2 replies to this topic

[Chris22]

Hi Everyone

I am working with a small protein excreted from a cell. I know the Amino Acid sequence of this protein, thats all.
The genome of the bacteria is not sequenced, nor any of the related genes.

I would like to look at this genes promoter/terminator sequence and need a way to do this.

If i were to design redundant primers for the sequence (based on the AA's), amplify this region, i would still only have the gene itself not the flanking Pro/Term. (because i can only design primers based on what i know )

Is it possible to take genomic DNA, design a forward primer based on the region i know, and run a PCR (high cycle number) using only this one primer so that it runs through to the terminator region. Would this create enough copies of the fragment to then send for sequencing using the same primer???

(Im aware that the PCR requires a reverse primer to increase exponentially but lets just say i just need the polymerase to run past the stop codon into the terminator sequence enough times to send off for sequencing)

This is only my playschool solution to it, im hoping that there is a proper way that i do not know about! Any help would be much appreciated!!

Many thanks in advance

C22

[phage434]

It is tempting today to say that you think about simply sequencing the genome using next generation sequencers such as Illumina or 454.

The tried and true method involves designing degenerate primers for a region of the gene.  Try to find Met or Trp AAs that provide unique codons for the 3' end of your primers.  For AAs such as gly or ala, you know the first two are gg or gc, so you can simply avoid the final base of the codon in a primer design.

Then, amplify a small region of the CDS, then sequence the PCR product.  Next, using your new sequence, design outward facing primers.  Cut genomic DNA with a frequent cutter, such as Sau3AI, then heat kill and ligate, forming circular fragments.  PCR with your outward directed primers and sequence the fragment.  You should now have flanking sequence (up to the GATC site cut by Sau3AI) which can be sequenced.  Rinse, lather and repeat as necessary to find what you want.

Once you have some good sequence, you may (depending on your genome length) be able to sequence directly from the genome, as an alternative.  Talk to your sequencing supplier.

[Chris22]

phage434, on Apr 8 2010, 07:58 PM, said:

It is tempting today to say that you think about simply sequencing the genome using next generation sequencers such as Illumina or 454.

The tried and true method involves designing degenerate primers for a region of the gene.  Try to find Met or Trp AAs that provide unique codons for the 3' end of your primers.  For AAs such as gly or ala, you know the first two are gg or gc, so you can simply avoid the final base of the codon in a primer design.

Then, amplify a small region of the CDS, then sequence the PCR product.  Next, using your new sequence, design outward facing primers.  Cut genomic DNA with a frequent cutter, such as Sau3AI, then heat kill and ligate, forming circular fragments.  PCR with your outward directed primers and sequence the fragment.  You should now have flanking sequence (up to the GATC site cut by Sau3AI) which can be sequenced.  Rinse, lather and repeat as necessary to find what you want.

Once you have some good sequence, you may (depending on your genome length) be able to sequence directly from the genome, as an alternative.  Talk to your sequencing supplier.

Thanks Phage434

This is actually only a side project of mine so i dont think i could justify the money involved in sequencing the genome.
But the inverse PCR you described sounds like the only other option apart from my own idea. I think i will try out the digestion with Sau3AI and hope for the best with the inverse primers, at the same time i will get in touch with MWG biotech (our sequencing provider) and ask about other options.
Thanks for your input, much appreciated!