BIOINFORMATICS -5

PRIMER DESIGNING
OUTLINE

• PCR
• PCR components
• PCR procedure
• Primer designing
PCR

• PCR is a technique invented by Kary Mullis (Noble prize -1993), used to make copies (amplify) of
DNA fragments

• It is an enzymatic replication of DNA without using a living organism.

• The technique allows a small amount of DNA to be amplified exponentially.

• The DNA fragment can be a gene, part of a gene or a non-coding sequence.

• PCR is commonly used in medical and biological research labs for a variety of tasks, such as the
detection of hereditary diseases, the identification of genetic fingerprints, the diagnosis of
infectious diseases, the cloning of genes, and paternity testing

• The PCR process is carried out in a thermal cycler, this is a machine that heats and cools the
reaction tubes to the exact temperature required for each step of the reaction
PCR COMPONENTS

• PCR, requires several basic components:

- DNA template, which contains the region of the DNA fragment to be amplified
- Two primers, which bind to the beginning and end of the two srands of the region to be amplified
- Taq polymerase a DNA polymerase which copies the region to be amplified
- Deoxynucleotide triphosphates, (dNTPs) from which the DNA polymerase builds the new DNA
- Divalent cation, magnesium or manganese ions
PRIMERS
• The DNA fragment to be amplified is determined by selecting primers. Primers are short, artificial
DNA strands — often not more than 50 and usually only 18 to 25 base pairs long — that are
complementary to the beginning or the end of the DNA fragment to be amplified

• They anneal by adhering to the DNA template at these starting and ending points, where the DNA

polymerase binds and begins the synthesis of the new DNA strand
PROCEDURE:
• Primers are what gives PCR its SPECIFICITY!!!
• Good primer design: PCR works great.
• Bad primer design: PCR works terrible.
Primer melting temperature (Tm):

The melting temperature (Tm) is the most important factor in

determining the optimal PCR annealing temperature (Ta).
TM CALCULATION
- Wallace rule:

Tm = 4 * (G + C) + 2 * (A + T)

CTGATCAAGTCGATGGCTTG
GOOD PRIMER CHARACTERISTICS

Primer length

18-24 bp
5’ 3’

Too short---less specific

Too long---wasting money
 BASE COMPOSITION
• Usually, average (G+C) content around 50-60% will give us the right

melting/annealing temperature for ordinary PCR reactions, and will

give appropriate hybridization stability.

5 GTGGATGTGGTGTCGATGGC 3
 3’END STABILITY
• The 3’ end stability is very important character of selected primers

5 GTGGATGTGGTGTCGATGGC 3

GC
5’ 3’
CG

3’

AT
5’
PRIMER DESIGNING
Tool name URL
CODEHOP http://blocks.fhcrc.org/codehop.html
Gene Fisher http://bibiserv.techfak.uni-bielefeld.de/genefisher/
DoPrimer http://doprimer.interactiva.de/
Primer3 http://frodo.wi.mit.edu/primer3/
Primer Selection Http://alces.med.umn.edu/rawprimer.html
Web Primer http://genome.www2.stanford.edu/cgi.bin/SGD/web.primer
PCR designer http://cedar.genetics.ston.ac.uk/public_html/primer.html
Primo pro 3.4 http://www.changbioscience.com/primo.html
Primo Degenerate http://www.changbioscience.com/primo/primod.html
3.4
PCR Primer Design http://pga.mgh.harvard.edu/serviet/org.mgh.proteome.primer
The Primer http://www.med.jhu.edu/medcenter/primer/primer.cgi
Generator
EPRIMERS http://bioweb.pasteur.fr/seqanal/interfaces/eprimer3.html
PRIMO http://bioweb.pasteur.fr/seqanal/interfaces/eprimo.html3
PrimerQuest http://www.idtdna.com/biotools/primer_quest/primer_quest.asp
MethPrimer http://itsa.uscf/~uralab/methprimer/index1.html
Rawprimer http://alces.med.umn.edu/rawprimer.html
MEDUSA http://www.cgr.ki.se/cgr/MEDUSA/
The Primer Prim’er http://www.nmr.cabm.rutgers.edu/bioinformatics/primer_primer_proj
Project ect/primer.html
GAP http://promoter.ics.uci.edu/primers/
PRIMER DESIGNING: PRIMER BLAST

https://www.ncbi.nlm.nih.gov/tools/primer-blast/
The input:
• Template sequence in FASTA format
• Or accession number
Range:
• Determine the site for forward and reverse primers in the template sequence
Primer parameters:
• Used to check already available primers, to BLAST these primers with the template sequence
Specificity checking parameter:
• Search mode: determine the mode to be used to check for primer specificity, (automatic, user
guided, No user guidance), the default is (Automatic)
Database:
• The database to be searched for primers target template sequences, the default is (Refseq mRNA)
Organism:
• The targeted organism, (Human = Homo sapiens)
Stringency:
• The number of mismatches with unintended sequences