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need some advise about PCR of PKD1-human gene


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#1 monamehr

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Posted 10 July 2014 - 09:06 AM

Hi everyone

Has anyone any experience with PCR of the PKD1-human gene and its pseudo genes?

How can I be sure that my primers don’t amplify pseudo genes, more than doing primer blast? And how can I find the exact direction and the +/- strand of these sequences in the genome?

Thnks

mehr


Edited by monamehr, 10 July 2014 - 09:09 AM.


#2 Ameya P

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Posted 11 July 2014 - 12:18 AM

HI Mona, 

 

Welcome to the forum! 

 

Regarding your question, I would suggest after designing your primers, you do an In-silico test for their specificity using this link. 

 

In case, the primers are amplifying more than one location, the software will give you more than 1 PCR product for your primer pair and then you can change your primers until you get a unique product with them. 

 

May I ask, what are you planning to do post your PCR. RFLP, Sequencing? 


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#3 monamehr

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Posted 11 July 2014 - 09:36 AM

hi Ameya

thanks for your reply

 

would you please sent me the software link again?

 

yes. I want to do sequencing after the PCR.but the problem is the high similarity (97%) of the psuedogenes sequence with the original gene.and I have no choice except designing primers at the regions with 1 or 2 mismatches from the psedogenes...

so I 'm not sure if the psuedogenes would amplify to some extent with the original fragment or not at the end

 

i noticed that the orientation of the original gene at GeneCards browser is:

 

minus strand (Start:    2,138,711 bp from pter     End:    2,185,899 bp from pter )

 

 and some psuedogenes are in:

 

plus strand (Start:    15,029,565 bp from pter     End:   up to 16,471,364 bp from pter)

 

does it mean that my forward and reverse primers from the original gene cant amplify these psuedogenes?

 

thanks again

mona



#4 Ameya P

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Posted 11 July 2014 - 09:34 PM

Hi Mona, 

 

Please click on the link .

 

I am not sure why it did not show up last time. 

 

I have done sequencing for a few genes like this and use the Exon Primer on the UCSC site to get my primers. 

I have not had any issues with pseudogenes amplifying, when I use these primers. 

 

Simply click on Exon Primer after you search for your Gene on UCSC database and enter reaction conditions that you are comfortable working with. 

 

Do you want to sequence the whole gene or just a few exons? 


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#5 monamehr

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Posted 12 July 2014 - 12:45 AM

hi Ameya

 

yes I want to sequenc all of the gene. thanks...I find that.

I have used USCS exon primer for other genes but here i think this is not so helpfull for this special gene!but thanks for reminding it to me

 

may i ask what are that genes?and have the same problem as PKD1?as I know there are a few genes with psuedogenes like that at the human genom



#6 Ameya P

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Posted 12 July 2014 - 10:12 PM

Mona, 

 

I had used it for Cytochrome P450 gene. Luckily my gene was small but with 46 exons, you have quite a bit of work to do. Is there is deadline for this? 


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#7 monamehr

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Posted 13 July 2014 - 08:36 AM

hi Ameya

unfortunately yes!! :(

 

I tried to find a way to do this...thanks for your participation :)



#8 Ameya P

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Posted 19 July 2014 - 10:39 PM

Hi Mona, 

 

Good to know that you have found a solution.

 

For the benefit of the forumers here, could you please share your method as well. 


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