Methods and compositions for sequencing nucleic acids using charge
The invention relates to methods and compositions, and systems for determining the identity of nucleic acids in nucleotide sequences, and in particular, sequences that contain consecutive repeats of a particular base. For example, a consecutive repeat of a particular base may be identified by a charged ion detection (e.g., hydrogen ion detection).
1. A method for sequencing nucleic acids comprising,
a) incorporating one or more nucleotides into a plurality of nucleic acids in one or more reaction chambers in contact with one or more charge detectors, wherein said nucleotides comprise a cleavable label and a 3′-OH blocking group,
b) cleaving said cleavable label under conditions such that a charged moiety is produced, wherein said charged moiety is a positively charged moiety or a negatively charged moiety, the nature of the charge being correlated with the identity of the base; and
c) detecting said charged moiety with said one or more charge detectors under conditions such that said one or more nucleotides are identified, wherein said positively charged moiety identifies a pyrimidine nucleotide.
2. The method of claim 1 , wherein there is a wash step prior to said cleaving which removes unincorporated nucleotides.
3. The method of claim 1 , wherein said cleavable label is cleaved enzymatically.
4. The method of claim 1 , wherein said cleavable label is cleaved chemically.
5. The method of claim 1 , wherein a portion of the sequence of said nucleic acid comprises consecutive identical bases of the formula Xn, where X is any base and n is a whole number between 3 and 10.
6. The method of claim 1 , wherein the cleavable label is attached through a cleavable linker to the base of said nucleotide.
7. A method for sequencing nucleic acids comprising,
a) incorporating one or more nucleotides into a plurality of nucleic acids in one or more reaction chambers in contact with one or more charge detectors, wherein said nucleotides comprise a cleavable label and a 3′-OH blocking group,
b) cleaving said cleavable label under conditions such that a charged moiety is produced, wherein said charged moiety is a positively charged moiety or a negatively charged moiety, the nature of the charge being correlated with the identity of the base; and
c) detecting said charged moiety with said one or more charge detectors under conditions such that said one or more nucleotides are identified, wherein said negatively charged moiety identifies a purine nucleotide.
8. The method of claim 7 , wherein there is a wash step prior to said cleaving which removes unincorporated nucleotides.
9. The method of claim 7 , wherein said cleavable label is cleaved enzymatically.
10. The method of claim 7 , wherein said cleavable label is cleaved chemically.
11. The method of claim 7 , wherein a portion of the sequence of said nucleic acid comprises consecutive identical bases of the formula Xn, where X is any base and n is a whole number between 3 and 10.
12. The method of claim 7 , wherein the cleavable label is attached through a cleavable linker to the base of said nucleotide.