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Sequencing question/primer design

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Sequencing question/primer design

Postby coppcw08 » Sun Nov 04, 2012 8:19 pm

My professor wants me to determine forward/reverse primers of a sequence (which I can do). However, he also wants me to determine sequencing primers (ones which will be in the middle of the sequence to provide complete gene coverage). There are 2 questions he is asking that confuses me.

1) Design a forward primer for XXX gene sequence that is in frame (in frame as in the Open reading frame w/ ATG?) and removes the signal peptide. Additionally, does XXX gene have a lipoprotein motif?

I'm not sure what he is referring to in that question. I know a signal peptide is attached to protein to transport it to where it needs to go but how do i know if it's there? Also, what is a lipoprotein motif?

2) For the primers I need to figure out have to decrease the "delta G" number for hairpins and dimers. How do I do that? My primers are around 40 bp long to make sure my Tm is about 58oC.

Anyone out there know primer design/sequencing that can help I would really really appreciate it.

-Clinton
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Postby JackBean » Sun Nov 04, 2012 11:19 pm

1) what he wants is primer for cloning, not for sequencing. Imagine you have sequence
ATG GTC ATC GTC TGA GTC
and first three amino acids belong to signal peptide (in reality it's much longer). In such case, you want your primer to start at GTC and go on further.
By lipoprotein motif he probably meant whether is it homologous to lipoproteins (you know, good and bad cholesterol, HDLP, LDLP). I don't know whether they have some conserved motif.

2) primers should be 20-25 bp long. That should be sufficient for Tm of 58°C.
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Re:

Postby coppcw08 » Mon Nov 05, 2012 12:27 am

JackBean wrote:1) what he wants is primer for cloning, not for sequencing. Imagine you have sequence
ATG GTC ATC GTC TGA GTC
and first three amino acids belong to signal peptide (in reality it's much longer). In such case, you want your primer to start at GTC and go on further.
By lipoprotein motif he probably meant whether is it homologous to lipoproteins (you know, good and bad cholesterol, HDLP, LDLP). I don't know whether they have some conserved motif.

2) primers should be 20-25 bp long. That should be sufficient for Tm of 58°C.


First, let me thank you for the help. You are right. It's cloning a sequence into a E. coli pET-102 directional TOPO vector. I have a couple more questions.

The sequence he gave me was: ATG NNN NNN...1300 NT long. There is nothing before the start codon. So, I assume that implies there is no signal peptide?

Also, my forward primer I ran every length between 18-30 in a program and none of the sequences gave me a Tm even close to 58oC. It took me 45 base pairs to finally get a Tm of 57oC w/ a G/C content of 28%.

Lastly, when I finally got my primers to a Tm of ~58% my delta G self-dimers (homo-dimers) was -9.5 kcal/mol.
Isn't the deltaG supposed to be about -6?
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Postby JackBean » Mon Nov 05, 2012 10:47 am

no, signal peptide is on protein sequence. You need to analyse it on-line to see whether there is some predicted signal peptide
http://www.cbs.dtu.dk/services/
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Re: Sequencing question/primer design

Postby citroenboom » Wed Nov 14, 2012 1:37 pm

Do not focus too much on Tm = 58. A primer of about 20 to 25 bases should do the trick. I always make sure there is a C or G at the end. Longer primers naturally give more trouble like hairpins. For checking my primers I like to use:
http://basic.northwestern.edu/biotools/oligocalc.html
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