homework

1. Primers must be outside of the targeted region of amplification.

2. Primer size should be 15-30 bp (smaller primers lacks specificity; larger primers decrease annealing efficiency). However, please note the following:

· If you plan to use the simplified formula to calculate Tm, the maximum primer length is 20 bp.

· If your primer is over 20 bp, you must also include buffer concentrations for the calculation of longer primer Tm.

· In the video (link provided on D2L), the primer length is stated as a range of 18-24, which is a very narrow guide. For the PCR primer design assignment, please use the 15-30 bp guide, but keep the two points immediately above this in mind.

3. GC content of 40-60% (Note: video is also more narrow on this range, with a 50-60% range)

4. Tm of primers should be within 5ºC of each other, but having them as similar as possible is better.

5. Tm of primers should be 50-65ºC (video states 55-65ºC, but again, use the broader range for your PCR primer design assignment).

6. 3’ end of primer should be a G or C (GC clamp). This stabilizes the primer binding at the 3’ end which is the most critical for polymerase to function.

7. Avoid stretches within your primers of G or C longer than 4 bases in a row.

8. Avoid long stretches of any one nucleotide.

9. Avoid intrastrand homologies within primer as this will lead to hairpin loop structures (which will decrease primer binding to target sequence).

10. Avoid interstrand homologies between primers as this will lead to primer dimer formation (which will decrease primer binding to target sequence).

11. Product size is typically 100-1000 bp. Larger amplicons can be designed; however, assays have to be carefully optimized to ensure efficient amplification. Smaller products are not recommend as this can interfere with distinguishing actual amplicon product versus potential primer dimer products.

12. Amplicon size can be easily calculated. This is done by subtracting the number of bases before the start of the forward primer from the starting bp (i.e. largest bp number) of the reverse primer. For example, if within a DNA sequence, the forward primer is from bp 105 – 123, and the reverse primer is from bp 425 – 404, then the calculation would be: 425-104. This calculation makes sure that the complete sequence of each primer is included in your amplicon size calculation.

13. In the PCR assay design, the temperature of the annealing step must be calculated (see item 14), and the time for each step of the cycle must be set. For our purposes, we will keep the denaturation step and elongation step at 94 ºC and 72ºC, respectively, so the annealing temperature is the only one that has to be calculated. For the length of each step of the cycle, you must commit to a specific time. You can’t program a PCR machine for a range of times or an approximate time. A specific time must be set. Depending on the result of the first test assay, the times and temperatures would then be adjusted, and a second test would be run. This would continue until a strong, specific amplicon product is obtained with the reaction.

14. The annealing temperature (Ta) of your first test assay using these primers should be approximately 5ºC below the Tm for the primers. If the Tm is different between the primers, this should be 5ºC below the lowest Tm of the primers.

As an FYI, by formal calculation for optimum Ta:

Optimal Ta = 0.3(Tm of primer) + 0.7(Tm of product) – 25

Tm of primer = Tm of less stable primer-template pair

Tm of product = Tm of the PCR amplicon product

As the simplified formula for calculating a Tm would not apply to the amplicon product (because it is too large), this is why we use the estimate of ~5 ºC below the primers Tm.