Probe composition containing a binding domain and polymer chain and methods of use

• US 5,703,222 A
• Filed: 11/21/1995
• Issued: 12/30/1997
• Est. Priority Date: 04/03/1992
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Perkinelmer Incorporated
• Inventors: Woo, Sam Lee, Menchen, Steven Michael, Fung, Steven, Grossman, Paul David, Winn-Deen, Emily Susan
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US08/561,478
• Time in Patent Office: 770 Days
• Field of Search: 435/6, 435/91.2, 536/24.3, 204/180.1, 204/182.8
• US Class Current: 536/24.3
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6862 Ligase chain reaction [LCR]
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2525/203 incorporating a composite nucleic acid containing a polypeptide sequence other than PNA
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2549/119 using nested primers
• CPC Class Codes: C12Q 2563/119 the label being proteinic
• CPC Class Codes: C12Q 2563/155 Particles of a defined size, e.g. nanoparticles
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides
• CPC Class Codes: C12Q 2565/125 Electrophoretic separation
• CPC Class Codes: C12Q 2565/131 Single/double strand conformational analysis, i.e. SSCP/DSCP
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: G01N 27/447 using electrophoresis
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Method and composition for detecting one or more selected polynucleotide regions in a target polynucleotide. In the method, a mixture of sequence-specific probes are reacted with the target polynucleotide under hybridization conditions, and the hybridized probes are treated to selectively modify those probes which are bound to the target polynucleotide in a base-specific manner. The resulting labeled probes include a polymer chain which imparts to each different-sequence probe, a distinctive ratio of charge/translational frictional drag, and a detectable label. The labeled probes are fractionated by electrophoresis in a non-sieving matrix, and the presence of one or more selected sequences in the target polynucleotide are detected according to the observed electrophoretic migration rates of the labeled probes in a non-sieving medium.

Claims

1. A probe composition for use in detecting one or more of a plurality of different target sequences in a polynucleotide sample, comprising

a mixture of sequence-specific probes, each capable of binding specifically to a different target sequence, wherein each probe is characterized by (a) an oligonucleotide binding polymer having a probe-specific sequence of subunits designed for base-specific binding of the polymer to one of the target sequences under selected binding conditions, and (b) attached to the binding polymer, a polymer chain which imparts to the probe a charge/translational frictional drag ratio that is distinctive relative to the charge/translational frictional drag ratios of the other probe(s) in said mixture.

2. The composition of claim 1, wherein said polymer chain is selected from the group consisting of polyethylene oxide, polyglycolic acid, polylactic acid, polypeptide, oligosaccharide, polyurethane, polyamide, polysulfonamide, polysulfoxide, and block copolymers thereof, including polymers composed of units of multiple subunits linked by charged or uncharged linking groups.

3. The composition of claim 1, wherein each sequence specific probe further includes a second binding polymer having a reporter label, where the first-mentioned and second binding polymers in each sequence-specific probe are effective to bind in a base-specific manner to adjacent and contiguous regions of a selected target sequence, allowing ligation of the two binding polymers when bound to the target sequence in a sequence-specific manner, and the polymer chain attached to the first binding polymer imparts to each ligated probe pair, a distinctive combined ratio of charge/translational frictional drag.

4. The composition of claim 1, wherein each sequence specific probe further includes a second binding polymer, where the first-mentioned and second binding polymers in a sequence-specific probe are effective to bind in a base-specific manner to opposite end regions of opposite strands of a selected duplex target sequence, allowing primer initiated polymerization of the target region in each strand, and the polymer chain attached to the first binding polymer imparts to each polymerized region, a distinctive combined ratio of charge/translational frictional drag.

5. The composition of claim 1, wherein each probe includes a reporter label.

6. The composition of claim 5, wherein the reporter label and polymer chain are both attached to a single subunit in the binding polymer.

7. The composition of claim 1, wherein each sequence-specific probe includes (1) a binding polymer composed of a first single-stranded DNA segment, and a second segment which includes single-stranded RNA, (2) a polymer chain attached to said first segment, and (3) a reporter attached to said second segment, and each polymer chain imparts to the probe, a distinctive ratio of charge/translational frictional drag.

8. A kit for detecting one or more of a plurality of different nucleotide sequences in a nucleic acid sample, comprising

a ligase enzyme, and

a mixture of sequence-specific probes, each capable of binding specifically to a different target sequence, wherein each probe is characterized by (a) an oligonucleotide binding polymer having a probe-specific sequence of subunits designed for base-specific binding of the polymer to one of the target sequences under selected binding conditions, and (b) attached to the binding polymer, a polymer chain which imparts to the probe a charge/translational frictional drag ratio that is distinctive relative to the charge/translational frictional drag ratio of the other probe(s) in said mixture,

wherein each sequence-specific probe further includes a second binding polymer, where the first-mentioned and second binding polymers in each sequence-specific probe are effective to bind in a base-specific manner to adjacent and contiguous regions of a selected target sequence, allowing ligation of the two binding polymers when bound to the target sequence in a sequence-specific manner, and the polymer chain attached to the first binding polymer imparts to each ligated probe pair, a distinctive combined ratio of charge/translational frictional drag.

9. The kit of claim 8, wherein each probe includes a reporter label attached to one of said binding polymers.

10. A kit for detecting one or more of a plurality of different nucleotide sequences in a nucleic acid sample, comprising

a polymerase enzyme, and

a mixture of sequence-specific probes, each capable of binding specifically to a different target sequence, wherein each probe is characterized by (a) an oligonucleotide binding polymer having a probe-specific sequence of subunits designed for base-specific binding of the polymer to one of the target sequences under selected binding conditions, and (b) attached to the binding polymer, a polymer chain which imparts to the probe a charge/translational frictional drag ratio that is distinctive relative to the charge/translational frictional drag ratios of the other probe(s) in said mixture,

wherein each sequence-specific probe further includes a second binding polymer, where the first-mentioned and second binding polymers in each sequence-specific probe are effective to bind in a base-specific manner to opposite end regions of opposite strands of a selected duplex target sequence, allowing primer-initiated polymerase extension of each binding polymer when bound to such target sequence, and the polymer chain attached to the first binding polymer imparts to each polymerized region a distinctive combined ratio of charge/translational frictional drag.

11. The kit of claim 10, wherein each probe includes a reporter label attached to one of said binding polymers.