Method for rapid amplification of DNA
AssigneeInvitrogen Corporation
InventorsWan Ji, Keqin Gregg, Bonnie Reus, Jon Kemppainen, Kristen Taylor, Scott Davis
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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.
Claims34
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.
A method of identifying a polymorphism, 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;
- (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.
A method of amplifying DNA, the method comprising:
- (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.
A method of identifying a polymorphism, the method comprising:
- (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.
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;
- 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.
Dependencies are inferred from the claim text.