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

• US 7,332,285 B2
• Filed: 10/31/2006
• Issued: 02/19/2008
• 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:
  US11/590,383
• Publication Number:
  US 20070054305A1
• Time in Patent Office: 476 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 one or more different target nucleotide sequences comprising:

providing a sample potentially containing one or more target nucleotide sequences comprising sequence 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;

performing a polymerase chain reaction to form extension products comprising the ligation product sequence and/or complements thereof; and

,

detecting the extension products to identify one or more target nucleotide sequences in the sample.

2. A method according to claim 1, wherein the ligation product sequence comprises a unique length, said method further comprising:

separating the extension products by size or electrophoretic mobility, wherein said detecting differentiates the extension products based on size.

3. A method according to claim 1, wherein the ligation product sequence can be distinguished from other nucleic acids in the polymerase chain reaction mixture, 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;

contacting the extension products with the solid support under conditions effective to hybridize the extension products to the capture oligonucleotides in a base-specific manner, wherein said detecting indicates the presence of extension products captured using the unique nucleotide sequence portions to identify one or more target nucleotide sequences in the sample.

4. A method according to claim 1, wherein one primer in the primer set comprises a detectable reporter label and the other primer comprises an addressable array-specific portion which is linked to the 5′

end of that primer and remains single stranded after the polymerase chain reaction, said method further comprising;

providing a solid support with different capture oligonucleotides immobilized at different particular sites, wherein at least one of the capture oligonucleotides has nucleotide sequences complementary to the addressable array-specific portion;

contacting the extension products with the solid support under conditions effective to hybridize the extension products to the capture oligonucleotides in a base-specific manner, wherein said detecting indicates the presence of extension products captured at at least one particular site to identify the target nucleotide sequence in the sample.

5. A method according to claim 1, wherein the ligation product sequence is substantially resistant to exonuclease digestion, said method further comprising:

subjecting the ligation product to exonuclease digestion after the ligase detection reaction; and

,

inactivating the exonuclease.

6. A method according to 5, wherein said performing a polymerase chain reaction on the ligation product sequence generates an extension product of unique length, said method further comprising:

separating the extension products by size or electrophoretic mobility, wherein said detecting differentiates the extension products which differ in size.

7. A method according to claim 1, wherein the ligation product comprises deoxy-uracil in place of deoxy-thymidine with the deoxy-uracil rendering the oligonucleotide probes and their ligation product sequences substantially sensitive to uracil N-glycosylase.

8. A method according to claim 1, wherein the ligase detection reaction comprises cycles from about 30 seconds to about five minutes long.

9. A method according to claim 1, wherein the ligase detection reaction is cycled for 2 to 50 times.

10. A method according to claim 1, wherein the ligase detection reaction is performed for a total of 1 to 250 minutes.

11. A method according to claim 1, wherein the ligase detection reaction is performed using a ligase selected from the group consisting of Thermus aquaticus ligase, Thermus thermophilus ligase, E. coli ligase, T4 ligase, and Pyrococcus ligase.