Isolation of DNA and genes Flashcards
lecture 12
Why do we isolate DNA?
For genetic manipulations and DNA analysis.
Applications include scientific research, medical diagnostics, forensic science, ecological studies, and archaeology.
What are the steps involved in DNA isolation?
Cell Lysis: Break cell membranes to release DNA.
DNA Purification: Remove proteins, lipids, and other contaminants.
Concentration: Precipitate and collect DNA.
Measure Purity: Use A260/A280 ratio (>1.8 indicates pure DNA)
What are the three main methods of cell lysis?
Biological: Enzymes (e.g., cellulase, lysozyme, saponin).
Physical: Osmotic pressure, freeze-thaw cycles.
Mechanical: Grinding, bead mills, vortexing, shearing.
What enzymes are used for biological lysis?
Cellulase: For plant cells.
Lysozyme: For bacterial cells.
Saponin: For eukaryotic cells.
What are two physical methods of cell lysis?
Osmotic Pressure: Cells swell and burst in hypotonic solutions.
Freeze-Thaw Cycles: Ice crystals rupture membranes.
What are examples of mechanical lysis techniques?
Pestle & Mortar: For plant cells and tissues.
Bead Mill: For tough samples.
Homogenizer: Shearing through narrow spaces.
Syringe-Needle: For long DNA fragments.
How can DNA be purified after lysis?
Phenol-Chloroform Extraction: Separates DNA from proteins.
Silica Membrane Columns: Bind DNA, wash away impurities, and elute pure DNA.
What does an A260/A280 ratio indicate?
> 1.8: Pure DNA.
<1.8: Contaminants like protein or RNA are present.
What are restriction endonucleases?
Enzymes that cut DNA at specific recognition sites.
Used in cloning, DNA fingerprinting, and mutation analysis.
How do restriction enzymes cut DNA?
They hydrolyze phosphodiester bonds at specific sequences (restriction sites).
Produce either sticky ends or blunt ends.
How are restriction enzymes named?
Based on the bacterial genus, species, strain, and type.
Example: EcoRI
Escherichia coli, strain R, type I.
How does agarose gel electrophoresis separate DNA fragments?
DNA moves through the gel based on charge, size, and shape.
Smaller fragments travel further toward the positive electrode.
How do you visualize DNA in agarose gel?
Use intercalating dyes (e.g., Nancy Red) that fluoresce under UV light.
How can DNA fragment size be determined using gel electrophoresis?
Compare fragment migration to a DNA ladder or use a standard curve to estimate size.
Why is efficient DNA isolation important?
Ensures reliable genomic testing, PCR, cloning, and forensic analyses.
Poor isolation compromises downstream results.
