Oligonucleotide ligation assay presentation

Factors, Principle and Phases of assay

1. By : S.Gowtham , III – B.Sc Microbiology. Oligonucleotide ligation assay

2. Introduction Principle Phases Ligation is regulated by 3 factors Content 2

3. Polymerase Chain Reaction- Oligonucleotide Ligation Assay (PCR-OLA) is a method to diagnose hereditary diseases caused by mutation not affecting restriction endonuclease sites. This method combines Polymerase Chain Reaction with the Ligation Assay. PCR-OLA distinguishes between the ligation and the absence of ligation of two oligonucleotides . A single nucleotide mismatch (at the site of hybridized oligonucleotides ) prevents ligation and hence we can distinguish between the wild and mutant genotype. PCR-OLA can be understood with the help of an example. PCR-OLA is a sensitive, rapid and highly specific method. Developed by  Grunstein and Hogness in 1975. Introduction 3

4. . Oligonucleotide ligation assay combined with polymerase chain reaction (PCR-OLA) is a technique which can be used for the detection of characterized sequence variations. Commonly known as the OLA, the Oligonucleotide Ligation Assay is an analyte specific reagent that is centered around the hybridization of a PCR primer with an exact match to a target sequence. The Oligonucleotide Ligation Assay is a genotypic assay for the detection of resistance-associated mutations to reverse transcriptase and protease inhibitors in Human Immunodeficiency Virus type1 subtype B. A pair of nucleotides designed to anneal to adjacent sequences within a PCR product – If perfectly hybridized – Joined by DNA ligase . Oligonucleotide complementary to normal and mutant sequences are differently labeled and the products are identified by a computer software. Introduction 4

5. Principle The target DNA sequence is PCR amplified prior to the ligation step of the annealed probes. One of the detection probes consists of a sequence complementary to the target sequence. The second detection probe is fully complementary to the target sequence and serves as a reporter. Detection probes bind next to each other. The product is captured by anti-TAG on the microsphere surface. 5

6. 6 Phases The OLA consists of Two phases, A multiplex PCR amplification A multiplex OLA, in a single tube format.

7. 7 A Multiplex PCR amplification In this reaction a PCR primer is hybridized to the target sequence. The primers are designed with either the normal or mutant nucleotide at the 3’ end and a tail of different lengths to distinguish various PCR products based on size at the 5’ end.

8. 8 A Multiplex OLA This reaction is a ligation reaction. A common primer contains a fluorescent dye marker at the 3’ end and meets the first primer right over the nucleotide position that will be altered in a mutant allele.

9. 9 Ligation is regulated by 3 factors The specificity of ligation is regulated by 3 factors The hybridization of oligonucleotide . The need for these primers to directly adjacent to one another. The requirement that the oligonucleotide have two bases perfectly complementary.

10. 10 PCR/OLA

11. 11 Normal Gene Normal gene sequence – say at position 50, nucleotide pair is A:T normal gene sequence

12. 12 Mutant Gene Mutant gene sequence – say at position 50, nucleotide pair is G:C

13. 13 Large Image Slide Any Queries…?