this slide will help you in designing the primmer for dna samples it will clearly explain the software names and method to perform primmer designing

1. PRIMER DESIGNING
BY INSILICO METHOD
BY
S .BOOBASH RAJ
3RD M .TECH
BIOTECHNOLOGY

2. Polymerase chain reaction
A simple rapid, sensitive and versatile in vitro method for selectively
amplifying defined sequences/regions of DNA/RNA from an initial
complex source of nucleic acid
Can amplify one molecule of DNA into billions of copies in a few
hours

3.  PCR methods rely on thermal cycling
 Thermal cycling exposes reactants to repeated cycles of heating and cooling to permit
different temperature-dependent reactions—specifically, DNA melting and enzyme-
driven DNA replication.
 Pcr employs two main reagents - primers and DNA POLYMERASE
 Pcr involves three steps
 Denaturation
 Primer annealing
 Primer extension

5. Ingredients of PCR
1. Template DNA
2. Primers
3. Master mix
4. Water

6. PRIMERS DESIGNING
Primers are designed to be exactly complementary to the template DNA
Primers are usually between 18-22 bp in length and should be made of approx.
equal no. of four bases
 sequences that are complementary to each other
 secondary structures
Palindromic sequences should be avoided
same base should be avoided
Primer melting temperature (Tm) between 55-60°C
Tm = (No of A+T)x2°C + (No of G+C)x°4C

7. PRIMER LENGTH
‣ Primer length determines the specificity and significantly affect its annealing to
the template
-Too short (resulting in non-specific amplification )
- Too long (decrease the template-binding efficiency at normal temperature)
‣ Primer length
Normally primer length should be
-18-24 bp for general applications
-30-35 bp for multiplex PCR
‣ Amplicon size
- 300-1000 bp
-50-150 bp

8. PRIMER MELTING TEMPERATURE
 Tm is the temperature at which 50% of the DNA duplex dissociates to become
single stranded
 Optimal melting temperature
- 52°C-- 60°C
- Tm above 65°C should be generally avoided -
-Higher Tm (75°C-- 80°C) is recommended for amplifying high GC
content targets.

10. OTHER CRITERIA FOR PRIMER DESIGN
• Primer G/C content
-Optimal G/C content: 45-55%
-Common G/C content range: 40-60%
• GC clamp
-The presence of G or C bases within the last five bases from the 3’ end of
primers (GC clamp) helps promote specific binding at the 3’ end due to the
stronger bonding of G and C bases.
• More than 3 g’s or c’s should be avoided in the last 5 bases at the 3’ end of the
primer.

11. PRIMER SECONDARY STRUCTURES
• Presence of the primer secondary structures produced by intermolecular or
intramolecular interactions can lead to poor or no yield of the product.
• Hairpins: It is formed by intramolecular interaction within the primer and should
be avoided.
• Self Dimer : A primer self – dimer is formed by intramolecular interactions
between the two (same sense) primers, where the primer is homologous to it self.
• Cross Dimer : Primer cross dimers are formed by intermolecular interaction
between sense and antisense primers, where they are homologous.

12. PRIMER DESIGNING TOOLS & SOFTWARE
• A plenty number of primer design tools are available that can assist in PCR
primer design
• These software are only online basis
-Primer3
-Primer3plus
-Primerz
-Perlprimer
-Vector ntiadvantage 10

13. RESOURCES FOR PCR PRIMER
• IDT oligoanalyzer 3.0
• NCBI blast