Primers, annealing and expression vectors

Designing a primer

The primers decide what PCR copies. A bad primer copies nothing, or copies the wrong thing. This lesson goes beyond NCERT, but A-Level, IB and the Olympiad ask these rules.

A good primer has these features.

• Length: 18 to 25 bases. A shorter primer pairs in too many places. A longer one is costly and pairs slowly.
• G and C: 40 to 60 per cent. G and C pair with three hydrogen bonds, and A and T with two.
• End: the 3 prime end should be a G or a C. This end must pair well, because the polymerase starts there.
• No self-pairing. The primer should not pair with itself, or fold into a hairpin, and the two primers should not pair with each other.
• Similar Tm. The two primers of a pair should have melting temperatures within about 5 degrees Celsius of each other.

The melting temperature

The melting temperature, Tm, is the temperature at which half of the primer is paired with its template. For a primer of more than 13 bases, a common estimate is

where $N$ is the length and $G + C$ is the number of G and C bases. This is an estimate. Real values also depend on the salts. The annealing temperature of the PCR is set a few degrees below the Tm, often about 5 degrees Celsius below. If it is too high, the primers do not pair. If it is too low, they pair in the wrong places.

Tails

A primer can carry extra bases at its 5 prime end that do not pair with the template. A common design is a short clamp, a few bases, then a restriction site, and then the part that pairs. The PCR copies the tails too. After the product is cut with the enzyme, it can be cloned. The Tm is worked out for the pairing part only, because the tail does not pair in the first cycles.

The time of the extension step

Taq polymerase adds about 1000 bases in a minute. This is a standard estimate. So the extension time is about 1 minute for each kilobase of the product. A product of 2400 bp needs about 2.4 minutes, or 144 seconds.

Expression vectors

A cloning vector only has to copy the gene. An expression vector must also make the protein. The foreign gene has to be placed in a cassette with the right signals. In a bacterium these are as follows.

• A promoter, where RNA polymerase starts. It is often a strong promoter that can be switched on by a chemical, such as the lac promoter, which is switched on by IPTG.
• A ribosome binding site just before the start, where the ribosome binds the mRNA.
• A start codon, ATG, and the gene in the right reading frame.
• A stop codon at the end.
• A terminator, which ends the transcription.
• An origin of replication and a selectable marker, as in every vector.

A gene from an animal has introns, and a bacterium cannot remove them. So the gene that is cloned is a DNA copy of the processed mRNA, the cDNA, or a synthetic gene.

The reading frame

The ribosome reads three bases at a time. Count the bases between the start codon of the vector and the first codon of the insert. If the number is not a multiple of 3, the frame shifts, and the protein is wrong. A site of 6 bases keeps the frame. Two extra bases from a bad primer shift it.

You should see that adding G or C to a primer of the same length raises the Tm. A longer primer with the same number of G and C has a lower Tm.

Try it in Python

The program checks three primers.

Practise with new numbers

Common mistakes

• Working out the Tm for the tail as well. The tail does not pair in the first cycles.
• Setting the annealing temperature above the Tm. The primers will not pair.
• Forgetting the reading frame. A shift of one or two bases ruins the protein.
• Putting a genomic animal gene in a bacterium. The introns are not removed, so a cDNA is used.
• Choosing primers with very different Tm. One of them will not pair at the temperature that suits the other.

Remember these

• A primer: 18 to 25 bases, 40 to 60 per cent G and C, a G or C at the 3 prime end, no self-pairing.
• Tm estimate: $64.9 + 41(G + C - 16.4)/N$. Annealing about 5 degrees Celsius below the Tm.
• Tails carry a clamp and a restriction site. The Tm is for the pairing part.
• Extension: about 1 minute for each kilobase.
• An expression vector: promoter, ribosome binding site, start codon in frame, gene, stop codon, terminator, ori, marker.

Check yourself

Try each one on your own first. If you are stuck, open a hint. Then check with Show the answer.

1. Why should the 3 prime end of a primer be a G or a C?

2. A primer has 20 bases and 10 of them are G or C. What is the share of G and C, and is it in the good range?

3. Why is the Tm of a primer with a tail worked out for the pairing part only?

4. How long is the extension step for a product of 3000 bp?

5. Why is the reading frame important when a gene is put into an expression vector?

References

• Sambrook J. and Russell D. W. (2001). Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, chapter 8.
• Brown T. A. (2016). Gene Cloning and DNA Analysis: An Introduction, 7th edition, Wiley, chapters 5 and 8.