DNA > Biotech > PCR Primers

PCR Diagram [use Firefox browser in older systems]
 
Sense primer
Direct primer, Forward primer
Left primer
"Bottom" primer
Primer 1		 Antisense primer
Reverse primer, Back primer
Right primer
"Top" primer
Primer 2

1) Strategy

Zone to be amplified? Check data banks :
Gene - gene data
OMIM - Online Mendelian Inheritance in Man - Data about allele sequence
Human Gene Mutation Database (HGMD) - Cardiff
Genome Data Viewer -  human genome map at NCBI

From RNA (choose a region including parts of at least two exons)

From DNA
 


2) Size of the region to be amplified

* min 100 bp

* max 2-4 kb (OK up to 1.5 kb)

* max 200-300 (400) bp if aimed at mutational screening

* max 700 bp if aimed at direct sequencing



3) Primer-BLAST software for primer design

Identify the region to be amplified by choosing the range in which to design each primer
Forward primer -  From... To...
Reverse primer  -  From... To...

Choose the range for the:
PCR product size

Indicate the length range for the primers (18-25 nucleotides):
Primer size

"Biochemical" parameters:

G or C at 3´ end; avoid T. Parameter:
GC Clamp (number of C/G at 3´ end)

GC content: ideally 45-55%. Parameter:
Primer GC%

Melting temperature should be >55°C, and similar for the two primers. Parameter:
Primer Tm
(4+2 rule: Tm=[(G/C x 4°C)+(A/T x 2°C)]

Advice: Tm=72°C, Ta=68°C

Reduce autocomplementarity, and inter-complementarity between the two primers. Parameter:
Max Self Complementarity (8; try lowering down to→5)
Max 3' Self Complementarity (3, try loweing down to→0)



4) "Biologic" primer sequence validation through sequence comparison - BLASTN
       to control for similarity with other sequences across the whole genome sequence.

 

5) Check the amplicon sequence

* digestion simulation (mutation search; cloning in a vector)

* hybridization simulation via BLAST (labeling and use as a probe in Northern or Southern blot)


[The small bioinformatician]

DNA