After amplification of the intergenic spacer region (IGS) of the ribosomal DNA from nematode species by PCR, gel electrophoresis reveals multiple bands. Why is this ?

 

Here is some information that may be relevant.

Depending on the species four to five bands can be observed which can have different intensities. The top band is usually the most intense

The primers used are designed based on the conserved regions of the flanking 18S and 28S.

Stringent PCR conditions of 60 C annealing temp and low MgCl conc employed to reduce non specific amplification.

The target is part of the rDNA which is a tandemly repeated unit.

 

I have two lines of thought on why this is occurring.

Initially I believed that the repeat copies of the IGS region were not homologous and therefore the primers produced multiple bands.

However when I gel purified the most intense band and used this as a template in a PCR a complex banding pattern was also observed. So I now think that the primers are binding to areas within the target sequence.

 

Any thoughts on why this is occurring would be very much appreciated.

A more detailed comment later but two corrections:

1)the use of the word homologous. I believe you refer to sequence similarity, however homology refers to genes that have been separated by speciation events (and this does not apply here).

2) bands are never 100% consisting of only one sequence. In fact, there are always low co-occurences of the others within the mixture. As such a re-amplification of a certain band can also result in a re-amplificaton of the rest. Depending on parameters and size event the low-abundant samples may be preferentially amplified.

 

It would be helpful if you could list up the expected sizes vs the amplified ones for each case.