DNA 1. enzymes2. : purified DNA or cDNA (complementary DNA)3. transformation, infection, electroporation, microinjection, gene gun4. : specific antibody screening, hybridization (radioactive isotope or fluorescent agent label),5. expression vectors, protein purification

DNA

The toolkit for DNA technology: 1.Restriction enzymes: Daniel Nathans, Hamilton Smith and Werner Arber (1978 Nobel Prize) 2.DNA vector3. Host organisms

Restriction enzymes: 1.Daniel Nathans, Hamilton Smith and Werner Arber (1978 Nobel Prize), DNA ligase

DNA

cDNA

DNA vector cloning vector Bacterial plasmidsBacteriophagesShuttle vectorsyeastRetroviruses

Host organisms: (1) Bacteria: (2) Yeast: (3) Animal or plant cell: DNA

Diagnosis of Genetic Diseases Human Gene Therapy Vaccines Pharmaceutical products Forensic Environment Agricultural

GMO

Diagnosis of Genetic Diseases Cystic fibrosis Huntington's disease

-1

-2

EnzymePolymerase bind single stranded DNA at a point adjacent to a double-stranded stretch, and binds to dNTPs (deoxynucleotide triphosphates)

Components of PCR reaction:Enzyme (Taq, Vent, Pfu)Appropriate buffersAmple dNTPsTemplate DNAPrimersCofactors (eg. Magnesium)

The cycle 1Denaturation94 or 92, 30 sec/incubationHalf activity after 30 incubations at 94 for 60 sec each.60-120 sec for the first step

The cycle 2AnnealingTm = 4 (GC bases) + 2 (AT bases)30-60 sec/incubation

The cycle 3Extension72, 500bp/30 sec; 500-1500/60 sec

CyclesExample: DenatureAnnealingExtensioncycles944572594557230-35945572 (10 min) 1

Condition of temperature

DNA

DNA

autosequencer

Human Gene Therapy SCID (severe combined immunodeficiency)1. retroviral vector + ADA()T lymphocyte intravenous injection2. ADA gene bone marrow cell

HDLs: LDLs:

Vaccines Protein coat of virus or bacteria.Modify pathogen's genome

Pharmaceutical products InsulinGrowth hormonesEPO (erythropoietin) Interferon

Forensic RFLPDNA fingerprinting

DNA

DNA

Environment Detoxification mining

Agricultural Transgenic animalsn enlarge valuable partsn resistant to pathagensn improved agricultural products, such as change AAs compositionsTransgenic plantsTi plasmid (Tumor-inducing plasmid)Agrobacterium tumefaciensAntisense genes

DNA