PCR mediated mutagenesis II Polymerase Chain Reaction Technique

PCR mediated mutagenesis 생화학 실험 II 이희두

Polymerase Chain Reaction Technique widely used in molecular biology With PCR it is possible to amplify a single or few copies of DNA across several orders of magnitude, generating millions or more copies of the DNA piece. Requirement components and reagent at a basic PCR DNA template A pair of primers (sense, antisense) Heat resistant DNA Polymerase Deoxynucleotide triphosphates (d. NTP) Polymerase buffer Thermocycler

Polymerase Chain Reaction Griffiths et al. (2000)

Application of PCR Sub-cloning DNA targets using PCR-meditated in vitro mutagenesis Site-directed mutagenesis Overlap Extension PCR Amplification of differentially expressed gene sequences RT-PCR Identifying genetic mutation Reverse Genetics Approach et cetera……

Overlap-extension PCR Genetic engineering technique allowing the construction of a DNA sequence with an alteration inserted beyond the limit of the longest practical primer length PCR-mediated INSERTION mutagenesis PCR-mediated DELETION mutagenesis Lee, J et al. Bio. Techniques 36, 398 -400 (2004)

Workflow of PCR mediated mutagenesis Primary PCR using chimeric primers ### oo o B A ## C # ooo D ### ooo D Primary PCR ### ooo A Ligation PCR A ### ooo D

Workflow of PCR mediated mutagenesis Primary PCR using chimeric primers Primer A + Primer B (chimeric primer with primer C sequence) Primer D + Primer C (chimeric primer with primer B sequence) Primer B COMPLEMENTARY SEQUENCE Primer C PCR construct purification through gel elution Intermediate PCR construct (A-B & C-D) Ligation PCR using outer primers (A and D) Using Intermediate PCR construct (A-B + C-D) as a DNA template Final product is mutagenized PCR construct which has deleted region

Procedure ; Primary PCR Ingredient for PCR reaction (Primary PCR) Ingredients Volume ( ul ) X 5 DNA template 0. 5 2. 5 10 x polymerase buffer 2 10 d. NTP 0. 6 3 Primer 0. 5 x 2 2. 5 x 2 Taq polymerase 0. 2 Distilled water 15. 7 78. 5 Total 20 99 Primary PCR condition Temp. (C) Time 95 5 min 95 1 min 57 1 min 72 30 sec 5 min 25 cycles

Confirmation Gel electrophoresis result AB CD AD

Primer production 5’ 5’ 3’ 3’ 3’ 5’ 5’

Primer production

Primer production 5’ 3. ATC ATG ATC CCC AAC CGT 1. ATG CAT CCA GGG GTC CTG 5’ 3’ 3’ 3’ 5’ 2. ACG GTT GGG GAT CAT GAT 5’ 4. TTA ACA CAA AAT GGA