Hi! everyone
I am trying to clone a specific gene directly from bacterial chromosome.regarding which I have following questions:
1) Is it correct to PCR amplify gene directly from chromosomal DNA or are there other ways to carry out such cloning?
2) I have protein sequence of the gene, so is it possible to design primers by converting this protein sequence into DNA sequence?
Hi Ravindra
1. Yes. You can isolate gDNA from your bacteria strain and perform a PCR.
2. Yes it is but be careful. You need to check the codon bias of the bacteria and it MIGHT be necessary to design degenerate primers.
The answer to your 1st question is yes. Since the bacterial gene usually does not contain intron, you can directly PCR amplify the DNA coding sequence from genomic DNA.
The answer to your 2nd question is no. Since each amino acid can be coded by several triplet codes, the converted gene coding sequence based on protein sequence could be very different from that in your bacterial genome and result a failed PCR amplification. If your bacterium specie has not sequenced, do a blast research with your protein sequence to find a gene coding sequence of a specie that is very close to yours and design your primers based on that sequence.
You can do colony PCR as suggested by Sebastian although sometimes it tends to be more difficult, if it fails then its much better to extract the gDNA check the concentration and proceed to amplification. With respect to the second question I suggest you do not convert the protein sequence to nucleotide sequence due to codon bias although you can take care of codon bias by understanding the codon choice of your bacteria.. Some strains of bacteria can be use to take care of some codon bias e.g Rosetta gami B DE3
1) Easiest way to clone is via direct PCR from colony (just pick a colony using a pipette tip and add it as template to your PCR mix). Best way to clone is from isolated/purified bacteria genomic DNA though.
2) I would suggest you run your protein sequence through NCBI tblastn. You may get some good possible predictions on the nucleotide sequence based on homology for your primer design.
That being said, you can test your primers using the direct colony PCR and do your actual cloning using purified extracted genomic DNA.
1) Genomic DNA (gDNA) can be difficult to obtain or handle, in which case Colony PCR (1 colony from an agar plate diluted in PCR-grade water, then preferably boiled, although this might not be necessary) is an interesting alternative as a PCR template. However, some bacteria will have very rigid colonies, which might require chemical or enzymatic treatment prior to their use, and colony PCR can be hard to replicate. It's up to you to decide which way is easier or more useful to you (gDNA can also serve many other purposes and can be stored for quite a while). To obtain the sequence of your gene, you could also synthesize it chemically; this option is generally expensive, but opens up interesting options, like changing the codon bias of the gene without changing the protein product.
2) I would not suggest starting from the protein sequence to design your primers; many amino acids are coded by more than one codon, it will be at best a gamble to predict which primers would be required to amplify your gene just from the amino acid sequence. Furthermore, it might yield unspecific amplification, which would complicate downstream use of the amplicon.
Did you check the NCBI/gene? Genome of your bacteria may have been sequenced.
If your gene is large (>2-3 kb) I would not use the colony PCR.
Thank you everyone for your suggestions. Your suggestions were of great help to me.
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