I would say there are 3 main probabilities

1

you are correctly amplifying 2 different length amplimers.

2 You are amplifying the amplimer that you want and also a non specific amplification not related to ITS from elsewhere on the genome

3

you are amplifying 2 or more very similar 600base fragments and the 700 base fragment is a heteroduplex of 2 different sequence amplimers running at a larger apparent size than the 600 that you expect due to the bubble formed by the mismatch of sequence within the double strand of the mixed amplimer

I think your colony is not pure or your sample is for a co-infection disease and another guess is contamination.

Hi Dear Kalpana Tyagi.

Possible causes of Several, non-specific amplification products in gel electrophoresis result may reflect many reasons:

1. Amplified heterozygous alleles, such as Rs and rs (reducing sugar alleles in carrots) in the Rs/rs individuals, the sequences of Rs and rs is the same except that rs has an extra insert in it

2. You may hit genes from a gene family, 2 genes have some conserved sequence part(s), but with different sizes.

3. You were PCRing a region with 'repeats'

4. PCR picked up (a) pseudo-gene(s).

5. You are PCRing a DNA fragment with one end can anneal F primer, and the other end with two regions which can anneal R primer (vise versa).

6. May be due to contamination or presence of two fungal spices in sample (mixed infection).

If it is unspecific, you can try to increase the annealing temperature. Some primers amplify non-specifically at low temperatures, but when you increase, they amplify a single specific product. Maybe do a gradient PCR (or several PCRs with different temperatures) to test the optimal temperature that gives you a single band.

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You used ITS4 and ITS 5 primer for fungal specie that replicated Internal transcribed spacer regions (ITS1-5.8S-ITS2).

The Size of this regions in fungal species are variable. It varies from 300 bp to about 900 bp.

In this case, Because you have 2 bands 600 bp and 700 bp, It seems to be caused by contamination during the DNA extraction process or PCR steps or presence of two fungal species in sample (mixed infection).

If your fungal isolate is yeast, for separating mixed infection, Please use CHROMagar Candida Medium to ensure purity of sample And try to subculture one colony in new media.

Also, make sure that the negative control shows no band to exclude contamination.

Good luck.

thanks to all for the information...your answers are very helpful...