Methods and reagents for improving nucleic acid detection

• US 20040023248A1
• Filed: 12/09/2002
• Published: 02/05/2004
• Est. Priority Date: 12/07/2001
• Status: Abandoned Application

• Current Assignee: Whitehead Institute for Biomedical Research
• Original Assignee: Whitehead Institute for Biomedical Research
• Inventors: OʼMalley, Shawn M.
• Application Number:
  US10/314,901
• Publication Number:
  US 20040023248A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 2525/313 Branched oligonucleotides
• 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 2563/137 Metal/ion, e.g. metal label
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C12Q 2565/543 characterised by the use of two or more capture oligonucleotide primers in concert, e.g. bridge amplification
• CPC Class Codes: C12Q 2565/628 being a surface plasmon resonance spectrometer

Abstract

The present invention features methods for enhancing nucleic acid molecule detection through the use of dendrimers. Novel dendrimers and methods of making such dendrimers are also features of the present invention.

Claims

1. A dendrimer comprising a nucleic acid molecule conjugated to a dendrimeric polymer, wherein said dendrimeric polymer is not a DNA dendrimeric polymer.

2. A method of detecting a target nucleic acid molecule, said method comprising the steps of:

a) contacting a target nucleic acid molecule with a bipartite capture probe comprising a target nucleic acid molecule capture region and a dendrimer capture region, under conditions that allow hybridization of said bipartite capture probe to said target nucleic acid molecule, thereby forming a target nucleic acid molecule bound capture probe;

b) contacting said target nucleic acid molecule bound capture probe with a detectably labeled dendrimer, said dendrimer comprising a dendrimeric polymer having one or more single stranded capture probe nucleic acid binding sequences attached to it, under conditions that allow hybridization of said dendrimer capture region of said bipartite capture probe to said single stranded capture probe binding sequence, thereby forming a labeled target nucleic acid molecule bound capture probe-dendrimer complex; and

c) detecting said labeled target nucleic acid molecule bound capture probe-dendrimer complex,

thereby detecting said target nucleic acid molecule.

3. A method of detecting a target nucleic acid molecule, said method comprising the steps of:

a) contacting a biotin-labeled target nucleic acid molecule with streptavidin under conditions that allow binding of said streptavidin to said biotin-labeled target nucleic acid molecule, thereby forming a biotin-labeled target nucleic acid molecule-streptavidin complex;

b) contacting said biotin-labeled target nucleic acid molecule-streptavidin complex with a biotin-labeled nucleic acid capture probe comprising a dendrimer capture region, under conditions that allow binding of said biotin-labeled capture probe to said biotin-labeled target nucleic acid molecule-streptavidin complex, thereby forming a biotin-labeled target nucleic acid bound capture probe;

c) contacting said biotin-labeled target nucleic acid bound capture probe with a detectably labeled dendrimer, said dendrimer comprising one or more single stranded capture probe nucleic acid binding sequences attached to it, under conditions that allow hybridization of said dendrimer capture region of said biotin-labeled target nucleic acid bound capture probe to said single stranded capture probe nucleic acid binding sequence, thereby forming a biotin-labeled target nucleic acid bound capture probe-detectably labeled dendrimer complex; and

d) detecting said biotin-labeled target nucleic acid bound capture probe-detectably labeled dendrimer complex,

thereby detecting said target nucleic acid molecule.

4. A method of detecting a target nucleic acid molecule, said method comprising the steps of:

a) contacting a hapten-labeled target nucleic acid molecule with a capture probe comprising a dendrimer capture region, said capture probe conjugated to an antibody that binds said hapten, under conditions that allow binding of said capture probe to said hapten-labeled target nucleic acid molecule, thereby forming a target nucleic acid bound capture probe;

b) contacting said hapten-labeled target nucleic acid bound capture probe with a detectably labeled dendrimer, said dendrimer comprising one or more single stranded capture probe nucleic acid binding sequences attached to it, under conditions that allow hybridization of said dendrimer capture region of said hapten-labeled target nucleic acid bound capture probe to said single stranded capture probe nucleic acid binding sequence, thereby forming a hapten-labeled target nucleic acid bound capture probe-detectably labeled dendrimer complex; and

c) detecting said hapten-labeled target nucleic acid bound capture probe-detectably labeled dendrimer complex,

thereby detecting said target nucleic acid molecule.

5. A method of detecting a target nucleic acid molecule, said method comprising the steps of:

a) contacting a biotin-labeled target nucleic acid molecule and a biotin-labeled dendrimer with a streptavidin molecule under conditions that allow binding of said biotin-labeled target nucleic acid molecule and said biotin-labeled dendrimer to same said streptavidin molecule, thereby forming a biotin-labeled target nucleic acid molecule-dendrimer complex; and

b) detecting said biotin-labeled target nucleic acid molecule-dendrimer complex,

thereby detecting said target nucleic acid molecule.

6. A method of detecting a target nucleic acid molecule, said method comprising the steps of:

a) contacting a biotin-labeled target nucleic acid molecule with streptavidin under conditions that allow binding of said streptavidin to said biotin-labeled target nucleic acid molecule, thereby forming a target nucleic acid molecule-streptavidin complex;

b) contacting said target nucleic acid molecule-streptavidin complex with a biotin-labeled nucleic acid capture probe comprising a first dendrimer capture region, under conditions that allow binding of said biotin-labeled nucleic acid capture probe to said target nucleic acid molecule-streptavidin complex, thereby forming a target nucleic acid molecule bound capture probe;

c) contacting said target nucleic acid molecule bound capture probe with a first detectably labeled dendrimer, said first dendrimer comprising one or more single stranded capture probe nucleic acid binding sequences and one or more single stranded second biotin-labeled dendrimer capture nucleic acid sequences, under conditions that allow hybridization of said first capture region of said target nucleic acid bound capture probe to said single stranded capture probe nucleic acid binding sequence, thereby forming a target nucleic acid molecule bound capture probe-first dendrimer complex;

d) contacting said target nucleic acid molecule bound capture probe-first dendrimer complex with a second detectably labeled dendrimer comprising one or more nucleic acid sequences that hybridize to the first dendrimer, under conditions that allow hybridization of said target nucleic acid bound capture probe-first dendrimer complex to said second detectably labeled dendrimer, thereby forming a target nucleic acid molecule bound capture probe-nested detectably labeled dendrimer complex; and

e) detecting said target nucleic acid molecule bound capture probe-nested detectably labeled dendrimer complex,

thereby detecting said target nucleic acid molecule.

7. A method of detecting a target nucleic acid molecule, said method comprising the steps of:

a) contacting a hapten labeled target nucleic acid with a capture probe comprising a dendrimer capture region, said capture probe conjugated to an antibody that binds said hapten, under conditions that allow binding of said capture probe to said hapten-labeled target nucleic acid molecule, thereby forming a target nucleic acid bound capture probe;

b) contacting said hapten-labeled target nucleic acid bound capture probe with a dendrimer, said dendrimer comprising one or more nucleic acid sequences, said nucleic acid sequences further comprising single stranded capture probe nucleic acid binding sequences attached to it, under conditions that allow hybridization of said dendrimer capture region of said hapten-labeled target nucleic acid bound capture probe to said single stranded capture probe nucleic acid binding sequence, thereby forming a hapten-labeled target nucleic acid bound capture probe-dendrimer complex; and

c) detecting said hapten-labeled target nucleic acid bound capture probe-dendrimer complex by amplifying a nucleic acid sequence on the dendrimer, and detecting said amplification products,

thereby detecting said target nucleic acid molecule.