1. Introduction to Bioinformatics II
Primer Designing
Lecture 10
By Shumaila Azam

2. Primer
A primer is a strand of nucleic acid that serves as a starting point for DNA synthesis.
It is required for DNA replication.
DNA polymerase can add only new nucleotide to an existing strand of DNA.
The polymerase starts replication at the 3’ end of the primer and copies the opposite strand.

3. Mechanism Leading strand has one primer to attach at 5’- 3’ end.
Lagging strand has to start replication in opposite direction as 3’-5’ end.
DNA polymerase III cannot synthesize DNA in the 3’-5’ end.
Shorts fragments re formed in the lagging strand called OKAZAKI FRAGMENTS.
Primase builds RNA primer.
Free 3’-OH on RNA primer to synthesize DNA from 5’-3’ end.

4. Mechanism
RNA primer are removed by DNA polymerase I for prokaryotes and by polymerase б for eukaryotes.
New nucleotides are added to fill the gaps where RNA was present.
DNA ligase then joins the nucleotides together completing the DNA synthesis.

6. Uses of primer Polymerase Chain Reaction (PCR)
RNA synthesis e.g Influenza virus

7. Polymerase Chain Reaction
Method for exponential amplification of DNA or RNA sequences
Basic requirements
template DNA or RNA
2 oligonucleotide primers complementary to different regions of the template
heat stable DNA polymerase
4 nucleotides and appropriate buffer

8. Basic Principles of PCR
1. Strands of template DNA (or RNA) are separated by melting
2. Forward Primer binds to one strand of template, Reverse Primer to other strand
3. DNA polymerase extends 3’ end of each primer, copying template
4. Strands are separated by raising temperature, allowing both original DNA and copies to act as templates
5. Repeat steps 2-4 many times

9. Temperature cycling
Annealing temperature (usually 45-60C) allows primers to hybridize to template
Extension temperature (usually 72C) allows polymerase to extend starting at the primer
Denaturation temperature (usually 95C) separates strands

10. Heat 1 copy Cool Heat 2 copies Cool Heat 4 copies Cool Heat etc.
+ and - strands of template
Cool
forward primer
reverse primer
amplified DNA 3’ 5’ 3’ 5’
Heat
2 copies 5’
Cool 3’ 5’ 3’
Heat
4 copies
Cool
Heat
etc.
8 copies

11. Primer Design Considerations
Primers must be specific for desired sequence to be amplified
primers should be long enough to ensure specificity (usually bases)
Primers must form stable duplex at annealing temperature
No complementarity between forward and reverse primers or primers and product

12. Initial primer selection criteria
Length (18-25 bases)
Base composition (45-55% GC)
Melting temperature (55-80C)
3’ terminal sequence
strong bonding base (G or C) at end
no runs (3 or more) of G or C at end

13. Primer complementarity criteria
Primer vs. self & forward vs. reverse
maximum number of consecutive bonds
maximum number of consecutive G-C bonds
Forward primer vs. Reverse primer
maximum number of consecutive bonds between the 3’ ends
Primer vs. product

14. Optimization criteria
Melting temperatures should be similar for both primers
Product should be as short as allowable