Capture of single stranded nucleic acids

• US 6,203,978 B1
• Filed: 05/30/1997
• Issued: 03/20/2001
• Est. Priority Date: 05/31/1996
• Status: Expired due to Fees

• Current Assignee: Du Vergier Ltd.
• Original Assignee: Du Vergier Ltd.
• Inventors: Bruce, Ian James, Davies, Martin J.
• Primary Examiner(s): ZITOMER, STEPHANIE W
• Application Number:
  US08/866,446
• Time in Patent Office: 1,390 Days
• Field of Search: 536/24.3, 536/22.1, 536/23.1, 435/6, 435/91.2, 935/77.78
• US Class Current: 435/6.12
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00459 Beads
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2549/126 using oligonucleotides as clamps
• CPC Class Codes: C40B 50/14 Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support

Abstract

A solid phase substrate derivatized with single stranded nucleic acids having random sequences of nucleotide bases and related method of capturing one or more single stranded nucleic acids by contacting with the solid phase substrate wherein the single stranded nucleic acids anneal to the nucleic acids of the substrate to result in the captured sequences.

Claims

1. A method of assaying labeled double stranded amplification products in a fluid medium expected to further contain labeled single stranded nucleic acids including primers, wherein the amplification product labels and the single stranded nucleic acid labels are the same, comprising:

(a) contacting the fluid medium with a solid phase substrate derivatized with single stranded nucleic acids having random sequences;

(b) capturing the labeled single stranded nucleic acids by hybridizing to the random sequence nucleic acids on the solid phase substrate;

(c) removing the solid phase substrate with the captured labeled single stranded nucleic acids;

(d) assaying the label remaining in the fluid medium to thereby assay the labeled double stranded amplification products.

2. The method according to claim 1, wherein said solid phase substrate is magnetizable and is removed by a magnet.

3. The method according to claim 1, wherein said captured single stranded nucleic acids are selected from the group consisting of oligonucleotides and polynucleotides.

4. The method according to claim 1, wherein said primers are polymerase chain reaction primers.

5. The method according to claim 1, wherein the label is provided by 5′

modification of the double stranded amplification products and primers or by introduction of the label at one of the nucleotides of the primers.

6. The method according to claim 1, wherein said single stranded nucleic acids having random sequences of nucleotides are prepared by automated synthesis.

7. The method according to claim 1, wherein the label is a fluorochrome attached to the 5′

terminus of the primer or to one of the internal nucleotides of the primer, and the amplification products are assayed by spectrophotometrically detecting the label.

8. The method according to claim 7, wherein said label is attached by means of a linking group constituted by a primary amine group.

9. The method according to claim 7, wherein said label is attached by means of a linking group constituted by an N-hydroxysuccinimide ester or its isothiocyanate derivative.

10. The method according to claim 1, wherein the solid phase substrate derivatized with random sequence single stranded nucleic acids is prepared by the steps of:

(i) adding biotin to the 5′

or 3′

terminus or to one or more of the internal nucleotides of said random sequence nucleic acids;

(ii) derivatizing the solid phase substrate with streptavidin;

(iii) conjugating the biotinylated random sequence nucleic acids to the streptavidin whereby the random sequence nucleic acids are attached to the substrate.

11. The method according to claim 1, wherein the solid phase substrate derivatized with random sequence single stranded nucleic acids is prepared by the steps of:

(i) adding a primary amine group to the 5′

or 3′

terminus or to one or more of the internal nucleotides of said random sequence nucleic acids;

(ii) derivatizing the solid phase substrate with carboxymethyl groups; and

(iii) conjugating the primary amine groups to the carboxymethyl groups whereby the random sequence nucleic acids are attached to the substrate.

12. The method according to claim 5, wherein said label is attached by means of a linking group constituted by a primary amine group.

13. The method according to claim 5, wherein said label is attached by means of a linking group constituted by an N-hydroxysuccinimide ester or its isothiocyanate derivative.