescent label A, fluorescent label B, fluorescent label C, or fluorescent label D has an excitation wavelength different from the excitation wavelength of the first fluorescently labeled sequence-specific probe.
5. The method of claim 1 , further comprising contacting at least four fluorescently labeled probes having, respectively, second, third, fourth, and fifth binding sequences to the single-stranded nucleic acid biopolymer.
6. The method of claim 5 , wherein the second binding sequence is constructed by eliminating the base at the 5′ end of the first binding sequence and adding a first replacement base to the 3′ end of the first binding sequence.
7. The method of claim 5 , wherein the third binding sequence is constructed by eliminating the base at the 5′ end of the first binding sequence and adding a second replacement base to the 3′ end of the first binding sequence.
8. The method of claim 5 , wherein the fourth binding sequence is constructed by eliminating the base at the 5′ end of the first binding sequence and adding a third replacement base to the 3′ end of the first binding sequence.
9. The method of claim 5 , wherein the fifth binding sequence is constructed by eliminating the base at the 5′ end of the first binding sequence and adding a fourth replacement base to the 3′ end of the first binding sequence.
10. The method of claim 5 , wherein the second fluorescently labeled sequence specific probe has an excitation wavelength different from that of the first fluorescently-labeled sequence-specific probe.
11. The method of claim 5 , wherein the second binding sequence differs from the first binding sequence.
12. The method of claim 11 , wherein the second binding sequence comprises from 4 to 6 nucleotides.
13. The method of claim 5 , further comprising illuminating the nucleic acid biopolymer so as to determine the presence or relative positions of the first terminator nucleotide, the second terminator nucleotide, the third terminator nucleotide, the fourth terminator nucleotide, or any combination thereof, adjacent to the second labeled sequence-specific probe.
14. The method of claim 1 , comprising determining the relative positions of two or more of the first terminator nucleotide, the second terminator nucleotide, the third terminator nucleotide, and the fourth terminator nucleotide on the linearized nucleic acid biopolymer.
15. The method of claim 1 , wherein said linearizing is in a channel.
16. The method of claim 15 , wherein the channel has dimensions at the same order as the persistence length of the nucleic acid biopolymer or below the persistence length of the nucleic acid biopolymer.
17. The method of claim 1 , wherein said linearizing is on a glass surface.