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 10 6 copies) – sequencing, alignment of all nucleotides – restriction digestion, testing specific nucleotides – reverse transcription – cDNA – qPCR – blotting – Southern (DNA) Northern (RNA) Western (protein)
PCR cycling (30 cycles) 1. n=2 1 2. n=2 2 3. n=2 3 Cycle: n = number of gene copies (exponential growing)
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 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 result under UV-light
Sequencing lines in vertical sequence gel: Genome sequencing
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 (cDNA) of eukaryotic organisms in laboratory, it results from reverse transcription in certain gene: cDNA < DNA It is quantified by qPCR - 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 BLOTTINGSOUTHERN BLOTTING: is used to locate and identify genes on DNA. 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