10-DNA Extraction and Purification.ppt

NEW CHAPTER DNA Extraction and Purification 1 * Learning objectives • Learn about the extraction and isolation steps of the cellular DNA. • Identification steps of the isolated DNA. • Restriction enzymes and their action. 2 DNA Extraction and Purification (Illustration) 3 Identification an...

NEW CHAPTER DNA Extraction and Purification 1 * Learning objectives • Learn about the extraction and isolation steps of the cellular DNA. • Identification steps of the isolated DNA. • Restriction enzymes and their action. 2 DNA Extraction and Purification (Illustration) 3 Identification and purity check of the extracted DNA: 1) Dische test: A diphenylamine (DPA) indicator will confirm the presence of DNA. The reaction requires a deoxyribose sugar and therefore is specific for DNA. This procedure involves chemical hydrolysis of DNA so the DNA is heated (e.g. ≥95 °C) in acid. Under these conditions, the 2-deoxyribose is converted to hydroxylevulinyl aldehyde, which reacts with the compound, diphenylamine, to produce a blue-colored compound. 2) Measuring the intensity of absorbance of the DNA solution at wavelengths 260 nm and 280 nm is used as a measure of DNA purity. DNA absorbs UV light at 260 and 280 nanometres, and aromatic proteins absorb UV light at 280 nm; a pure sample of DNA has the 260/280 ratio at 1.8 and is relatively free from protein contamination. A DNA preparation that is contaminated with protein will have a 260/280 ratio lower than 1.8. 4 Restriction Endonucleases • One of the major obstacles to molecular analysis of genomic DNA is the immense size of the molecule involved. A special group of bacterial enzymes, called restriction endonucleases (restriction enzymes), which cleave double-stranded (ds) DNA into smaller, more manageable fragments. 5 • A DNA sequence that is recognized by a restriction enzyme is called a restriction site. These sites are recognized by restriction endonucleases that cleave DNA into fragments of different sizes. For example, an enzyme that recognizes a specific four-base-pair sequence produces many cuts in the DNA molecule. In contrast, an enzyme requiring a unique sequence of six base pairs produces fewer cuts and, hence, longer pieces. Hundreds of these enzymes, having different cleavage specificities. 6 Restriction sites 7 * Atlas of Biochemistry by J Koolman, 2005. p. 259. 8