D. Darban, Ph.D Department of Microbiology School of Medicine Alborz University of Medical Sciences 1 Probe and Primer Design

Primer sequence shall be unique in the template DNA (BLAST search ) 2

PCR primers are typically 16–28 nucleotides long Length is too short difficult to design gene-specific primers choose optimal annealing temperature Very long oligos increase Oligo synthesis cost Increasing Tm More likely to form secondary structures Result in decreased PCR efficiency Promote primer dimer formation 3

In most PCR applications primer GC content 35% and 65% If GC content is too high Mispriming frequently results Very low GC content Poor primer binding Leading to decreased PCR efficiency 4

3’ end residues contribute strongly to non-specific primer extension by Taq DNA polymerase Therefore, primers with very high 3’ terminal stability should be rejected. Low specific binding at the 3' end lower GC content to avoid mispriming 5

5’3’ 5’ 3’ 6

Low-complexity sequences discard likelihood of mispriming with such primers. Some types of low sequence complexity pose a challenge for oligonucleotide synthesis 7

T anneal = T m_primer – 4C Annealing Temperature, T anneal temperature at which primers anneal to template DNA. It can be calculated from T m An ideal PCR reaction should have forward and reverse primers with similar Tm values. Each G/C contributes 4ºC Each A/T contributes 2ºC 8

If primers can anneal to themselves, or anneal to each other rather than anneal to the template, PCR efficiency will be decreased dramatically. Harmless : For example, some dimers or hairpins form at 30 C while during PCR cycle, the lowest temperature only drops to 60 C. 9

Since PCR efficiency is one of the most important factors for accurate expression quantification, the amplicon should be smaller than 250 bp. Typically the size range is 50–250 bp. 10

TaqMan Primers * equal Tm ( C) * bases in length * G+C content % * no runs of four or more Gs (any nucleotide) * no more than two G+C at the 3’ end * no G at the 5' end (A or C preferred) * amplicon size bp (max 250) * span exon-exon junctions in cDNA 11

TaqMan Probes * Tm value 10 0 C higher than primers * no runs of identical nucleotides (no consecutive Gs) * G+C content 30-80% * more Cs than Gs * no G at the 5' end 12