Chemical method for the analysis of DNA sequences

• US 5,534,424 A
• Filed: 04/18/1995
• Issued: 07/09/1996
• Est. Priority Date: 05/12/1992
• Status: Expired due to Term

• Current Assignee: Cemu Bioteknik AB
• Original Assignee: Cemu Bioteknik AB
• Inventors: Uhlen, Mathias, Lundeberg, Joakim
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): REES, DIANE
• Application Number:
  US08/331,552
• Time in Patent Office: 448 Days
• Field of Search: 435/6, 435/5, 435/91.5, 435/91.2, 536/24.3, 536/24.33
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6869 Methods for sequencing

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.

Claims

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.

2. The method of claim 1, wherein, in step (A), said sample DNA is amplified by an in vitro amplification reaction using a first primer which is immobilised on a solid support or which is provided with means for immobilisation on a solid support.

3. The method of claim 2, wherein said first primer includes a non-hybridising region which upon chain extension creates, in double-stranded form, a recognition site for a DNA-binding protein carrying a label, and wherein, in step (F), the formation of double stranded DNA by chain extension is identified by adding the labeled DNA-binding protein to said aliquots and determining the binding of said labeled DNA-binding protein to the DNA in each of said aliquots.

4. The method of claim 2, wherein said first primer carries biotin as means for immobilisation.

5. The method of claim 2, wherein:

in step (A), said in vitro amplification reaction further uses a second primer for introducing a loop structure into the amplified DNA, wherein said second primer hybridises to a region A at the 3'"'"' terminus of said sample DNA and/or to a region B extending 3'"'"' from said region A, said second primer having a 3'"'"' terminal sequence which hybridises to at least a portion of regions A and/or B of said sample sequence while having at its 5'"'"'-end a sequence substantially identical to A, whereby said amplification reaction of step (A) produces double-stranded sample DNA having at the 3'"'"'-end of the sample sequence, in the following order, the region A, a region capable of forming a loop, and a sequence A'"'"' complementary to sequence A;

in step (B), said amplified, double-stranded DNA, if not already immobilised, is immobilised on a solid support at one end of one strand of said double-stranded DNA, thereby forming an immobilised strand an a non-immobilized strand, and is subjected to strand separation, whereby said non-immobilised strand is liberated and immobilised single-stranded DNA remains; and

in step (C), said extension primer is provided by permitting or causing said region A'"'"' of said immobilised strand to hybridise to region A of said immobilised strand at a temperature above the self-annealing temperature of said second primer, thereby forming said loop, whereby the hybridised portion of said loop forms said extension primer.

6. The method of claim 1, wherein, in step (F), the formation of double stranded DNA is identified by detecting pyrophosphate liberated in the extension reaction.

7. The method of claim 6, wherein said pyrophosphate is detected by reacting said aliquots with luciferase in the presence of luciferin, wherein emission of light is an indicator or measure of pyrophosphate.