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Primers for a gene that is present more than 1x in the genome

Primers for a gene that is present more than 1x in the genome - Real-Time PCR and Quantitative PCR Forum

Primers for a gene that is present more than 1x in the genome - Real-Time PCR and Quantitative PCR Forum


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Old 02-24-2010, 05:01 PM
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Default Primers for a gene that is present more than 1x in the genome



Hi,

I have a question. If you have a gene that has several duplicated forms in the genome and you make primers, specifically to the conserved regions of the gene, would that be a good idea?
The chance that the primers find a binding place is much higher that way, but does that not give problems in creating amplicons of different sizes which would completely destroy your real-time pcr experiment (quantitation, efficiency,...)?

What is your opinion on that?
Help much appreciated!!
Wozzels
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Old 02-24-2010, 06:06 PM
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Default Re: Primers for a gene that is present more than 1x in the genome

I remember doing a diagnostic PCR on a virus, that had the target gene duplicated at each end of the genome.

The duplicates were exact and the PCR gave one single band. The PCR produced plenty of amplicon and I was able to keep the number of cycles pretty low (30 or 35) without losing sensitivity.

I just kept a mental note that each virus genome would give me twice as much signal than other diagnostic pcr's.
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Old 02-25-2010, 07:53 AM
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Default Re: Primers for a gene that is present more than 1x in the genome

Yes, thank you,

but if you have the situation where both duplicated genes lie directly downstream of each other on the same chromosome, would you not get pcr-products much bigger than the desired amplicon (the forward primer binds to the amplicon in the first gene and the reverse primer binds to the amplicon on the second gene)? As a consequence the efficiency of your reaction would be smaller and this way a lot of nucleotides are wasted...
I think I just have to check for bands an a post-pcr electrophoresis.

Greetings!!
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Old 02-25-2010, 04:40 PM
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Default Re: Primers for a gene that is present more than 1x in the genome

I was facing identical problem once, but figured out the DNA fragments I tried to amplify were identical, even if duplicated on the genome. So I was in a similar situation to that of danfive, but the genes I amplified were downstream of each other, like you described.

I suggest to check up the sequences carefully and perform a standard PCR with electrophoretic analysis. In PCR, and especially Real-time PCR, polymerases tend to amplify shorter fragments more efficiently. If you set up proper timing for each step of your reaction then you should get only the shorter products.
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