The prokaryotic gene I'm looking at has conserved DNA sequences/ sequence motifs, but do not inlcude the start and stop codons within them although the motifs are part of the CDS. So if I were to design my primers for future expression studies, should I go for the sequence with the start and stop codons, or should I design primers to flank the conserved sequences instead? Better yet, would cloning the whole gene itself be a good choice?
If you design primers only to flank the conserved sequences (motifs) [as you stated in the question], the product you obtain will be just a part of the gene of your interest, not the full length of the gene. Seems to me, you are worried that the start codon is too far away (upstream) and hard for you to PCR the whole gene (including start and stop codons) for cloning purpose. Is that correct? How big is the gene? How far upstream is the start codon from the motif?
hi
if your gene is from eukaryote so you have to clone its cDNA
otherwise if your gene is from prokaryote so you can clone the gene directly >
I'm not quite sure I understand what you mean when you say there are conserved regions in the coding sequence not close to the start and stop codons...
If you're looking to clone a gene for, say, sticking it into an expression vector, you should clone from the cDNA, with your primers spanning the full length of the coding sequence.
I apologize for not stating the question more clearly. The gene is of prokaryotic origin, and by conserved DNA sequences, I meant sequence motifs. The start codon is apparently positioned quite far upstream of the motif. The expression plasmid I'm using has a 5' tag.
Okay, It means you have already initiation codon and RBS present in your vector. Make sure that your gene sequences should be in frame with vector tag sequences and with start codon. You can add stop codon in your reverse primer although it may not be necessary.
My quick answer, is yes, you must include the start and stop codons for proper expression. However, that being said, many expression vectors with N-terminal Myc or FLAG tags have their own start codon and Kozak sequence, so if you put your ORF in frame with the N-terminal tag, you will not need the gene's ATG. Similarly, many of these expression vectors have stop codons in all three reading frames at the end of the multiple cloning site (MCS). If you do not want these additional codons (depending on the where the 3' end of your gene is in the MCS) translated, you can include the stop codon at the end of your gene.
Just cloned the coding sequence, it is important the start codon for appropriate translation, and the signal for stop to be sure you are working with the whole gene,
If you design primers only to flank the conserved sequences (motifs) [as you stated in the question], the product you obtain will be just a part of the gene of your interest, not the full length of the gene. Seems to me, you are worried that the start codon is too far away (upstream) and hard for you to PCR the whole gene (including start and stop codons) for cloning purpose. Is that correct? How big is the gene? How far upstream is the start codon from the motif?
As suggested by Gaurav, It will be better to first look at vector and restriction site availabe, Different companies have different vectors, You should confirm the frame of both start and stop codon of vector along with your gene. Otherwise you may need to provide the start or stop codon
In my opinion to use primers that flank the CDS region of gene and if you worry about the start codon you can insert this CDS region in expression vector downstream the promotor in the open reading frame & also, you can use tags such as his tag as to purify your expressed protein.
In the first try, i would clone the whole gene. You don't express the conserved sequences but the whole protein, so you need the whole coding sequence.
In case you don't use a vector's promoter and RBS, then clone the gene with its native promoter sequence and clone it till the end.
If you use a vector's promoter and RBS, make sure that your PCR and ligation result in correct coding sequence (in frame), otherwise, the expression will not work.
Dear Avin Koh
I suggest you to please go for the in silico analysis of your gene of interest, software you can use for this process are viector NTI provided by the Invitrogen and BioEdit.
In general you can clone your CDS in to the expression vector by using the apmlicon with specific primer with start and stop codon sequences. There is no more need to clone the whole gene.
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