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 composition for amplification and sequencing of DNA templates, comprising at least two of the following three nucleotides:
a. 2′-deoxyinosine-5′ triphosphate,
b. 5-propynyl-2′-deoxycytidine-5′-triphosphate, and
c. 8-oxo-2′-deoxyguanosine-5′-triphosphate.
2. The composition of claim 1 , comprising all three of said nucleotides.
3. The composition of claim 1 , wherein each of said nucleotides present in the composition is present in the range of 1 μM to 5 mM.
4. The composition of claim 1 , comprising 2′-deoxyinosine-5′ triphosphate and 8-oxo-2′-deoxyguanosine-5′-triphosphate in a ratio of 100:1 to 2:1.
5. The composition of claim 1 , comprising 2′-deoxyinosine-5′ triphosphate and 5-propynyl-2′-deoxycytidine-5′-triphosphate in a ratio between 100:1 to 2:1.
6. The composition of claim 1 , wherein the composition promotes amplification and/or sequencing of circular or linear DNA.
7. The composition of claim 6 , wherein the circular or linear DNA contains one or more of: a GC-rich region, a repetitive sequence, or a hairpin secondary structure.
8. The composition of claim 1 , further comprising the following components:
a sequence-specific primer;
a mixture of nucleotides dATP, dCTP, dITP and dUTP;
a mixture of fluorescently labeled nucleotides ddATP, ddCTP, ddGTP and ddTTP;
at least one thermostable DNA polymerase;
a thermostable pyrophosphatase;
a buffer; and
a magnesium salt.
9. The composition of claim 8 , wherein one of the at least one thermostable DNA polymerases is Taq polymerase.
10. The composition of claim 8 , wherein the thermostable pyrophosphatase is recombinant Thermus thermophiles (rTth) pyrophosphate.
11. The composition of claim 8 , wherein the composition is a direct DNA sequencing composition.
12. The composition of claim 1 , further comprising the following components:
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.
13. The composition of claim 12 , wherein the DNA polymerase is phage 29 (Phi29) DNA polymerase.
14. The composition of claim 12 , wherein the composition is a composition for rolling circle DNA amplification or rolling circle DNA amplification followed by DNA cycle sequencing.