DNA Isolation Techniques

• DNA isolation is a key process in molecular biology and diagnostics
• This process frees DNA from the cell, separating it from other cellular components
• It is essential for genetic studies, forensic analysis, and medical diagnostics

Basic Steps

• Lysis involves breaking open the cell and nucleus to release the DNA
• Precipitation involves separating DNA from other cellular components
• Purification ensures a high yield and purity of DNA for downstream applications

Lysis Methods

• Mechanical methods use a tissue homogenizer, mortar and pestle, or cutting tissue into small pieces
• Chemical methods use detergents and enzymes like Proteinase K to dissolve proteins and free DNA

Precipitation Methods

• Sodium ions neutralize DNA's negative charge, making it less water-soluble and more stable
• Add ethanol to precipitate DNA

Substances for DNA Extraction

• Tris maintains pH of solution at pH=8, interacts with LPS and permeabilizes cell membrane for easy lysis
• EDTA is a chelating agent that blocks the activity of DNase enzyme
• SDS is an anionic detergent that denatures membrane protein and breaks open the cell membranes and nuclear envelopes
• NaCl prevents the denaturation of DNA
• MgCl2 protects DNA from mixing with other cell organelles
• Phenol is a nonpolar solvent that unfolds and precipitates protein impurities
• Chloroform sharpens the fuzziness between organic and aqueous layers
• Isoamyl alcohol prevents foaming between interphase and is an anti-foaming agent
• Sodium Acetate precipitates DNA through interaction with PO3-
• Ethanol/isopropanol assists with DNA precipitation by interacting with water through hydrogen bonds
• TE buffer dissolves DNA

DNA Extraction Protocol

• Materials include: blood specimen, DNA extraction kit, ethanol, centrifuge, micropipettes, and vortex
• The procedure involves isolating the buffy coat from a blood sample
• Lyse cells using Proteinase K and buffer followed by precipitating DNA using ethanol
• Purification of DNA is achieved with spin columns and then stored at -20°C

Isolating Buffy Coat from EDTA Tubes

• Centrifuge EDTA tubes with collected blood at 4,500 rpm for 15 minutes
• Three layers become visible after centrifugation
  • The top layer is plasma
  • The middle (white) layer is the buffy coat
  • The bottom layer is red blood cells
• Use a 1,000 pL pipette to aspirate the plasma layer and transfer it in 1.5 mL microcentrifuge tubes
• Aspirate until plasma is 1-0.5 cm above the buffy coat
• Prior to aspirating the buffy coat, cut at least 3 mm off the yellow tip to widen the orifice
• Place the cut yellow tip on top of the buffy coat layer and slowly aspirate while doing a circular motion

Protocol steps

• Pipette 20µl protease, add 200µl sample, and add 200µl Buffer AL
• Mix by pulse-vortex for 15s and incubate at 56°C for 10 minutes
• Briefly centrifuge and add 200µl 96-100% ethanol then mix by pulse-vortex for 15s
• Apply the mixture to the Mini spin column and centrifudge at 8000 rpm for 1 minute
• Discard the collection tube, place the column in a new 2ml collection tube and add 500µl buffer AW1
• Centrifuge at 8000 rpm for 1 minute, place the column in a new 2ml collection tube
• Add 500µl buffer AW2 and centrifuge at 14000 rpm for 3 minutes. Place the column in a new 2ml collection tube and centrifuge at 14000 rpm for 1 minute
• Place the column in a new 1.5ml tube, add 200µl buffer AE, and incubate at room temperature for 1 minute
• Centrifuge at 8000 rpm for 1 minute and quantify the DNA concentration
• For a 3X dilution, add 400µl buffer AE to dilute the eluted DNA

Commercial DNA Extraction Kit B

• Materials & Equipment: Similar to Kit A with slight variations
• Procedure Differences: Uses different lysis and binding buffers and utilizes PureLink® Genomic Lysis/Binding Buffer
• Elution step with PureLink® Genomic Elution Buffer

DNA Extraction from Sperm Cells

• Materials: Lysis buffer, Proteinase K, and phenol/chloroform/isoamyl alcohol
• Lyse cells using detergents and Proteinase K
• Remove proteins using phenol/chloroform extraction, precipitate DNA using sodium acetate and ethanol
• Purify and store DNA appropriately

DNA Isolation via Salting Out

• Materials: RBC lysing buffer, Phosphate Buffered Saline (PBS), Sodium Dodecyl Sulphate (SDS), Proteinase K, NaCl, and ethanol
• Uses high salt concentration to precipitate proteins, leaving DNA in solution for further purification

Measuring DNA Quality and Concentration

• Spectrophotometry measures absorbance at 260 nm and 280 nm
• A 260/280 ratio of 1.8-2.0 indicates high purity
• A ratio of less than 1.8 indicates there may be proteins and/or other UV absorbers
• A ratio higher than 2 indicates to reprecipitate, can be contaminated with chloroform or phenol
• Gel Electrophoresis is used to confirm free of RNA and determine fragment sizes

Procedure for Spectrophotometry

1. Dilute the genomic DNA sample with sterile distilled water or a working buffer (optional)
2. Use either sterile distilled water or the working buffer as a blank for the spectrophotometer
3. Measure the absorbance at 260 nm and 280 nm

Spectrophotometry Computations

• Concentration of DNA (pg/ml) = A260 reading × 50 pg/ml. x dilution factor
• Dilution Factor = Total volume / volume of sample
• Total DNA Yield (ug)= DNA concentration x total sample volume (mL.)
• DNA purity= A260 / A280 ratio

Purified DNA Applications

• Can be used for diagnosis of diseases like sickle cell anemia
• Can be used for Forensic applications such as paternity testing
• Applied in Molecular biology research

Molecular Techniques Using Purified DNA

• Polymerase Chain Reaction (PCR)

• Southern blotting

• Restriction Fragment length polymorphism (RFLP)

• DNA isolation is a fundamental skill in molecular biology

• Understanding various methods helps select the best approach for specific applications

• Proper technique ensures high DNA yield and purity