Did you try a touchdown PCR program? it should help you avoiding non-specific bands.

You can as well try the following primer combinations suggested in Nubel et al., (1997), I used it successfully to identify unialgal cultures of Phormidium spp.

Forward primer: CYA359F GGG GAA TYT TCC GCA ATG GG

Reverse primer: CYA781R

Reverse primer CYA781R is an equimolar mixture of CYA781R(a) and CYA781R(b).

CYA781R(a) GAC TAC TGG GGT ATC TAA TCC CAT T

CYA781R(b) GAC TAC AGG GGT ATC TAA TCC CTT T

Amplicon size: 450bp

Dear Dinesh,

Thanks for your reply. I do have CYA359F primer. I will try with Touch down PCR method. My query is, when we CYA 359F and CYA 781 R, the expected amplification size will be 450bp. Do you feel, is it enough to classify Cyanobacteria at Species level? 

Please let me know whether your cultures are free of bacterial contamination. So that I can suggest some alternatives.

FYI, 450bp is not enough for species level identification.

Unfortunately, my culture is mixed culture... It is an marine samples which contains bacteria and some Eukaryota algae too. 

Still yet to purify to isolate single Cyano species. It would be great if you can give some methods to purify Cyano. I am intend to use my Antibiotic and serial dilution to isolate single colony. Is that will work?

Thanks for the mention of the article, George.  I uploaded it in the box above.

Venkatachalam, the above article is a great introduction to the ITS region, but we have moved forward since then.  In that article it mentions primers 1 and 2.  I have found it relatively easy to amplify this region, and it is nice because it includes a large portion of the 16S rRNA (1161 nucleotides) and all of the ITS.  Because of the problem with operons, I clone all my PCR reactions and then submit 3-4 clones with internal primers so that I get the whole read.  ITS will not sequence well without cloning.

The ITS has been used recently to separate cyanobacterial species.  The secondary structure is useful, and as we get more taxon sampling we are using this region in other ways too. I am attaching a recent paper that applies the ITS in some interesting ways.  If you contact me directly (my email is in the article), I can give you access to a lot more.

Good luck.

Dear George,

Thanks for your reply and the articles. I have tried  with all changes, then only come to Research gate forum. If i am correct, I believe, since it is a Metagenomic template, the primer  CYA 106F was binding to some other position, may be in chloroplast or green algal DNA. But, i tried to use some other primer data set. Thanks for your all suggestions!

Regards

Venkat

Dear Jefferey,

Thanks for your suggestion and your recent  article which is very interesting.  I am very happy to interact with you regarding Cyano classification. As soon as i get some back ground studies , i will contact you directly to get more information's.

Thanks once again,

Cheers

Venkat

I am a remote sensng geophysicist and I don't know much about the genes, but I was able to find a way of mapping cyanobacteria (microcystis, planktothrix, and a few others) with LANDSAT satellite data by looking for phycocyanin, the blue pigment that makes these cyanobacteria blue-green in color, because they also produce Chl-a pigment, which is green.  The blue pigment, phycocyanin, is more nearly unique to cyanobacteria than Chl-a, which is produced by most  plankton.  For your work, then, I suggest looking for a gene that is responsible for producing phycocyanin.  Sometimes the macroscopic world can be useful to the microscopic world, and I have found the reverse of that to be true and helpful for my work in macroscopic world.  Multidisciplinary science requires learning more than staying within your own training knowledge, but if you read in areas related to your work that you weren't trained it, you can sometimes find wonderful and helpful solutions to some important problems that have been under-addressed because of the "disciplinary walls" that are built around "pillars" of science.  I like problem solving, and that requires me to stretch into areas that I have never had a course in, such as microbiology and genetics.  I hae B.S. degrees in math and physics, a master's degree in physics, and a Ph.D. in Geology (the latter from U. of Michigan in Ann Arbor).  Yet I have read a lot about cyanobacteria, originally because, in addition to my graduate courses in geological, biological, and environmental remote sensing, I taught Geology 100 at the General Science level, which made me realize that cyanobacteria have been around for 2.5-3.0 Billion years and put most of the oxygen in our Earth's atmosphere, making mammals possible on Earth, among many other things.    Cyanobacteria can change their buoyancy (by expulsing and storing gas in vacuoles), so that they can lower themselves to a few meters when sea state is rough, and rise when sea states are calm and it is sunny.  Other "green" algae have to stay at the surface and don't live as long.  I find cyanobacteria very interesting because in geology, rocks change VERY SLOWLY, over thousands and millions of years, but cyanobacteria display exponential growth over hours and days.  It is truly a wonderful planet on which we live.