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

• US 20060024731A1
• Filed: 09/16/2005
• Published: 02/02/2006
• Est. Priority Date: 05/29/1996
• Status: Active Grant

• Current Assignee: Cornell Research Foundation Incorporated (Cornell University)
• Original Assignee: Francis Barany, George Barany, Matthew Lubin, Robert P. Hammer
• Inventors: Barany, George, Barany, Francis, Hammer, Robert P., Lubin, Matthew
• Granted Patent: US 7,429,453 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. A method for identifying a target nucleotide sequence comprising:

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.

2. The method according to claim 1, wherein at least one primer in the PCR mixture comprises a label.

3. The method according to claim 1, wherein the ligation product comprises a unique sequence that can be distinguished from other nucleic acid molecules in the PCR mixture.

4. The method according to claim 3 further comprising:

providing a solid support with different capture oligonucleotides immobilized at different particular sites, wherein at least one of the capture oligonucleotides has a nucleotide sequence complementary to the unique nucleotide sequence of the ligation product;

contacting the PCR mixture with the solid support under conditions effective to hybridize the amplified ligation product to the capture oligonucleotides in a base-specific manner; and

detecting the presence of amplified ligation product captured at a particular site.

5. The method according to claim 1, wherein the ligation product comprises a blocking group outside of its non-ligating ends with the blocking group rendering the ligation product sequence substantially resistant to exonuclease digestion.

6. The method according to claim 5 further comprising:

subjecting the ligase detection reaction mixture to exonuclease digestion after the forming of the ligation product.

7. The method according to claim 6, wherein the ligation product comprises deoxy-uracil in place of deoxy-thymidine with the deoxy-uracil rendering the ligation product substantially sensitive to uracil N-glycosylase.

8. The method according to claim 7 further comprising:

blending the ligase detection reaction mixture, after forming a ligation product and before amplifying the ligation product, to form an amplified ligation product in a PCR mixture, with a downstream primer complementary to the ligation product, and a polymerase, to form an extension mixture;

subjecting the extension mixture to a hybridization treatment, wherein the downstream primer hybridizes to the ligation product and extends to form an extension product complementary to the ligation product;

inactivating the polymerase;

blending the extension mixture, after said inactivating, with uracil N-glycosylase to form a uracil N-glycosylase digestion mixture;

subjecting the extension mixture to uracil-N-glycosylase digestion substantially to destroy ligation product and extension product generated from original target without destroying the extension product generated from the ligation product;

blending, after said subjecting the extension mixture to uracil N-glycosylase digestion, a polymerase with the uracil N-glycosylase digestion mixture to form the PCR mixture; and

subjecting the PCR mixture to one or more PCR cycles to form an extension product in the first cycle which is substantially the same as the ligation product sequence except comprising deoxy-thymidine in place of deoxy-uracil.

9. The method according to claim 1, wherein the ligase is selected from the group consisting of a Thermus aquaticus ligase, a Thermus thermophilus ligase, an E. coli ligase, T4 ligase, and a Pyrococcus ligase.