PCR Magnesium chloride 5 2 5 m M

PCR απαιτούνται: • Magnesium chloride: . 5 -2. 5 m. M • Buffer: p. H 8. 3 -8. 8 • d. NTPs: 20 -200µM • Primers: 0. 1 -0. 5µM • DNA Polymerase: 1 -2. 5 units • Target DNA: 1– 10 μg/ml

Cloning and selection markers Antibiotics

The problem of self ligation -Need for additional selection markers

Generation of engineered restriction sites on plasmids on selection markers

Lac. Z, Blue-white selection + INSERT

Lac. Z, Blue-white selection

Applications of PCR Basic Research • Genotyping • Genomic cloning • Sequencing Applied Research • Detection of pathogens • Pre-natal diagnosis • Cancer research-Mutation detection • Molecular Epidemiology +Real time PCR +c. DNA synthesis and quantitative RT-PCR

Basic Research_Molecular cloning

Short tandem repeat

DNA FRAGMENT ANALYSIS STR 1 STR 2 STR 3 STR 4

DNA-Based Human Identification

PRC diagnostics RT-PCR can detect specific RNA sequences within a sample. Example: Retroviruses have an RNA genome. Retroviral RNA can be detected by RT-PCR to diagnose retroviral infections. Viruses • HIV, SARS, H 5 N 1 Bacteria • meningococcus, legionellosis Analysis for resistant genes • MRSA, VRE

Molecular Identification: Detection Of Pathogens Sensitivity of detection of PCR-amplified M. tuberculosis DNA. (Kaul et al. 1994)

Mutation detection

Sanger Sequencing Genomic PCR amplification

Sanger sequencing

c. DNA synthesis

RECOMBINANT PROTEINS

Production of recombinant Insulin

p. ET System T 7 RNA Polymerase is an RNA polymerase from the T 7 bacteriophage that catalyzes the formation of RNA in the 5'→ 3' direction.