Primer design for PCR cloning special case for when the insert is to be cloned PCR also used for diagnostic (is gene present) PCR also used to quantitate gene/transcript copy #

PCR cloning TA cloning, utilizing a T or A single base overhang formed by Taq polymerase (Thermotoga aquaticus) “Tailed Oligo” technique using oligonucleotides having short regions of target gene non- complementarity and encoding restriction sites compatible with cloning sites on the plasmid vector

A B A B PCR product plasmid ABCDE ligate restriction digest prepare PCR product prepare vector

Example of a restriction enzyme cutting site 5’-XXXGGATCCXXX-3’ 3’-XXXCCTAGGXXX-5’ 5’-XXXG 3’-XXXCCTAG BamHI GATCCXXX-3’ GXXX-5’ (colored sequence is the overhang)

Oligo design guidelines Tm is temp where 50% of oligos are annealed to target Tm should be between 55°C and 80°C Tm= (%GC) - 675/N-%mismatch (N=length) Tm= 4(G+C) + 2(A+T) Ta is annealing temp annealing temp should be 5°C below Tm too high=low product too low=non-specific annealing & spurious products

Oligo design guidelines primers should have 17 to 28 nt complementarity 50-80% GC (can be tough) not longer than base pairs (due to errors in synthesis) 3’ end should end in at least one G or C (better if there are 2)

Oligo “tails” The 5’ tail encodes restriction site, which when cut yields a compatible end to vector tail should also have 5-8 nt beyond restriction site for enzyme to “grab onto” primer for 5’ end of gene (upstream or forward primer) may encode an ATG primer for 3’ end of gene (downstream or reverse primer) may need to encode a stop codon. NdeI and NcoI encode ATG in their restriction sites pay attention to frame don’t use enzyme that is also found in coding sequence of insert!

Restriction enzyme considerations many enzymes that have different cleavage sites that yield compatible cohesive ends (tables in NEB catalog) e.g. NdeI yields AT overhang compatible with AseI for double digests, try to choose enzymes that will function in the same buffer, otherwise do digests sequentially and change/alter buffer for each enzyme

5’ atgaccatga ttacggattc actggccgtc 3’ tactggtact aatgcctaag tgaccggcag 5’ end of lacZ gene atg acc atg att acg gat tca ctg gcc gtc M T M I T D S L A V

5’ gctaccatta ccagttggtc tggtgtcaaa aataa 3’ 3’ cgatggtaat ggtcaaccag accacagttt ttatt 5’ 3’ end of lacZ gene cag ttg gtc tgg tgt caa aaa taa Q L V W C Q K *

Software to help you Vector NTI (Mac/PC) DNA Strider (Mac only)