Methods and compositions for amplification and sequencing of difficult DNA templates
This disclosure provides methods and compositions for amplification and sequencing of DNA templates, comprising at least two of: 2′-deoxyinosine-5′ triphosphate, 5-propynyl-2′-deoxycytidine-5′-triphosphate, and 8-oxo-2′-deoxyguanosine-5′-triphosphate. Incorporation of these promoting nucleotides into amplification and sequencing reactions improves the amplification and sequencing of difficult-to-sequence DNA regions such as a GC rich regions or GT rich regions; repetitive sequences, including dinucleotide, trinucleotide, direct, inverted, Alu, poly A or poly T repeats; and hairpin or other secondary structures.
1. A method for amplifying a DNA template, comprising:
a. providing a DNA template and an exonuclease-resistant random hexamer primer;
b. denaturing said DNA template;
c. annealing said DNA template with said primer; and
d. incubating said template and said primer with a nucleotide composition comprising at least two of: 2′-deoxyinosine-5′ triphosphate, 5-propynyl-2′-deoxycytidine-5′-triphosphate, and 8-oxo-2′-deoxyguanosine-5′-triphosphate, for a time sufficient for said template to be amplified.
2. The method of claim 1 , wherein the DNA template is a circular DNA template.
3. The method of claim 2 , wherein said circular DNA template comprises at least one of the following: a GC rich or GT rich sequence, a homopolymer repetitive sequence; or a hairpin secondary structure.
4. The method of claim 1 , wherein said circular DNA template is provided in step (a) in an amount of 1 pg to 500 ng.
5. The method of claim 1 , wherein said template and said primer are incubated in step (d) at 28-32° C. for 2-18 hours.
6. The method of claim 1 wherein in step (d) said template and said primer are incubated with a composition comprising:
a mixture of nucleotides dATP, dCTP, dGTP and dTTP,
DNA polymerase with strand displacement activity,
a buffer,
a magnesium salt, and
one or more ammonia sulfate salts,
for a time sufficient for said template to be amplified.
7. The method of claim 1 , wherein the nucleotide composition of step (d) comprises all three of 2′-deoxyinosine-5′ triphosphate, 5-propynyl-2′-deoxycytidine-5′-triphosphate, and 8-oxo-2′-deoxyguanosine-5′-triphosphate.