Question

Juan a molecular biology student works at a lab and was asked to develop a multiplex reaction to detect different strains of pathogenic E.coli .However, his PCR results showed smearing and faint bands toward the bottom of the gel.Describe the factors which are responsible for the poor PCR results .

Explain what troubleshooting steps juan could have taken to address these problems.

Answer 1

In multiplex PCR, different combination of PCR is used to detect different PCR products in same reaction.

Smearing and nonspecific faint bands at end of gel in PCR can due to following reasons: The template is degraded. As a result, only short fragments of the DNA template are present. This leads to amplification of only these fragments, leading to smaller fragments. The degradation of template DNA also leads to smearing. The primer concentration of each primer is also no optimized for each reacttion. Cycling conditions (annealing, denaturation and extension temperatures and time) are also not optimal for each of the PCR reaction.

The primers used may of inferior quality or present in low quantity for efficient PCR reaction. There may have been too much DNA polymerase used. Increase amount of DNA polymerase used will also lead to smearing. Increase template DNA will affect the polymerization reaction, which is an enzyme catalyzed reaction.

Trouble shooting:

1. A two-step PCR can be used rather than a 3 step PCR. Further, number of cycles can be reduced by 2-5 cycles.

2. Template DNA should be checked for degradation. The template DNA has to be placed on ice to prevent its degradation. RNase is added to prevent DNA degradation.

3. Fresh primers can be used. Primer concentration should be optimized in individual single PCR reaction prior to multiplexing. Decreasing primer concentration may also help. If the primers are of poor quality, the primers need to be redesigned.

4. The template concentration should be reduced. Optimal enzyme concentration that are used for multiplex reactions are 0.5–0.8U/50 mL reaction