Chemical method for the analysis of DNA sequences
First Claim
1. A method of identifying the base in a target position in a DNA sequence of sample DNA, wherein:
- (A) said sample DNA is subjected to amplification, thereby forming amplified, double-stranded DNA;
(B) said amplified, double-stranded DNA is immobilised on a solid support at one end of one strand of said double-stranded DNA, thereby forming an immobilised strand and a non-immobilised strand, and then subjected to strand separation, whereby said non-immobilised strand is removed and immobilised single-stranded DNA remains;
(C) an extension primer is provided, which hybridises to said immobilised single-stranded DNA at a position immediately adjacent to said target position;
(D) each of four aliquots of said immobilised single-stranded DNA is subjected to a polymerase reaction in the presence of a dideoxynucleotide, each aliquot using a different dideoxynucleotide, whereby only in the aliguot using the dideoxynucleotide complementary to the base at said target position will said dideoxynucleotide become incorporated into said immobilised single-stranded DNA to form dideoxy-blocked DNA;
(E) each of said four aliquots are subjected to extension in the presence of all four deoxynucleotides, whereby the DNA which did not form dideoxy-blocked DNA is extended to form double-stranded DNA, while the dideoxy-blocked DNA remains as non-extended DNA; and
(F) the formation of double-stranded DNA and/or non-extended DNA is identified to indicate which dideoxynucleotide was incorporated into said immobilised single-stranded DNA, and hence which base was present at said target position.
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Accused Products
Abstract
The invention provides a method of identification of the base in a target position in a DNA sequence wherein sample DNA is subjected to amplification; the amplified DNA is immobilised and then subjected to strand separation, the non-immobilised strand being removed and an extension primer, which hybridises to the immobilised DNA immediately adjacent to the target position, is provided; each of four aliquots of the immobilised single stranded DNA is then subjected to a polymerase reaction in the presence of a dideoxynucleotide, each aliquot using a different dideoxynucleotide whereby only the dideoxynucleotide whereby only the dideoxynucleotide complementary to the base in the target position becomes incorpored; the four aliquots are then subjected to extension in the presence of all four deoxynucleotides, whereby in each aliquot the DNA which has not reacted with the dideoxynucleotide is extended to form double stranded DNA while the dideoxy-blocked DNA remains as non-extended DNA; followed by identification of the double stranded and/or non-extended DNA to indicate which dideoxynucleotide was incorporated and hence which base was present in the target position.
171 Citations
7 Claims
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1. A method of identifying the base in a target position in a DNA sequence of sample DNA, wherein:
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(A) said sample DNA is subjected to amplification, thereby forming amplified, double-stranded DNA; (B) said amplified, double-stranded DNA is immobilised on a solid support at one end of one strand of said double-stranded DNA, thereby forming an immobilised strand and a non-immobilised strand, and then subjected to strand separation, whereby said non-immobilised strand is removed and immobilised single-stranded DNA remains; (C) an extension primer is provided, which hybridises to said immobilised single-stranded DNA at a position immediately adjacent to said target position; (D) each of four aliquots of said immobilised single-stranded DNA is subjected to a polymerase reaction in the presence of a dideoxynucleotide, each aliquot using a different dideoxynucleotide, whereby only in the aliguot using the dideoxynucleotide complementary to the base at said target position will said dideoxynucleotide become incorporated into said immobilised single-stranded DNA to form dideoxy-blocked DNA; (E) each of said four aliquots are subjected to extension in the presence of all four deoxynucleotides, whereby the DNA which did not form dideoxy-blocked DNA is extended to form double-stranded DNA, while the dideoxy-blocked DNA remains as non-extended DNA; and (F) the formation of double-stranded DNA and/or non-extended DNA is identified to indicate which dideoxynucleotide was incorporated into said immobilised single-stranded DNA, and hence which base was present at said target position. - View Dependent Claims (2, 3, 4, 5, 6, 7)
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Specification