Detection of nucleic acid sequence differences using coupled ligase detection and polymerase chain reactions

• US 20070048783A1
• Filed: 10/31/2006
• Published: 03/01/2007
• Est. Priority Date: 05/29/1996
• Status: Active Grant

• Current Assignee: Cornell Research Foundation Incorporated (Cornell University)
• Original Assignee: Cornell Research Foundation Incorporated (Cornell University), Board of Supervisors of Louisiana State University Agricultural & Mechanical College (Louisiana State University System), Regents of The University of Minnesota (University of Minnesota)
• Inventors: Barany, George, Barany, Francis, Lubin, Matthew, Hammer, Robert
• Granted Patent: US 7,312,039 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6862 Ligase chain reaction [LCR]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2521/531 Glycosylase
• CPC Class Codes: C12Q 2525/125 incorporating agents resulting in resistance to degradation
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2535/131 : Allele specific probes
• 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 2545/114 : involving a quantitation step
• CPC Class Codes: C12Q 2561/125 : Ligase Detection Reaction [LDR]
• CPC Class Codes: C12Q 2600/156 : Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/16 : Primer sets for multiplex assays

Abstract

The present invention relates to a method for identifying a target nucleotide sequence. This method involves forming a ligation product on a target nucleotide sequence in a ligase detection reaction mixture, amplifying the ligation product to form an amplified ligation product in a polymerase chain reaction (PCR) mixture, detecting the amplified ligation product, and identifying the target nucleotide sequence. Such coupling of the ligase detection reaction and the polymerase chain reaction permits multiplex detection of nucleic acid sequence differences.

Claims

1-9. -9. (canceled)

10. A method for identifying a target nucleotide sequence comprising:

forming a ligation product on a target nucleotide sequence in a ligation detection reaction mixture, wherein the ligation product comprises an upstream primer portion and a downstream primer portion, wherein the upstream primer portion and the downstream primer portion are not complementary with the target nucleotide sequence;

amplifying the ligation product to form an amplified ligation product in a polymerase chain reaction (PCR) mixture, wherein the PCR mixture comprises an upstream primer and a downstream primer; and

detecting the amplified ligation product to identify the target nucleotide sequence.

11. The method according to claim 10 wherein the ligation detection reaction mixture comprises adjacently hybridizing ligation probes.

12. The method according to claim 10 wherein the ligation product results from the ligation of two ligation probes, wherein the two ligation probes are two different molecules.

13. The method according to claim 10, wherein the upstream primer or the downstream primer contains a label.

14. The method according to claim 10, wherein the ligation product can be distinguished from other nucleic acids in the PCR, said method further comprising:

providing a solid support with different capture oligonucleotides immobilized at different particular sites, wherein the capture oligonucleotides have nucleotide sequences complementary to a unique nucleotide sequence of the extension products.