Sequencing DNA; a modification of the polymerase chain reaction

• US 5,817,797 A
• Filed: 06/01/1988
• Issued: 10/06/1998
• Est. Priority Date: 06/01/1988
• Status: Expired due to Fees

• Current Assignee: United States Department Of Health And Human Services
• Original Assignee: United States Department Of Health And Human Services
• Inventors: Mitchell, Lloyd G., Merril, Carl R.
• Primary Examiner(s): Wilson, James O.
• Application Number:
  US07/200,876
• Time in Patent Office: 3,779 Days
• Field of Search: 435/6, 435/172.3, 435/810, 435/91, 435/803, 435/17, 435/3, 536/26, 536/27, 536/28, 536/29, 536/25.3, 536/25.4
• US Class Current: 536/25.3
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

The invention provides a process wherein a biotinylated oligonucleotide primer and an oligonucleotide primer which has not undergone biotinylation are used when amplifying a DNA sequence to facilitate separation of the DNA strands following the polymerase chain reaction process. The biotinylation/PCR product is then exposed to a support which will selectively bind the biotinylated strand to allow selective elution of the product.

Claims

1. A process for preparing DNA amplified by a polymerase chain reaction which comprises:

binding biotin to an oligonucleotide to produce a biotinylated oligonucleotide primer for use in a polymerase chain reaction,

amplifying a nucleic acid by a polymerase chain reaction by use of said biotinylated oligonucleotide primer and a second non-biotinylated oligonucleotide primer,

reacting the thus produced amplified double-stranded DNA with a solid support to bind said amplified DNA to said solid support through said biotin,

denaturing the DNA to separate from said double-stranded DNA the biotin-free single DNA strand which does not have biotin bound thereto, and

recovering said biotin-free DNA strand.

2. A process for the preparation of single-stranded nucleic acid which comprises:

i) conducting a polymerase chain reaction amplification of a nucleic acid template by utilizing a first primer which is an oligonucleotide having biotin bound thereto and a second primer which is an oligonucleotide free of biotin to produce a double-stranded nucleic acid product having said first primer incorporated into only one of the two strands,

ii) contacting said double-stranded nucleic acid product with a solid support including a moiety capable of specifically binding to biotin whereby said double-stranded nucleic acid product binds to said solid support;

iii) exposing said solid support to a buffer capable of separating the strands of said double-stranded nucleic acid product without disrupting the binding of said biotin to said solid support; and

iv) separating said buffer containing single-stranded nucleic acid free of biotin from said solid support having the biotinylated strand bound thereto.

3. A process for the preparation of single-stranded nucleic acid that comprises:

i) selecting a solid support that is derivatized with a moiety that specifically binds to biotin;

ii) covalently modifying an oligonucleotide primer with biotin;

said

iii) utilizing said biotin-modified oligonucleotide and a second oligonucleotide free of biotin as the primers in a polymerase chain reaction amplification of a nucleic acid template;

iv) contacting the products of the polymerase chain reaction of step (iii) with said solid support;

v) purifying the double-stranded product incorporating the biotin-modified oligonucleotide of step (ii) by washing said polymerase chain reaction products bound to said solid support with a buffer that does not denature complementary nucleic acid strands;

vi) separating the complementary nucleic acid strands by exposing the solid support, with the double-stranded polymerase chain reaction product attached, to a buffer which will denature complementary nucleic acid strands, but which will not disrupt the specific binding of the biotin-binding moiety to biotin;

vii) separating the solid support with the attached, biotin-labelled denatured polymerase chain reaction product from the denaturing buffer; and

viii) collecting the single-stranded polymerase chain reaction product from the buffer.

4. A process for sequencing a nucleic acid amplified by a polymerase chain reaction which comprises:

i) binding biotin to an oligonucleotide to produce a biotinylated oligonucleotide primer for use in a polymerase chain reaction,

ii) amplifying a nucleic acid by a polymerase chain reaction by use of said biotinylated oligonucleotide primer and a second non-biotinylated oligonucleotide primer,

iii) reacting the thus produced double-stranded DNA with a solid support to bind said double-stranded DNA to said solid support through said biotin,

iv) denaturing the double-stranded DNA to separate from said double-stranded DNA the biotin-free single DNA strand which does not have biotin bound thereto,

v) recovering said biotin-free DNA strand, and

vi) determining the sequence of nucleotides comprising said biotin-free DNA strand.

5. A process for sequencing a nucleic acid that comprises:

i) conducting a polymerase chain reaction amplification of a nucleic acid template by utilizing a first primer which is an oligonucleotide having biotin bound thereto and a second primer which is an oligonucleotide free of biotin to produce a double-stranded nucleic acid product having said first primer incorporated into only one of the two strands,

ii) contacting said double-stranded nucleic acid product with a solid support including a moiety that specifically binds to biotin thereby binding said double-stranded nucleic acid product to said solid support;

iii) exposing said solid support to a buffer capable of separating the strands of said double-stranded nucleic acid product without disrupting the binding of said biotin to said solid support;

iv) separating said buffer containing single-stranded nucleic acid free of biotin from said solid support having the biotinylated strand bound thereto; and

v) determining the sequence of nucleotides comprising the single-stranded nucleic acid in said buffer.

6. A process for sequencing a single-stranded nucleic acid that comprises:

i) selecting a solid support that is derivatized with a moiety capable of binding to biotin;

ii) covalently modifying an oligonucleotide primer with biotin;

iii) utilizing said biotin-modified oligonucleotide with a second oligonucleotide free of biotin as the primers in a polymerase chain reaction amplification of a nucleic acid template;

iv) contacting the products of the polymerase chain reaction of step (iii) with said solid support;

v) purifying the double-stranded product incorporating the biotin-modified oligonucleotide of step (ii) by washing said polymerase chain reaction products bound to said solid support with a buffer that does not denature complementary nucleic acid strands;

vi) separating the complementary nucleic acid strands by exposing the solid support, with the double-stranded polymerase chain reaction product attached, to a buffer which will denature complementary nucleic acid strands, but which will not disrupt the specific binding of the biotin-binding moiety to biotin;

vii) separating the solid support with the attached, biotin-labelled denatured polymerase chain reaction product from the denaturing buffer;

viii) collecting the single-stranded polymerase chain reaction product from the buffer; and

ix) determining the sequence of nucleotides comprising said biotin-free DNA strand.

7. A process for sequencing a nucleic acid that comprises:

i) conducting a polymerase chain reaction amplification of a nucleic acid template by utilizing a first primer which is an oligonucleotide having biotin bound thereto and a second primer which is an oligonucleotide free of biotin to produce a double-stranded DNA product having said first primer incorporated into only one of the two strands,

ii) contacting said double-stranded DNA with a solid support including a moiety that specifically binds to biotin whereby said double-stranded DNA binds to said solid support;

iii) exposing said solid support to a buffer capable of separating the strands of said double-stranded DNA without disrupting the binding of said biotin to said solid support;

iv) separating said buffer thus containing single-stranded DNA free of biotin from said solid support having the biotinylated strand of DNA bound thereto; and

v) determining the sequence of nucleotides comprising the single-stranded DNA in said buffer.