Primer design for PCR cloning
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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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Presentation Transcript

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


ABCDE

prepare PCR product

prepare vector

plasmid

PCR product

B

A

restriction

digest

A

B

ligate


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)


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


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)


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’ 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


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 *


Software to help you

• Vector NTI (Mac/PC)

• DNA Strider (Mac only)


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