Introduction to Bioinformatics II Primer Designing Lecture 10 By Shumaila Azam

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.

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.

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.

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

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

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

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

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

Primer Design Considerations Primers must be specific for desired sequence to be amplified primers should be long enough to ensure specificity (usually 18-30 bases) Primers must form stable duplex at annealing temperature No complementarity between forward and reverse primers or primers and product

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

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

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