Methods for attaching polynucleotides to supports

• US 4,797,355 A
• Filed: 06/13/1985
• Issued: 01/10/1989
• Est. Priority Date: 06/13/1985
• Status: Expired due to Term

• Current Assignee: Amgen Inc.
• Original Assignee: Amgen Inc.
• Inventors: Stabinsky, Yitzhak
• Primary Examiner(s): NOT, DEFINED
• Assistant Examiner(s): NOT, DEFINED
• Application Number:
  US06/744,507
• Time in Patent Office: 1,307 Days
• Field of Search: 435/6, 435/7, 436/501, 536/28, 536/29, 935/77, 935/78
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation

Abstract

A probe is attached to a stock support to which is bound a single-stranded polynucleotide by hybridizing both the probe and the bound polynucleotide to a connecting strand with which they are both complementary and then ligating an end of the bound polynucleotide and the probe. After denaturation, the probe remains attached to the support by way of the bound polynucleotide. A target polynucleotide is then hybridized with the support-bound probe and with a labelled probe to provide a detectable hybridization complex.

Claims

1. A method for a hybridization assay comprising the steps of:

attaching a first single-stranded polynucleotide to a support so that the first single-stranded polynucleotide has a first free end;

hybridizing a first portion of a second single-stranded polynucleotide with the first single-stranded polynucleotide so that the second single-stranded polynucleotide has a free single-stranded end;

connecting by hybridization a third single-stranded polynucleotide to a second portion of the second single-stranded polynucleotide, separate and distinct from the first portion of the second single-stranded polynucleotide, so that the third single-stranded polynucleotide has a first end adjacent the first end of the first single-stranded polynucleotide;

ligating the first end of the first single-stranded polynucleotide to the first end of the third single-stranded polynucleotide to form a probe polynucleotide bound to the support;

denaturing the second single-stranded polynucleotide from the probe polynucleotide; and

immobilizing a fourth polynucleotide constituting a target analyte by hybridization to the probe polynucleotide.

2. The method as recited in claim 1 further comprising the steps of:

prior to the step of denaturing the second single-stranded polynucleotide from the probe polynucleotide,

providing the second single-stranded polynucleotide with a free single-stranded end having a first nucleotide sequence;

hydrogen bonding at least a portion of the first nucleotide sequence to a complementary sequence on a single-stranded end of a fifth polynucleotide; and

joining the fifth polynucleotide to the third single-stranded polynucleotide by ligation to form a probe polynucleotide bound to the support.

3. The method as recited in claim 2 wherein the fifth polynucleotide is a double-stranded polynucleotide and further comprising the step of removing one strand of the fifth polynucleotide by denaturation.