Method of amplification of GC-rich DNA templates
AssigneeAsuragen, Inc.
InventorGary J. Latham
Life of this patent
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Abstract
Methods are provided for increasing the processivity of DNA polymerases on GC-rich templates. The methods relate to providing enhancers and biased ratios of dNTPs, and may be used in DNA amplification reactions. The methods are useful for detecting genotypes associated with GC-rich repeats, including Fragile X Syndrome.
Claims27
A method of increasing the processivity of one or more DNA polymerases on at least one GC-rich DNA template having a GC-richness of at least 51%, the method comprising performing a DNA amplification reaction in an aqueous solution comprising the at least one GC-rich DNA template having a GC-richness of at least 51%;
- at least two distinct primers;
- at least one enhancer;
- and dNTPs in a GC/AT ratio of 1.2 or higher.
A method of increasing the processivity of one or more DNA polymerases on at least one GC-rich DNA template, the method comprising performing a DNA amplification reaction in an aqueous solution comprising the at least one GC-rich DNA template;
- dNTPs in a GC/AT ratio of 1.2 or higher;
- and at least one enhancer from each of (a) and (b):
- a. a first enhancer, which is betaine or a betaine analog;
- and
- b. at least one additional enhancer selected from the group consisting of DMSO, a neutral detergent, and 7-deaza-GTP.
A method of increasing the processivity of one or more DNA polymerases on at least one GC-rich DNA template comprising CGG repeats of the 5′ UTR of FMR1, the method comprising amplifying the template comprising CGG repeats of the 5′ UTR of FMR1 by PCR in an aqueous solution comprising:
- a. dNTPs in a GC/AT ratio between 2 and 10;
- b. at least one enhancer chosen from betaine, DMSO, and a neutral detergent;
- c. at least one DNA polymerase;
- d. a total magnesium concentration of 1.5-2 mM;
- and
- e. a total dNTP concentration of 0.7-0.9 mM.
A method of detecting a genotype associated with a GC-rich trinucleotide repeat disorder, comprising performing a DNA amplification reaction using at least two distinct primers on at least one genotype associated with the GC-rich trinucleotide repeat disorder, in which the processivity of one or more DNA polymerases is increased by providing an aqueous solution comprising dNTPs in a GC/AT ratio of 1.2 or higher.
A method of detecting a genotype associated with Fragile X Syndrome, Fragile X-associated tremor ataxia syndrome, and/or Fragile X-associated primary ovarian insufficiency, comprising performing a DNA amplification reaction using at least two distinct primers on at least one GC-rich DNA template associated with Fragile X Syndrome, Fragile X-associated tremor ataxia syndrome, and/or Fragile X-associated primary ovarian insufficiency, in which the processivity of one or more DNA polymerases is increased by providing an aqueous solution comprising dNTPs in a GC/AT ratio of 1.2 or higher.
Dependencies are inferred from the claim text.