Question

When I checked the gel for pcr ynder uv light , there are some samples have bands while others are not . What does that mean . If there is a band is that mean the primers bind to the DNA or the primer will bind any way to all samples ?

Answer 1

Ans. During denaturation step (94-98⁰C) of PCR, the two strands of the sample dsDNA are separated into individual ssDNA templates. It is the annealing step (55-65⁰C) during which the primers complementarily bind to the template ssDNA strands.

# Your Observations:

#I. Some sample show bands: These samples must have complementary sequences to the primers added in PCR mix. Since the primers complementarily bind to these sample DNA templates, they would be amplified in next step, and successive PCR cycles. Therefore, the bands of these samples would be observed under UV light.

#II. Some sample do NOT show bands: If any sample DNA does not have sufficiently large complementary sequences to the primers, the primers won’t pair with them because the higher annealing temperature hinders non-complementary binding between the template DNA and primers. Such DNA samples won’t be amplified during PCR cycles because absence of primer from template DNA does not facilitate DNA replication.

# Conclusion: Primers bind only to the DNA samples which have sufficiently large complementary sequences to the primer at specified temperature. The primers do NOT bind to a DNA sample while have no complementary sequences to the primer or complementary sequence is too short to make a stable template DNA- Primer pairing at the high annealing temperature.