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Primer design for PCR cloning

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

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Primer design for PCR cloning

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  1. 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 #

  2. 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

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

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

  5. Oligo design guidelines •Tm is temp where 50% of oligos are annealed to target •Tm should be between 55°C and 80°C •Tm=81.5 + 0.41(%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

  6. Oligo design guidelines •primers should have 17 to 28 nt complementarity •50-80% GC (can be tough) •not longer than 35-40 base pairs (due to errors in synthesis) •3’ end should end in at least one G or C (better if there are 2)

  7. 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!

  8. 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

  9. 5’ end of lacZ gene 5’ atgaccatga ttacggattc actggccgtc 3’ tactggtact aatgcctaag tgaccggcag atg acc atg att acg gat tca ctg gcc gtc M T M I T D S L A V

  10. 3’ end of lacZ gene 5’ gctaccatta ccagttggtc tggtgtcaaa aataa 3’ 3’ cgatggtaat ggtcaaccag accacagttt ttatt 5’ cag ttg gtc tgg tgt caa aaa taa Q L V W C Q K *

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

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