Primer design with a tag - (Feb/04/2011 )
This is the first time that I am designing primers and would like some help specifically with the forward one. I want to add an HA tag at the N-terminus. The order of the forward primer is as the following:
Restriction enzyme - Kozak - HA - gene
Regarding the kozak sequence and the HA tag, do they have to be in frame with my gene? My understanding is that the upstream sequence of the gene should always be in frame with the ATG! Does this always apply?
I have also realized that most of the tags do not start with an ATG, will this fact affect the gene expression?
Hi, the Kozak sequence is something like GCCACCATG(G), where the ATG is the start codon of the gene or tag you're adding. This should make it "in-frame" with the tag, and the tag should definitely be in-frame with the gene otherwise either the gene or the tag will not express! It is commonplace to add a spacer between the tag and the gene which can be done in 3bp increments so as to keep the tag and gene in the same coding frame.
You need to have an ATG at the start of the tag or it will not be expressed.
Thank you! This is helpful.
Hi to everybody, I'm new in this forum and I'm also new in molecular biology.
I work on marine bacteria involved in Nitrogen Cycle and from environmental samples I have to do PCR on specific functional genes. I have to work both with DNA than cDNA and on these selected amplicons do pyrosequencing 454. To distinguish the samples from DNA and cDNA I should add a tag of 5 nucleotids for each primer (forward and reverse) that needs to be different for DNA and cDNA. So when I'll have my sequences I know from which samples they come.
Probably i didn't explain it very well.... sorry!!!!
My questions are:
1. With these tags, since the lenght is higher, is it necessary to modified the annealing temeperature???
2. The tags mustn't do dimers and secondary structures with the sequence. How I have to choose the correct nucleotides to put at the end of each primers? The company where I order them can do this? Or I have to do by myself? and How?
thank you in advance
I am not entirely sure what you are trying to say, but it seems that you have to work both with (transfected?) DNA and native cDNA from the marine bacterium?
You will not be able to distinquish between the sequences if both the DNA and the cDNA are native, unless your genes have introns and exons (highly unlkiely given that it is a bacterium). However it is probably possible to transfect in a tagged DNA from a plasmid of some sort and have that express, and you can identify the tag so as to see if the DNA is transfected or not.
However, I know very little about 454 sequencing so there may be some trick that I am missing there that will work in the native system.
1) if the tags are binding to something, then they should be included in the annealing temperature. If they are not binding, then they should not be counted in calculating annealing temperature.
2) If your tags are only 5 bp or so, there shouldn't be a problem with them forming secondary structure unless they can bind to other parts of the primer. Avoid runs of any nucleotide.
The most common companies for buying primers are Invitrogen and Sigma-Aldrich. For both of these compaines, you have to design the primers yourself and then order them.
Primer3 is a good free website for primer design if you know your genome and genes are annotated, but not much use if you need to design lots and lots of primers.