Polymerases for analyzing or typing polymorphic nucleic acid fragments and uses thereof
First Claim
1. A method of identifying, analyzing or typing a polymorphic DNA fragment in a sample of DNA, said method comprising:
- (a) contacting said sample of DNA with one or more DNA polymerases, wherein said one or more DNA polymerases comprise one or more mutations or modifications in the O-helix of said one or more DNA polymerases which reduce their ability to add one or more non-templated nucleotides to the 3′
terminus of a synthesized DNA molecule;
(b) amplifying said polymorphic DNA fragment within said sample to produce a population of amplified DNA fragments, wherein less than about 50% of said amplified DNA fragments have one or more non-templated 3′
nucleotides compared to amplification products produced by Taq DNA polymerase assayed under the same conditions; and
(c) analyzing said amplified polymorphic DNA fragment.
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Abstract
The present invention provides methods for use in identifying, analyzing and typing polymorphic DNA fragments, particularly minisatellite, microsatellite or STR DNA fragments. In particular, the invention provides methods using DNA polymerases, more particularly thermostable DNA polymerases, and most particularly Thermotoga polymerases or mutants or derivatives thereof, whereby minisatellite, microsatellite or STR DNA molecules may be amplified and analyzed for polymorphisms. The invention also relates to polymerases having reduced, substantially reduced or eliminated ability to add non-template 3′ nucleotides to a synthesized nucleic acid molecule. In accordance with the invention, such reduction or elimination may be accomplished by modifying or mutating the desired polymerase.
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Citations
42 Claims
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1. A method of identifying, analyzing or typing a polymorphic DNA fragment in a sample of DNA, said method comprising:
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(a) contacting said sample of DNA with one or more DNA polymerases, wherein said one or more DNA polymerases comprise one or more mutations or modifications in the O-helix of said one or more DNA polymerases which reduce their ability to add one or more non-templated nucleotides to the 3′
terminus of a synthesized DNA molecule;(b) amplifying said polymorphic DNA fragment within said sample to produce a population of amplified DNA fragments, wherein less than about 50% of said amplified DNA fragments have one or more non-templated 3′
nucleotides compared to amplification products produced by Taq DNA polymerase assayed under the same conditions; and(c) analyzing said amplified polymorphic DNA fragment. - View Dependent Claims (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 29, 30, 33, 34, 35, 36, 42)
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2. A method of producing amplified copies of a polymorphic DNA fragment, said method comprising:
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(a) contacting a DNA sample with one or more DNA polymerases, wherein said one or more DNA polymerases comprise one or more mutations or modifications in the O-helix of said one or more DNA polymerases which reduce their ability to add one or more non-templated nucleotides to the 3′
terminus of a synthesized DNA molecule; and(b) amplifying said polymorphic DNA fragment within said DNA sample to produce a population of amplified DNA fragments, wherein less than about 50% of said amplified DNA fragments have one or more non-templated 3′
nucleotides compared to amplification products produced by Taq DNA polymerase assayed under the same conditions.
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19. A kit comprising one or more DNA polymerases, wherein said one or more DNA polymerases comprise one or more mutations or modifications in the O-helix of said one or more DNA polymerases which reduce their ability to add one or more non-templated nucleotides to the 3′
- terminus of a synthesized DNA molecule, and wherein amplification of a polymorphic DNA fragment with said DNA polymerase produces a population of DNA fragments in which less than about 50% of said DNA fragments have one or more non-templated 3′
nucleotides compared to amplification products produced by Taq DNA polymerase assayed under the same conditions. - View Dependent Claims (20, 21, 22, 23, 24, 25, 26, 27, 31, 32, 37, 38, 39, 40, 41)
- terminus of a synthesized DNA molecule, and wherein amplification of a polymorphic DNA fragment with said DNA polymerase produces a population of DNA fragments in which less than about 50% of said DNA fragments have one or more non-templated 3′
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28. A method for amplifying a double stranded DNA molecule, comprising:
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(a) providing a first and second primer, wherein said first primer is complementary to a sequence at or near the 3′
-terminus of the first strand of said DNA molecule and said second primer is complementary to a sequence at or near the 3′
-terminus of the second strand of said DNA molecule;(b) hybridizing said first primer to said first strand and said second primer to said second strand in the presence of one or more DNA polymerases, wherein said one or more DNA polymerases comprise one or more mutations or modifications in the O-helix of said one or more DNA polymerases which reduce their ability to add non-templated 3′
nucleotides to a synthesized nucleic acid molecule under conditions such that a third DNA molecule complementary to said first strand and a fourth DNA molecule complementary to said second strand are synthesized;(c) denaturing said first and third strands, and said second and fourth strands; and (d) repeating steps (a) to (c) one or more times to produce a population of amplified DNA fragments, wherein less than about 50% of said amplified DNA fragments have one or more non-templated 3′
nucleotides compared to amplification products produced by Taq DNA polymerase assayed under the same conditions.
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Specification