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 (dNTP) Polymerase buffer Thermocycler

Polymerase Chain Reaction Griffiths et al. (2000)

Application of PCR  Sub-cloning DNA targets using PCR  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 Lee, J et al. BioTechniques 36, (2004) PCR-mediated INSERTION mutagenesisPCR-mediated DELETION mutagenesis

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

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)  Primary PCR condition IngredientsVolume ( ul ) DNA template x polymerase buffer 2 dNTP 0.6 Primer 0.5 x 2 Taq polymerase 0.2 Distilled water 15.7 Total 20 Temp. (C)Time 95 5 min 95 1 min 57 1 min sec 72 5 min 25 cycles X x

Confirmation  Gel electrophoresis result ABCDAD

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

Primer production

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