Chain-terminating nucleotides for DNA sequencing methods
First Claim
Patent Images
1. A method for sequencing DNA, comprising:
- contacting a polymerase selected from the group consisting of T4 polymerase, T2 polymerase, T6 polymerase and E. coli DNA polymerase II, with a primed DNA strand to be sequenced in the presence of dATP, dGTP, dCTP, dTTP, a first chain-terminating nucleotide, a second chain-terminating nucleotide, a third chain-terminating nucleotide and a fourth chain-terminating nucleotide; and
allowing said contacting to proceed under reaction conditions to maintain polymerase activity for a period of time sufficient to obtain sequencing information, wherein said first chain-terminating nucleotide is 3'"'"'-amino-2'"'"',3'"'"'dideoxy-ATP, said second chain-terminating nucleotide is 3'"'"'-amino-2'"'"',3'"'"'dideoxy-GTP, said third chain-terminating nucleotide is 3'"'"'-amino-2'"'"',3'"'"'dideoxy-CTP and said fourth chain-terminating nucleotide is 3'"'"'-amino-2'"'"',3'"'"'dideoxy-TTP.
3 Assignments
0 Petitions
Accused Products
Abstract
There are provided variant family B DNA polymerases having no 3'"'"'→5'"'"' exonuclease activity. These variant polymerases have utility as DNA sequencing polymerases. Methods for DNA sequencing with family B DNA polymerases and chain-terminating nucleotides not previously used for sequencing have been developed. The methods disclosed involve the use of family B DNA polymerases not known heretofore to have utility in DNA sequencing, such as variant or wild type forms of phage T4 DNA polymerase or Escherichia coli DNA polymerase II, with novel combinations of deoxynucleotides and chain-terminating nucleotides.
130 Citations
6 Claims
-
1. A method for sequencing DNA, comprising:
-
contacting a polymerase selected from the group consisting of T4 polymerase, T2 polymerase, T6 polymerase and E. coli DNA polymerase II, with a primed DNA strand to be sequenced in the presence of dATP, dGTP, dCTP, dTTP, a first chain-terminating nucleotide, a second chain-terminating nucleotide, a third chain-terminating nucleotide and a fourth chain-terminating nucleotide; and allowing said contacting to proceed under reaction conditions to maintain polymerase activity for a period of time sufficient to obtain sequencing information, wherein said first chain-terminating nucleotide is 3'"'"'-amino-2'"'"',3'"'"'dideoxy-ATP, said second chain-terminating nucleotide is 3'"'"'-amino-2'"'"',3'"'"'dideoxy-GTP, said third chain-terminating nucleotide is 3'"'"'-amino-2'"'"',3'"'"'dideoxy-CTP and said fourth chain-terminating nucleotide is 3'"'"'-amino-2'"'"',3'"'"'dideoxy-TTP. - View Dependent Claims (2, 3, 4, 5, 6)
-
Specification