Method for rapid amplification of DNA
First Claim
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1. A method of amplifying DNA, the method comprising:
- (a) providing a single reaction mixture comprising;
(i) a DNA sample;
(ii) a first primer comprising a random sequence of nucleotides at its 3′
end and a generic sequence 5′
of the random nucleotides;
(iii) a second primer comprising the generic sequence of the first primer and lacking the random sequence of the first primer; and
(iv) a heat-stable DNA polymerase;
(b) subjecting the DNA sample to DNA amplification wherein the first primer anneals to the DNA to allow the heat-stable DNA polymerase to produce a DNA product; and
(c) subjecting the DNA product of step (b) to DNA amplification wherein the second primer anneals to the DNA product.
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Abstract
The present disclosure relates to methods of DNA amplification with a first primer that has a random sequence of nucleotides at its 3′ end and a generic sequence 5′ of the random nucleotides, as well as a second primer with the generic sequence of the first primer. The disclosure further relates to a method of precipitating DNA on a solid medium to improve DNA amplification. In a preferred embodiment, the presently disclosed methods are used for high-throughput genotyping of DNA samples, such as bloodstains or trace amounts of DNA.
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Citations
34 Claims
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1. A method of amplifying DNA, the method comprising:
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(a) providing a single reaction mixture comprising;
(i) a DNA sample;
(ii) a first primer comprising a random sequence of nucleotides at its 3′
end and a generic sequence 5′
of the random nucleotides;
(iii) a second primer comprising the generic sequence of the first primer and lacking the random sequence of the first primer; and
(iv) a heat-stable DNA polymerase;
(b) subjecting the DNA sample to DNA amplification wherein the first primer anneals to the DNA to allow the heat-stable DNA polymerase to produce a DNA product; and
(c) subjecting the DNA product of step (b) to DNA amplification wherein the second primer anneals to the DNA product. - View Dependent Claims (2, 3, 4, 5, 6)
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7. A method of identifying a polymorphism, the method comprising:
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(a) providing a single reaction mixture comprising;
(i) a DNA sample;
(ii) a first primer comprising a random sequence of nucleotides at its 3′
end and a generic sequence 5′
of the random nucleotides;
(iii) a second primer comprising the generic sequence of the first primer and lacking the random sequence of the first primer; and
(iv) a heat-stable DNA polymerase;
(b) subjecting the DNA sample to DNA amplification wherein the first primer anneals to the DNA to allow the heat-stable DNA polymerase to produce a DNA product;
(c) subjecting the DNA product of step (b) to DNA amplification wherein the second primer anneals to the DNA product to allow the heat-stable DNA polymerase to produce amplified DNA products; and
(d) analyzing the amplified DNA products of step (c) to identify a polymorphism. - View Dependent Claims (8, 9)
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10. A method of amplifying DNA, the method comprising:
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(a) providing a single reaction mixture comprising;
(i) a DNA sample to be amplified;
(ii) a first primer comprising a random sequence of nucleotides at its 3′
end and a generic sequence 5′
of the random sequence;
(iii) a second primer comprising the generic sequence and lacking the random sequence of the first primer; and
(iv) a heat-stable DNA polymerase;
(b) heating the single reaction mixture to a temperature that denatures the DNA to be amplified;
(c) cooling the single reaction mixture to a temperature that allows the random sequence of the first primer to hybridize to its complement DNA and incubating the reaction mixture to allow synthesis of a DNA product by the heat-stable DNA polymerase;
(d) repeating steps (b) and (c) at least one time.
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11. A method of identifying a polymorphism, the method comprising:
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(a) precipitating a DNA sample on a solid medium;
(b) providing a single reaction mixture comprising;
(i) the DNA sample;
(ii) a first primer comprising a random sequence of nucleotides at its 3′
end and a generic sequence 5′
of the random nucleotides;
(iii) a second primer comprising the generic sequence of the first primer and lacking the random sequence of the first primer; and
(iv) a heat-stable DNA polymerase;
(c) subjecting the DNA sample to DNA amplification by the heat-stable DNA polymerase wherein the first primer anneals to the DNA to allow the heat-stable DNA polymerase to produce a DNA product;
(d) subjecting the DNA product of step (c) to DNA amplification by the heat-stable DNA polymerase wherein the second primer anneals to the DNA product to allow the heat-stable DNA polymerase to produce a second DNA product; and
(e) analyzing the second DNA product to identify a polymorphism. - View Dependent Claims (12)
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13. A method of amplifying DNA, the method comprising:
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(a) providing a single reaction mixture comprising;
(i) a DNA sample;
(ii) a first primer comprising a random sequence of nucleotides at its 3′
end and a generic sequence 5′
of the random nucleotides; and
(iii) a second primer comprising the generic sequence of the first primer and lacking the random sequence of the first primer;
(b) subjecting the DNA sample to DNA amplification by a heat-stable DNA polymerase wherein the first primer anneals to the DNA to produce a DNA product. - View Dependent Claims (14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)
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Specification