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

• US 7,364,858 B2
• Filed: 10/31/2006
• Issued: 04/29/2008
• Est. Priority Date: 05/29/1996
• Status: Expired due to Fees

• 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, Hammer, Robert P., Lubin, Matthew
• Primary Examiner(s): Horlick; Kenneth R.
• Application Number:
  US11/590,529
• Publication Number:
  US 20070042419A1
• Time in Patent Office: 546 Days
• Field of Search: 435/6, 435/91.1, 435/91.2
• US Class Current: 435/6.12
• 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 copy number of a target nucleotide sequence comprising:

providing a sample potentially containing one or more target nucleotide sequences comprising one or more copy number differences;

performing a ligase detection reaction to form a ligation product comprising a first target specific portion complementary to the target nucleotide sequence and a second target specific portion complementary to the target nucleotide sequence, wherein the ligation product further comprises a 5′

upstream primer-specific portion and a 3′

downstream primer-specific portion, wherein the first target specific portion and the second target specific portion are located between the 3′

downstream primer-specific portion and the 5′

upstream primer-specific portion;

providing an oligonucleotide primer set comprising (a) an upstream primer containing the same sequence as the 5′

upstream primer-specific portion of the ligation product sequence and (b) a downstream primer complementary to the 3′

downstream primer-specific portion of the ligation product sequence;

providing a polymerase;

blending the ligation product, the oligonucleotide primer set, and the polymerase to form a polymerase chain reaction mixture;

subjecting the polymerase chain reaction mixture to a polymerase chain reaction to form an extension product comprising the ligation product sequence and/or complements thereof;

detecting the extension product;

and determining the copy number of the target nucleotide sequence in the sample based on said detecting the extension product.

2. The method according to claim 1, wherein the upstream primer or the downstream primer contains 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 extension product to the capture oligonucleotides in a base-specific manner; and

detecting the presence of the extension product captured at a particular site.

5. The method according to claim 1, wherein the ligation product comprises a blocking group, wherein the blocking group renders the ligation product substantially resistant to exonuclease digestion.

6. The method according to claim 5 further comprising:

subjecting the ligation product to exonuclease digestion.

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. A method for identifying copy number of a target nucleotide sequence comprising:

providing a sample potentially containing one or more target nucleotide sequences comprising one or more copy number differences;

performing a ligase detection reaction to form a ligation product comprising a first target specific portion complementary to the target nucleotide sequence and a second target specific portion complementary to the target nucleotide sequence, wherein the ligation product further comprises a 5′

upstream primer-specific portion and a 3′

downstream primer-specific portion, wherein the first target specific portion and the second target specific portion are located between the 3′

downstream primer-specific portion and the 5′

upstream primer-specific portion;

providing a first oligonucleotide primer set, wherein one or both probes contain deoxy-uracil in place of deoxythymidine;

subjecting the ligation product, in the presence of the first oligonucleotide primer set, to an amplification procedure to form a first extension product mixture;

subjecting the first extension product mixture to uracil-N-glycoslylase digestion;

providing a second oligonucleotide primer set comprising (a) an upstream primer containing the same sequence as the 5′

upstream primer-specific portion of the ligation product sequence and (b) a downstream primer complementary to the 3 downstream primer-specific portion of the ligation product sequence;

blending a polymerase and the second oligonucleotide primer set with the uracil N-glycosylase digested first extension product mixture to form a polymerase chain reaction mixture;

subjecting the polymerase chain reaction mixture to a polymerase chain reaction to form a second extension product;

detecting the second extension product; and

determining the copy number of the target nucleotide sequence in the sample based on said detecting the second extension product.