PRIMER DESIGN

Primers

• Single-stranded sequence of nucleotides designed to attach to a specific part of the DNA or RNA

• Complementary to the beginning and end of the target

Forward Primer

• 5’ primers

• bind to the antisense strand

Reverse Primer

• 3’ primers

• bind to the sense strand

Melting Temperature (Tm)

• Is temperature at which the Hydrogen bonds can be dissociated thus releasing each other

• 50% of the primer is bound to its complement while the other 50% is dissociated

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

Formula for Melting Temperature

Annealing Temperature (Ta)

• Is temperature at which the primers will anneal to the target strand

  • If Ta is too high, primers may not bind to the template

  • If Ta is too low, non-specific binding and dimer-formation can occur

Ta C = Tm C - (5 to 10)

Formula for Annealing Temperature

Hairpin

Self-Dimer

Cross-Dimer

Types of Primer Dimer

Hairpin

Interaction within the primer

Self-dimer

interaction between two same sense primers

Cross-dimerPCR

interaction between sense and antisense primers

PCR-Based Cloning

• A technique where a target DNA fragment with added restriction sites is amplified then introduced to a vector (usually a plasmid) for cloning

• The result is a recombinant DNA molecule which can be introduced to host cells through the process of transformation