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regarding primer - PCR - Polymerase Chain Reaction Forum

regarding primer - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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  #1  
Old 04-26-2010, 06:32 AM
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Default regarding primer



Hi, I am trying to isolate an unknowon gene from an organism, from which the gene has never been sequenced and cloned. It is a fungus. Actually sometimes ago, i have designed degenerate primers for that and did 3'RACE. surprisingly I got a band in place and got 2-3 nested right, but when I sequenced it, it showed a completely different gene, though the functions are related. My question is, I think the primers were not that much specific. Can anybody tell me, that what are the criterias for desigining primers in this case? I want a detailed instruction regarding that. Thanx in advanced.
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Old 04-26-2010, 04:14 PM
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Default Re: regarding primer

Hi,

can't you do restriction digests, TOPO TA cloning, PCR amplification (to screen for best size matches for target gene) then send them off to sequencing.
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Old 04-29-2010, 04:57 AM
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Default Re: regarding primer

Thank you sir for ur valuable suggestion.
But surprisingly, I have done restriction analysis of the band, TA cloning I did and then send it for sequencing. But it resulted in a different gene. And I other than that i have used 8-10 primers, but they are not giving bands. Sometimes I get bands, but they are of different sizes and not a good band, something like heazy or zmear like. I think the primer designing is a problem. thats why I wanted to know detailed about primer designing. And while doing 3' RACE, how I can calculate the size of the expected band?As there will be presence of 3' UTR, the size of which is unknown to me. All these are problems I am having. Thanx in advance. Expect a solution from your side. otherwise it is a big problem for me. Thanx again.
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Old 04-29-2010, 03:52 PM
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Default Re: regarding primer

Confirm the speceficity of primer after you design your primers. Use Primer design software like primer3
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Old 04-30-2010, 03:57 AM
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Default Re: regarding primer

Hi, thanx again for ur reply. But I am designing primers from other related organisms. It is not possible to check the specificity of the primer when the actual sequence of the gene from which the primer is designed is not known. I have no idea or data regarding the gene from that particular organism.
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Old 05-07-2010, 04:49 PM
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Cool Re: regarding primer

Quote:
Originally Posted by prabuddhasarkar View Post
Thank you sir for ur valuable suggestion.
But surprisingly, I have done restriction analysis of the band, TA cloning I did and then send it for sequencing. But it resulted in a different gene. And I other than that i have used 8-10 primers, but they are not giving bands. Sometimes I get bands, but they are of different sizes and not a good band, something like heazy or zmear like. I think the primer designing is a problem. thats why I wanted to know detailed about primer designing. And while doing 3' RACE, how I can calculate the size of the expected band?As there will be presence of 3' UTR, the size of which is unknown to me. All these are problems I am having. Thanx in advance. Expect a solution from your side. otherwise it is a big problem for me. Thanx again.
1.No I meant to suggest you take genomic DNA, not the RACE band, and digest, clone, send to sequencing. basically you produce a plasmid DNA genomic library of X, X1, and X2 restriction digests.
2. PCR amplify to screen based on size if you want.
3. You then sequence with the plasmid primers (M13F*R or whatever).
4. You then can run homology analyses with your other better-known species data to find the target gene.

Designing degenerate primers for a different species, you gotta ask, are these two species so closely related that this is a good strategy???
I've actually done it (the PCR amplification) with algae species and it all worked, but I ended up implementing the above strategy anyway because one gene, just doesn't give that much info anyway.
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Old 05-07-2010, 04:53 PM
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Wink Re: regarding primer

Quote:
Originally Posted by prabuddhasarkar View Post
Thank you sir for ur valuable suggestion.
But surprisingly, I have done restriction analysis of the band, TA cloning I did and then send it for sequencing. But it resulted in a different gene. And I other than that i have used 8-10 primers, but they are not giving bands. Sometimes I get bands, but they are of different sizes and not a good band, something like heazy or zmear like. I think the primer designing is a problem. thats why I wanted to know detailed about primer designing. And while doing 3' RACE, how I can calculate the size of the expected band?As there will be presence of 3' UTR, the size of which is unknown to me. All these are problems I am having. Thanx in advance. Expect a solution from your side. otherwise it is a big problem for me. Thanx again.
In my experience, one band doesn't mean one PCR amplicon, could be a population of different pcr products at each band (especially w/degenerate primers, etc). The smears are also OK with me, I would totally go after those smears, clone them, sequence them---but in masse. I wouldn't just pick one or two colonies I'd clone a large number of them.
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Old 05-08-2010, 06:25 AM
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Default Re: regarding primer

Thank you sir. Actually now I get the thing. I was designing degenerate Hi
Sir,
primers from other seven related species gene sequence. But still didnt get the thing. Now I can understand, that there are several other factors for which the sequence of any particular organism can change due to evolution. And classification search really doesnt confirm that thing while searching for related oeganisms. Another thing I'd like to ask, when anybody get any gene sequence from a neq soursce, do they follow the strategies which u have said all the time? (i.e. genomic sequencing and then homology search and mRNA sequence)? Because I am realising that by doing RACE with degenerate primers to isolate a gene from new organism is very difficult. As the related organism may not be that much related which we think, so that they can produce perfect primer for new organism. Another thing is that, generally how many bands are seen when we prepare genomic DNA digested with 3 different RE? Because if it produce 40-50 or more bands, then it is very difficult to sequnce all of those here. Anyways, thanx again for your valuabe time and suggestions. Will always expect suggestionss and encouragements from a respected person like you.
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Old 05-08-2010, 06:39 AM
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Default Re: regarding primer

Sir,
Thank you for ur seggestions. Actually now I can understand the procedure u have said. That is a much more easy process than the one I was following. Actually the question came in my mind also that are the related orgaisms wwhich I selected from the classification close enough to produce a good primer so that it can bind and give desired products? And I also thought that there are several other factors which determines that, how much a gene can diverge in evolution. Another question is that when anybody isolates a gene from a new organism, do they follow the strategies which u said(i.e. genomic DNA sequencing and all) or RACE as it is very difficult to get it by RACE. And can you please tell me that when a geomic DNA is digested with 3 RE, generally how much bands can be formed? As it is very difficult to sequence a large number of DNA. Orelse I can follw the strategies readily.Thanks again for your valuable time and suggestions. Hope to get more suggestions and encouragements from a respected person like you.
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Old 05-11-2010, 06:15 PM
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Default Re: regarding primer

Quote:
Originally Posted by prabuddhasarkar View Post
Sir,
Actually the question came in my mind also that are the related orgaisms wwhich I selected from the classification close enough to produce a good primer so that it can bind and give desired products?
Are they close enough, I don't even know what the species are.
But I know how to find out.
Perform a Dig-labelling PCR reaction using those related species(DNA) and primers (you'll get double stranded probes or single stranded probes)---then use the Digoxygenin-labeled probes and react them to a dot blot of your "unknown" species. Use the known relatives as positive controls; also use low annealing temps and optimize your way up. If the dot blots are positive, then there is positive sequence homology. (Perhaps there is gene homology but not primer homology, thus the degenerate primers aren't working).

Quote:
And I also thought that there are several other factors which determines that, how much a gene can diverge in evolution.
Yes there are a lot. That's why I think you should just break all the genomic DNA into strands smaller than 1000bp, ligate them into plasmids, clone them, then use plasmid DNA primers to create your sequence data of this unknown. Once you have new sequence data, you can FASTA/BLAST the sequence and find out what species indeed has gene homology.

Quote:
Another question is that when anybody isolates a gene from a new organism, do they follow the strategies which u said(i.e. genomic DNA sequencing and all) or RACE as it is very difficult to get it by RACE.
My suggestion is for getting the genome of the unknown organism sequenced, not just 1 or 2 genes.

Quote:
And can you please tell me that when a geomic DNA is digested with 3 RE, generally how much bands can be formed?
If your DNA quality is high then, too many to count.
If your DNA quality is low then a good amount.

Quote:
As it is very difficult to sequence a large number of DNA. Orelse I can follw the strategies readily.
If you only care about 1 or 2 genes, not the genome, then consider the dot blot solution.

You can also alter that, and probe with labelled primers.

The way this would work, you get the best performing PCR primers for all related species, plus a nice housekeeping gene, ribosomal RNA works great, as quality/positive control.
Just order labeled primers, lyse your unknown species, isolate DNA, transfer equal amounts to blot paper, then use your labeled primer to probe. The ones with positive signal are the primer you will use for gradient PCR, at this point gradient PCR is better than RACE.

Last edited by danfive; 05-11-2010 at 06:19 PM.
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