Tissue type and age

Good quality: Young, healthy and tender Etiolated young leaves Growing the source plant for 34 days in dark before harvesting Bad quality: Mature leaves (high concentrations of polyphenols, tannins, polysaccharides and other 2nd metabolites). Collected fresh and directly subjected to processing Stored at 18oC, 80oC Sotred in liquid nitrogen Stored in saturated cetyltrimethylammonium bromide Dried with silica gel and then stored to avoid contamination Stored in the form of lyophilized tissue Reduce viscosity caused by high molecular weight (carbohydrates) in cell wall. Grinding mechanically: mortar and pestle, steel or glass rods, glass beads, etc. Griding in iquid nitrogen using a flash freezing procedure (prefered) hydrolyzing enzymes (cellulases, pectinases) Cell wall macerases to dissolve the cell wall Sodium/potassium ethyl xanthogenate

The source material and processing Problem: Difficulty in obtaining contamination free, highquality genomic DNA. Contaminated nucleic acid cant give precise, reliable and reproducible results. Removal of cell walls/ membranes Separation of DNA from all other cell components Maintenance of integrity of DNA during the procedure
About DNA isolation

Because of a rigid polysaccharide cell wall

Collection and storage

Homogenization

Methodology Major factors influencing isolation methodology

Gel electrophoresis, spectrometric analysis, restriction digestion, PCR amplification, and chromatographic techniques. Spectrometric analysis: ratio of absorbance at 260/280 nm is 1.8 for pure DNA

Plant genomic DNA isolation

Assessment of DNA quality and yield Presence of contaminants

Polysaccharides

Prime interferers: they are undetectable and hard to re-move Makes the tissue homogenate very viscous Interfere with the activity of enzymes (polymerases, ligases, endonucleases) Render the DNA unsuitable for downstream processing To remove them from DNA solution High concentration of NaCl CTAB (Forms a complex with DNA and make it precipitate) NaCl with detergent CTAB (forms insoluble complexes with proteins) Hydrolytic enzymes Oxidized polyphenols causes enzymatic browning of the DNA pellet Solution: add antioxidants to the extraction buffer to prevent the oxidation of phenols (PVP, PVPP) RNA purged using DNase free pancreatic RNase Proteins removed with chemicals that destroy their structure (SDS, DTT, mercaptoethanol) Protein-hydrolyzing enzymes (proteinase K) Organic solvents (phenol/chloroform) Homogenizing plant tissue in a isolation buffer with osmoprotectants (glucose, sucrose) Nuclear membrane stabilizers Anonionic detergent Triton X-100

Polyphenols

Rapid and less expensive Equipped with mechanical cell disruptors , lysis buffers, RNase. Proteins and polysaccharides are removed by salt precipitation and centrifugation.

Proteins and RNA DNA isolation kits

Non-nuclear DNA (chloroplast and mitochondrial DNA)