Biotechnology DNA technology DNA diagnostics DNA therapy DNA

Biotechnology

DNA technology • DNA diagnostics • DNA therapy

DNA isolation • • Cell lysis Removal of proteins DNA precipitation by ethanol DNA dilution in water or buffer

DNA diagnostics – PCR (amplifying part of DNA up to 106 copies) – sequencing, alignment of all nucleotides – restriction digestion, testing specific nucleotides – reverse transcription – c. DNA – q. PCR – blotting – Southern (DNA) Northern (RNA) Western (protein)

PCR Polymerase Chain Reaction

1. 2. 3.

PCR cycle

PCR cycling (30 cycles) Cycle: 1. n=21 2. n=22 n = number of gene copies (exponential growing) 3. n=23

DNA Visualization and Separation Gel sieve structure of polymer molecules with pores Fluorescence dye binding to DNA and excites photons under UV-exposure

DNA Visualization and Separation Gel Electrophoresis result under UV-light • the movement of charged molecules in electric field • the movement direction from – to + • DNA-rate in gel depends on DNA-fragment length in indirect proportion

Sequencing Genome sequencing lines in vertical sequence gel:

Sequencing

Restriction digestion Bacterial restriction enzymes recognize palindromic sequence in DNA.

Restriction digestion Detection of mutation § mutative DNA – digestion (two small fragments) § normal DNA – non- digestion (one large fragment) results after gel electrophoresis read under UV-light

Complementary DNA (c. DNA) of eukaryotic organisms • in laboratory, it results from reverse transcription • in certain gene: c. DNA < DNA • It is quantified by q. PCR - marker of gene expression

CELL DIFFERENTIATION: • arises because cells make and accumulate different sets of RNA and protein molecules • that is, they express different genes DNA of all cells in the body of one individual is identical ! ! !

Blotting: transfer a substance from gel to membrane Mechanisms: § Capillary attraction § electrophoretic transfer

Blotting: a basic molecular biology technique originally created by Edwin Southern (1975) for locating gene specific sequences on DNA fragments. SOUTHERN BLOTTING: is used to locate and identify genes on DNA restriction fragments are electrophoretically separated. The fragments are blotted onto membranes, where the DNA bonds. Hybridization with labeled DNA probes & localizing target DNAs. NORTHERN BLOTTING: a variation on Southern blotting. RNAs are separated by electrophoresis, transferred to membranes, and probed with a labeled DNA probes. WESTERN BLOTTING: another variation on Southern blotting. Proteins are separated by electrophoresis, transferred to membranes, and probed with an antibody that binds to the protein you are interested in locating.

DNA therapy Bacteria or yeast produce human proteins coding by human genes: - coagulation factor VIII - insulin - growth hormone

DNA cloning

Cloning organisms • Reproductive • Therapeutic

Literature Biology, eighth edition, Campbell, Reece Unit three: Genetics Chapter 20: Biotechnology Pages 396 – 425