Genetic mutations lead to the production of defective genes. Such genetic mutation can affect the restriction endonuclease site sometimes but not in all cases. 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. Assume that in

Normal gene sequence — say at position 50, nucleotide pair is A:T

Mutant gene sequence — say at position 50, nucleotide pair is G:C

We are aware of the nucleotides on both sides of position 50. Therefore, we synthesize two oligonucleotide sequences which are complementary to normal and mutant gene sequence (one of the two native strands). These two oligonucleotide sequences will act as probe. One of the oligonucleotide sequences, say probe 1, is labeled at its 5’end with biotin (B) and its 3’end has nucleotide

Adenine, A (nucleotide complementary to position 50 in the normal gene sequence). Another oligonucleotide, say probe 2, is labeled at its 3’end with digoxigenin (D, an antibody binding indicator) and its 5’end has nucleotide complementary to position 51 (adjacent to mutated nucleotide) in the mutant gene sequence, say cytosine (C).

Normal gene

Target DNA

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PCR

↓

Amplified normal gene sequence

↓

Add probe 1 and probe 2

Probe 1 — Nucleotide at 3’end base pairs with nucleotide at position 50 of normal gene sequence

Probe 2 — Nucleotide at 5’end base pairs with nucleotide at position 51 of normal gene sequence

↓

Add DNA ligase

↓

Probe 1 and probe 2 are joined covalently

Mutant gene

Target DNA

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PCR

↓

Amplified mutant gene sequence

↓

Add probe 1 and probe 2

↓

Probe 1—-Nucleotide at 3’end is a mismatch. Nucleotide at this position will bulge out

Probe 2-Nucleotide at 5’end base pairs with nucleotide at position 51 of normal gene sequence

↓

Add DNA ligase

↓

No ligation of Probe 1 and probe 2

(Because of single nucleotide misalignment)

Now, we have to determine whether ligation has taken place between the two probes in both cases (normal and mutant gene sequence) or not. After hybridization and ligation,

DNA is denatured

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Hybridized probes are released

Reaction mixture is transferred to wells coated with streptavidin

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Unbound material is washed away

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Biotin-labeled probe DNA is present only in the wells

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Antidigoxigenin antibodies conjugated to alkaline phosphatase (AP) are added

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Then, unbound Antidigoxigenin antibodies – AP are washed away

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Chromogenic substrate is added

If colored product- Ligation has occurred -Normal genotype (because digoxigenin labelled probe has ligated to biotinylated probe and digoxigenin has in turn bound to antidigoxigenin antibodies)

↓

If no colored product – No ligation – Mutant genotype