Question

In a typical microbiology laboratory, reasons for no bands from a gel of a polymerase chain reaction may be due to errors relating to omission of ingredients in the reaction mix and absence of the target sequence in the template DNA. Based on (i) primer problem and (ii) purity/potential contamination of the target sequence, explain the reasons for non-appearance on bands.

Answer 1

PCR or polymerase chain reaction is the inviitro amplification of DNA sequence. The reaction requires Various things to be mixed properly and then the reaction are allowed to run in PCR machine. Reason for no bands in the gel may be many like omission of ingredients in the reaction mix or can be the absence of target sequence in the template DNA and many more.

i. Primer problem arises like wrong concentration of primer, too little primer give won't see any products and too much primer give primer dimerization and not give enough amplification. Another problem like poor primer designing, errors like self complimentary, complementary between paired primers or excessively long oligonucleotide give wrong result or no results.

ii. ) The non purity of target sequence in a PCR sequence is a much factor of contaminants in the reaction. If the target sequence is not pure or not potential it gives wrong result or no results. So, at the time of purification of DNA, the main thing to be kept in mind that it should be properly purified. Chemical like chloroform, phenol, EDTA, ionic detergents etc. can inhibit PCR. So, extra clean step on the template is required so that the PCR gives result confirmly and the bands appear on gel.