Question

What is ligation-mediation PCR? Explain what it is used for and how it works

Answer 1

When we have to amplify an unknown fragment of DNA or have knowledge of the sequence of only one end of DNA, we can make use of ligation-mediated PCR (LM-PCR). LM-PCR is used in genomic typing techniques which are used to identify new species, strains, or subspecies of already known bacteria due to various processes like intragenic recombination, horizontal gene transfers in bacteria, etc.

How it works;

Symbols-

Red lines - primer with a complementary sequence to restrictions digested cohesive ends.

Yello lines- complementary sequence to red primer sequence.

Green lines- DNA to be amplified

Step 0 - Digestion of target DNA with a restriction enzyme with a known cohesive end sequence.

Step 1- Annealing of primers (red color) with a complementary sequence to the cohesive ends generated (that still have longer overhangs after ligation due to increased primer length).

Step 2- Extension of the primer overhang region using a DNA polymerase.

Step 3- Now the yellow strands are generated which have a complementary sequence to the red primer sequence.

Step 4- Denature DNA with high temperature. Now the red primers can again bind to the yellow region that got extended due to complementarity. This can be used to amplify the DNA further.

Stem 5- Amplify and generate as many copies as required.