PRIMER DESIGN QUIZZ

Basing on the properties of primer for PCR process, a primers are designed to satisfy most of the requirements to be a good primer:
The forward primer: 5’- VR THGAGTWYTA YRTYRTVG - 3’, the template from nucleotide 399 to 418

The revere primer: 3’- G TGAARTYVCG HACCSKN - 5’, the template from nucleotide 609 - 627

1) The reason for the region chosen:
There are two basic reason for the region chosen for primer design:
- Firstly, in this region, there are a high similarities of the DNA sequence of 4 organisms.
- Secondly, the percentage of G and C are satisfied in the range of 40 - 60%.

2) The forward primer is VR THGAGTWYTA YRTYRTVG and have the direction of 5’ end to 3’ end: 5’- VR THGAGTWYTA YRTYRTVG - 3’
The reverse primer has an opposite direction which is 3’- G TGAARTYVCG HACCSKN - 5’

3) The ideal length of PCR primer for general application should be between 18 to 24 bp (Dieffenbach, Lowe, & Dveksler, 1993). In reality, if the primer is too short, it will possess low specificity result in non-specific amplification. However, in another case that it is too long, it will decrease the template-binding efficiency at normal annealing temperature due to the high potential of forming secondary structure such as hairpins.
The designed primer has the length of 20 bp with forward primer and 18 bp with reverse primer. This length is in the optimal condition for PCR primer.

4) The primers are always specified 5’ to 3’, and in the PCR process, the extension is depends on a good match at the 3’ end. From this reason, the 3’ end should be designed with G or C due to the fact the bond between G-C is stronger and tougher than A-T (There is 3 hydrogen bonds in bind of G and C compared to 2 bonds in A-T). In the designed forward primer: 5’- VR THGAGTWYTA YRTYRTVG - 3’, the 3’ end is G In the designed reserve primer: 3’- G TGAARTYVCG HACCSKN - 5’, the 3’ end is G

5) The Melting temperature (Tm) is the temperature at which one half of DNA duplex will dissociated into single stranded and it is used for the duplex stability’s indication (Dieffenbach, Lowe, & Dveksler, 1993). Primer with the melting temperature in the range of 52 - 58 oC generally produces the best result. Moreover, the melting temperature (Tm) is calculated by the equation:
Tm = 4x (G+C) + 2 x (A+T). In other words, the temperature gets two degrees for each A:T pair and 4 degrees for each C:G
Consequently, the temperature for the designed primers are:
Tm (Forward primer) = 55oC (for Humicolaxyn 2); 54.9 oC (for TRC30XYN1); 52.9 oC (for TRC30XYN2) and 45.5 oC (for DICTYXYNB).
Tm (Reverse primer) = 58 oC, 60 oC, 58.2 oC, 57.3 oC respectively

6) The secondary structure of primers could be hairpins, self-dimer or cross dimer (Borah, 2011).
- To calculate the possibility of secondary structure formation, At the 3’ end of primer value of the ∆G (Gibbs Free Energy) at -3Kcal/mol for avoiding internal hairpin.
- For avoiding self-dimer (the intermolecular interaction formation between two homologous primers and have same sense) and cross dimer (the intermolecular interaction formation between two homologous primer, sense and antisense primers) ∆G value should be at -6kcal/mol.

7) The length of amplified region is determined by the position of the start base of forward primer and reverse primer. In this quiz, the designed forward primer start at position 399 and reverse primer start at position 627. The length of amplified region is :
Product length = (Position of antisense primer-Position of sense primer) + 1. = ( 627 - 399) + 1 = 229 basepairs. 8) a PCR primer sequence is called degenerate if some of its position have several possible base. The degeneracy of the primer, consequently, is the number of unique sequence combinations it contain (Shamir & Linhart, 2005).
The genetic code for base is stated as follow: B = C or G or T (3 possibilities) | D = A or G or T (3 possibilities) | H = A or C or T (3 possibilities) | K = G or T (2 possibilities) | M = A or C (2 possibilities) | N = A or C or G or T (4 possibilities) | R = A or G (2 possibilities) | S = C or G (2 possibilities) | V = A or C or G (3 possibilities) | W = A or T (2 possibilities) | Y = C or T (2 possibilities) |

In this design:
The forward primer is: 5’- VR THGAGTWYTA YRTYRTVG - 3
The degeneracy for this primer is = 3*2*1*3*1*1*1*1*2*2*1*1*2*2*1*2*2*1*3*1 = 3456 possible primers

The reserve primer is: 3’- G TGAARTYVCG HACCSKN - 5’
The degeneracy for this primer is = 1*1*1*1*1*2*1*2*3*1*1*3*1*1*1*2*2*4 = 576 possible primers

Bibliography
Borah, P. (2011). Primer designing for PCR. Science Vision, 11(3), 134-136.
Dieffenbach, C. W., Lowe, T. M., & Dveksler, G. S. (1993). General concepts for PCR primer design. Genome Research, 3, S30-S37.
Shamir, R., & Linhart, C. (2005). The Degenerate Primer design problem: Theory and applications. Journal of computational biology, 12(4), 431-456.