How many bp is too many away from the start/stop codon to be a good primer? How many is too few?
The short answer is… it depends. It depends on your template and what you want to do with the PCR product. Let’s say you are amplifying from genomic DNA – including additional bp up and downstream of the start/stop codon will increase the likelihood that your amplified product will include regulatory elements that help fine-tune gene expression. You might want that if you are studying the way that gene is normally expressed in cells. You might NOT want that if you know you want to engineer your gene into a plasmid/vector to express a protein for a functional purpose (ex: to purify the protein to use as a drug). Now. let’s say that you want to generate a fusion protein. You’ll learn more about these later, but briefly, GST-CaMPARI is an example of a fusion protein. GST (glutothione-s-transferase) and CaMPARI are alone functional proteins, and when linked together into the same amino acid chain, they form a single protein that performs BOTH functions. The key here is that they have to be linked into a single amino acid chain… this means that there can be only ONE stop codon. So, if you are cloning a fusion protein using PCR, you might want to exclude the stop codon, or use your PCR primer to introduce a single base pair change that will switch the stop codon into an amino-acid coding codon.
Correct me if I’m wrong on primers – There can be many “correct” answers; Just because it works doesn’t mean it’s the best.
You are correct smile MANY primers will work, but the most important property of the best pair is that they will form just ONE PCR product from the desired template. I use NIH’s PrimerBlast to check for off-target binding when amplifying a gene from genomic DNA or cDNA (this is spliced mRNA that has converted to DNA; useful because it does not have the introns).
If you can’t find an ideal 20bp long, 50/50 GC/AT primer sequence, is it better to keep the length or the ratio? Does it all depend on how close to the ideal melting temperature you can get?
Melting temperature is more important than length and GG/AT ratio. As an aside, when you calculate the melting temperature of a primer that has a non-homologous end (such as when you add a restriction site), only the homologous portion contributes to the melting temperature of the primer… it has to be part of a dsDNA complex to need to melt.
How are primers made? Is it hard to make your own?
In a word – chemistry. It’s not really hard in theory… but it’s cheap enough that it’s not efficient to make primers on your own. Here are some references from companies that sell synthetic single-stranded oligos and double stranded DNA. These technologies have recently advanced to the point that you can buy a 3kb gene for ~$500. In contrast, a single primer for PCR is ~$4-6, often even less if you buy a large number of them all at once.
https://www.idtdna.com/pages
https://www.sigmaaldrich.com/technical-documents/articles/biology/dna-oligonucleotide-synthesis.html
https://www.genewiz.com/en/Public/Services/Gene-Synthesis
https://www.thermofisher.com/us/en/home/life-science/oligonucleotides-primers-probes-genes.html
What is the annealing temperature of dNTPs? Can annealing of free nucleotides interfere with primer annealing?
Since a dNTP only needs to form 2-3 hydrogen bonds, it has a very low annealing temp, but the interaction is also not very strong… when primers bind, they do compete with free dNTPs, but the primer-template interaction is a much lower energy state (stronger) than template-dNTP.
Is there a trick for quickly preparing PCR primers?
If your sequence of 20 is 50% GC, then it will automatically have a 60C melting temp… you can save a little time confirming melt temp calculations that way.
Why is the forward primer the same as the sense strand?
The forward primer anneals to the antisense strand and has the same 5’-3’ sequence as the sense strand. The reverse primer anneals to the sense strand and has the same 5’-3’ sequence as the anti-sense strand. If you are given the sense strand and need to find the sequence of the anti-sense strand, write the sense sequence in the opposite direction, then write the complementary base pairs. This is called “finding the reverse complement”. For example, if the sense strand is 5-ATGCGT-3, the reverse sequence is 3-TGCGTA-5 and the reverse complement is 5-ACGCAT-3.
Is there a limit to the length of a primer?
There is no specific limit, but long primers are more likely to self-anneal or have problems with secondary structures.
Does the restriction site incorporated into the primer overhang not bind to the DNA?
It does not – the restriction site is incorporate at the 5’ end. The rest of the primer anneals to the template, and DNA polymerase adds nucleotides directly to the primer in the 5’->3’ direction (making covalent bonds as each new nucleotide is incorporated) using the complementary sequence as a template.
What does the “oligo” portion of the word “oligonucleotide” mean?
“Oligo” is a prefix meaning “a few”, like in oligarch.
Why does the restriction site incorporated into the primer overhang not bind to the DNA?
The restriction site overhang is not complementary to the template sequence (if it were complementary, then we wouldn’t call it an overhang). During PCR, DNA polymerase binds at the double stranded complementary sequences and extends 5′ – 3′ to form a new piece of DNA that is composed of the primer and new nucleotides that are complementary to the template strand. The product from the very first cycle of PCR is a hybrid single and double stranded molecule where the overhang from the primer is still single stranded but the rest of the extension product is double stranded. At the next denaturation step, these strands separate and what you’re left with are two molecules – the original template and one new molecule containing the full primer and new DNA. The next PCR cycle will make a new molecule that is complementary to this sequence. So, in future PCR cycles, the whole primer sequence can bind to the second cycle products.
