Primer Design
[via FISH441 Lab / TAs: M. Gavery & C. Storer]
Primers, or oligonucleotides (oligos), are short stretches of synthetic DNA that are used most commonly for PCR and DNA sequencing. They direct DNA polymerases to specific regions on larger DNA molecules for amplification. They are designed in pairs to amplify DNA in the forward and reverse directions. Oligos are custom synthesized by various manufacturer’s to contain the precise sequence requested by the customer. For a good introduction to the theory of primer design procede to this link.
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Photo source: http://bioweb.uwlax.edu/genweb/molecular/seq_anal/primer_design/primer_design.htm
Here is a breif list of things to take into consideration when designing primers. Although none of these are absolute, they will help ensure your primers will hybridize to your target sequence with the best efficiency.
1. Design your primers to be within 18-30 bases in length.
2. The melting temperature (Tm) of primers should be within 2C of each other.
3. Avoid primer dimers and primer hairpins
4. Avoid high G/C stretches, particularly at the 3’ end
5. G/C clamp at 3’ end of primers.
Primer design is most commonly done via computerized means and the algorithms used take the above rules into consideration. Of course, the user always has the opportunity to adjust the parameters that define how primers are designed by the software. There is a great deal of software available for primer design. Two commonly used primer design tools are NCBI Primer and Primer 3. The software will allow you to enter a full DNA sequence and then define what region(s) you would like to amplify, the ideal size of the amplicon (PCR product), the ideal length of the oligos, etc. However, often the preset conditions are already optmized to Additionally, after you have selected some proposed primers, you can compare melting temps, G/C content, primer dimer/hairpin probabilitites, etc.
We will go over the process of designing primers in more detail in lab.
Extraction Again
There were some recent educational posts, but I just remember we have some great products from FISH441 that Mac created and Caroline touched up this year. Here you can read everything and more about RNA and Protein Extraction, as well as reverse transcription and PCR, so have a look.
More foundation
As a follow-up to the prior post on extraction/RT/PCR, I thought I would provide some other resources for those just starting out. There is some great stuff in the FISH546: Bioinformatics for Environmental Sciences lectures. The first one (<-downloads PDF) has some fundamental molecular biology (plus PLEs!). Image at left is from this lecture. The second lecture is more about sequence databases, primers, and BLAST. The third one recaps some of the cool stuff available on the NCBI site and gives a start at NGS.
Fresh Tissue
Summer quarter is upon us, and with an influx of new persons, I thought I might take the time to talk about we like to call the transcriptional trinity. That is to say almost everything can be broken down to 1) RNA extraction, 2) cDNA synthesis, and 3) PCR. Yes, all of you science nerds are saying “What about epigenetics?”. True, but most newbies are going to start with fresh tissue and want to find out how much of gene X is being expressed under a given set of conditions.
Most, if not all of our protocols can be found on the wiki, but I will give you the run-down on the big 3. For RNA extraction we use Tri-Reagent. The manufacturer’s protocol is found here. It always good to look at the manufacturer’s protocol no matter what you are up to. Our slightly modified protocol is available on the wiki. Highlights are that Tri-Reagent and chloroform are dangerous, and RNA is very, very labile. Be careful, do not degrade the samples (nor the common stocks of solution).
For reverse transcription of said RNA, we commonly use Promega’s MMLV. (wikiful version) This is pretty straight forward and scalable.
Then all is left is PCR of the now more stable product / cDNA. Primers are needed for PCR and those can be designed based on your target of interest at IDT or NCBI. We happen to order our primers from IDT. Things might get more specialized however it good for newbies to do what one might call a conventional PCR and run an agarose gel to visualize.
Of course there are little details that you will not learn until you fail, but this should get most started. Always follow best practices by keeping a competent notebook, documenting your primers, and whine about what is wrong.
