Question

What are six possible reasons for a PCR not generating an amplified DNA product?

Answer 1

polymerase chain reaction is an amplification technique that generates a large supply of a particular segment of DNA.When PCR fails it leads to the generation of many non specific segments of DNA of varying sizes that appear as smear of bands on agarose gels.

somtimes mutations are unintentionally introduced in the amplicons, resulting in the heterogeneous population of PCR products.

the possible reasons for a PCR not generating a desired amplified DNA product are:

1. the optimised ampount of DNA input is necessary as the higher amount of DNA may increase the risk of non spec ific amplification while low amounts of DNA may reduce the yields.

2. amplification efficiency of the specific template amount is highly dependent upon reaction components and parameters.it also depends on the sensitivity of DNA polymerase. the selected DNA polymerase should be able to give results for controlled low level of residual DNA to minimise false signals in PCR.

3.primers used in the process should be optimally designed ie the first primer should be identical to the target sequence while second primer should be the reverse compliment ofthe target, secondly there should be no chance for hairpinning or primer dimerisation.

4. the reagents used in the PCR reaction should be checked and DNA sholud be added at the end to prevent its contamination

5. the concentration of all the reagents should be optimum as higher concentrations would disrupt the process while lesser concentration would reduce the yield.

6.the final step is to check all the machine parameters.