Bioinformatics. Technique to design PCR primers using BLAST. Primer designing. Technology related

1. PCR & Primer
Designing
PRESENTED BY: MUZNA KASHAF
Roll no. : 16261514-030

2.  What Is A Primer?
 PCR Primer Design
 Primer Blast
 Primer3
 Oligonucleotide Physiochemical Parameter
 PCR Primer Based On Protein Sequence
 PCR Primer Based Upon Multi-ligaments

3. A primer is a short synthetic oligonucleotide used in many
molecular techniques.
Primers are designed to have sequence which is the reverse
compliment a region of template or target DNA to which we wish
the primer to anneal.

6. Only one target site in the template DNA where the primer binds,
which means the primer sequence shall be unique in the template
DNA.
Uniqueness can be determined by BLAST
Length of 18-24 bases.
40-60% GC content.
Melting temperature, Tm (the temperature at which half the DNA
strands are single stranded and half are double stranded) of 50-60°C.
Primer pair should have Ta (annealing temp.) within 5°C of Tm.
Primer pairs should not have complimentary regions.

7. Runs of three or more Cs or Gs at the 3’ends of primers may promote misprinting at G or C
rich sequence and should be avoided.
3’ ends of primers should not be complimentary.
Primer self-complimentarity should be avoided.
CONTINUE…

8. Excellent site for designing PCR primers.
Developed at NCBI to help users make primers that are specific to
the input PCR template.
It uses primer 3 to design PCR primers and then submits them to
BLAST search against user-selected database.
The BLAST results are then automatically analyzed to avoid
primer pairs that can cause amplifications of targets other than the
input template.

9. This site has very powerful PCR primer design program permitting
one considerable control over the nature of the primers, including
size of product desired, primer size and Tm range, and presence
/absence of a 3’ GC clamp.
PRIMER3PLUS:
A new improved web interface to the popular Primer3 primer
design program.

10. Net Primer:
Net Primer combines the latest primer analysis algorithms with a web-based
interface allowing the user to analyze primers over the Internet.
All primers are analyzed for primer melting temperature using the nearest
neighbor thermodynamic theory to ensure accurate Tm prediction.
DNA MATE:
It calculates a consensus Tm for short DNA sequence using a merged method
that is based on 4 different thermodynamic tables .
Oligocalc:
It is an online oligo-nucleotide properties calculator.

11. PCR AND CLONING:
AMUSER:
It offers quick and easy PCR primers optimized for various USER
cloning based DNA engineering.
It facilitates DNA assembly and introduction of virtually any type
of site-directed mutagenesis by designing optimal PCR primers for
the desired genetic changes.

12. The PCR Suite:
This is a suite of 4 programs based upon primer3 for genomic
primer design.
All offer considerable control on primer properties:
 Overlapping Primers :- Creates multiple overlapping PCR
products in one sequence.
Genomic Primers:- Designs primers around exons in genomic
sequence.
SNP Primers:- Designs primers around every SNP in a GenBank
file.
cDNA Primers:- Designs primers around open reading frames.

13.  F Miura, C Uematsu, Y Sakaki, T Ito -
Bioinformatics, 2005 - academic.oup.com
 Howley,P.M., Israel,M.F., Law,M.F., and
Martin,M.A., 1979, J Bio Chem 254:4876-
4883.
 Panjkovich,A., Norambuena,T. and
Melo,F., 2005,Mate.
 PM Vallone, JM Butler - Biotechniques,
2004 - Future Science.