DNA EXTRACTION Flashcards
DNA EXTRACTION
• Goal:
To obtain pure and good quality DNA
• separates DNA from cellular fluid & cellular debris such as proteins, lipids, polysaccharides & other compounds.
DNA EXTRACTION
• the technique used to isolate DNA in a biological sample.
DNA EXTRACTION
Genetic analysis:
Scientific research
Medical diagnostics
Forensic
■ Gene cloning – introduction of DNA into cells (introduction of DNA into cells (humans, animals or plants) for diagnostic purposes
Scientific- Research
Medical- Diagnostics
■ Genome sequencing (outbreak analysis)
■ Hereditary & infection disease
■ Vaccine development
Forensic-
Paternity, etc.
DNA Isolation:
aims to collect DNA by physical or
chemical means.
DNA Purification:
the process of eliminating
contamination from isolated DNA.
DNA Extraction:
achieve both purification and isolation.
Where do we obtain DNA from? From all living things such as:
● Human cell
● Plant cell
● Bacterial cell
Basic Steps
Cell lysis
Precipitation
Purification
- breaking cells to release DNA from nucleus
Cell Lysis
- Separates DNA to cellular debris
a. Such as proteins, RNAs, other macromolecules
Precipitation
- to remove remaining unwanted material
Purification
General Steps for DNA Extraction (5)
1- Tissue Homogenization / Cell Disruption / Cell Lysis
2- Denaturation and Separation of Other Biomolecules from the Nucleic Acid
3- Precipitation of Nucleic Acids
4- Washing of Precipitated Nucleic Acids
5- Drying of Pellet and Dissolution of Dried Pellet
TISSUE HOMOGENIZATION OR CELL DISRUPTION OR LYSIS
• Mechanical Methods
• Chemical Treatment
• Enzymatic Treatment
• Thermal
MECHANICAL METHODS (MVSHF)
• Mortar and pestle
• Vortexing with beads
• Sonication
• High pressure
• French press
CHEMICAL TREATMENT
• Denaturants
• Reducing agents
• Buffer
• Cell Lysis agents
• Salt
• ENZYMATIC TREATMENT (PCLL)
• Protienase K: animal cells
• Cellulase: Plant cells
• Lyticase: Yeast
• Lysozyme: Bacteria
(With the use of Temperature)
THERMAL
Factors that Influence the Disruption Strategy (SSPCC)
• Stability of Molecules
• Size of the Sample
• Presence of Inhibitors
• Cohesion of Cells
• Cell Membrane Type
Denaturation and Separation of Other Biomolecules from the Nucleic Acid (CEC)
Chemical Treatment
Enzymatic Treatment
Centrifugation
Denaturation and Separation of Other Biomolecules from the Nucleic Acid
Chemical Treatment (SPP)
○ Sodium dodecyl sulfate
○ Phenol and chloroform
○ Protein denaturation
Denaturation and Separation of Other Biomolecules from the Nucleic Acid (CEC)
● Enzymatic Treatment
○ Protease; Proteinase
Precipitation of Nucleic Acids
MONOVALENT CATIONS
Ammonium, Potassium or Sodium
Acetate or Lithium or Sodium
Chloride Alcohol Precipitates Nucleic Acid
ETOH (95% Absolute)
Isopropanol
________, like sodium ions, neutralize the negatively charged phosphate groups on nucleic acids, overcoming electrostatic repulsion, enabling
the aggregation of nucleic acid molecules, and ultimately promoting their precipitation for
subsequent purification.
Monovalent cations
Precipitation of Nucleic Acid
Alcohol precipitates nucleic acid
○ ETOH (95% - Absolute)
○ Isopropanol
Washing of Precipitated Nucleic Acids (2)
● 70-80% ETOH
● Centrifugation
Drying of Pellet and Dissolution of Dried Pellet
Drying: (2)
● Dissolution in_____ or ____
● Room temperature or at____
● Air drying or vacuum crying
● sterilized molecular grade water or TE
● 50-55 C water bath
• Resuspend in TE
• Neutral pH
• Chelating Agent
Nucleic Acid Stabilization
Nucleic Acid Stabilization
• Store at______
• Aliquot sample
• AVOID:______
• Use_____ free reagents and plastic ware
- 20 C or - 70 C
freeze-thaw cycles
nuclease
PHENOL CHLOROFORM EXTRACTION
• The nucleic acid solution is extracted by successively washing with a volume of_______; a volume of______:______:______, and_____:_____
• Centrifugation is performed intermittently and the upper aqueous phase is transferred to a new tube while avoiding the interphase
• The contaminants are denatured and accumulate in the organic phase and the nucleic acids are preserved in the aqueous phase Anotherway of removing proteins is by using the enzyme proteinase K
phenol
phenol: chloroform: isoamyl alcohol (25:24:1)
chloroform: isoamyl alcohol (24:1)
PRECIPITATION OF NUCLEIC ACIDS
•____ precipitation
• Diluting nucleic acid with a_____, adding alcohol to it and mixing gently
• The precipitated DNA is pelleted by____
• The salts and alcohol remnants are removed by washing with______
• _____ and______are the standard alcohols used for
Nucleic Acid extraction
Alcohol
monovalent salt
centrifugation
70% alcohol
Ethanol and Isopropanol
RESUSPENDING DNA
• Nucleic Acid pellet resuspended in either_____ or_____
sterile distilled water or
Tris-EDTA (10mM Tris: 1mM EDTA)
PURIFICATION OF DNA
• DNA is purified by incubating the Nucleic Acid solution with _______ at_____ and reprecipitation following________ extraction to remove the RNAse
RNASe A (10mg/mL) at 37C
phenol: chloroform
SPECIFIC PROCEDURES
- Disrupt the Cell
- Disrupt Protein Disulphide Bonds
- Denature Proteins Further, Remove Lipids and some Polysaccharides
- Precipitate Nucleic Acids
- Washing Nucleic Acid
- Remove RNA and Elute
- Resuspend DNA
- DNA Measurement
- Disrupt the Cell
• Preheat CTAB isolation buffer to_____ in a water bath
• Weigh 0.1g fresh, dried, or frozen material
• Place in a______
• Add _____and grind to fine powder.
• Optional: Add______ and grind further
• Transfer powdered sample to 2mL tube microfuge tube
65C
mortar and pestle
liquid nitrogen
40mg PVPP(Polyvinylpolypyrrolidon)
- Disrupt Protein Disulphide Bonds
• Add 800uL CTAB isolation buffer
• Vortex to mix
• Incubate at 65C for 1 hour with occasional swirling or shaking
- Denature Proteins Further, Remove Lipids and some Polysaccharides
• Add 800 uL______
• Mix gently for 10 minutes at room temperature
• Centrifuge at room temperature (6000 g for 10 minutes)
• Pipet the upper aqueous phase with wide bore pipet
• Place in a clean 2mL microfuge tube
chloroform-isoamyl alcohol (24:1)
- Precipitate Nucleic Acids
• Add 600uL cold_____
• Mix gently by inverting tubes several times
• Incubate several hours to overnight at room temperature
• Centrifuge for 1-2 minutes at 12000 rpm
• Gently pour off the supernatant
isopropanol
- Washing Nucleic Acid
• Add 800uL____ directly to pellet. Swirl gently. Leave for at least 20 minutes with gentle swirling.
• Centrifuge at 12000 rpm for 3 minutes.
Pour off supernatant carefully.
• Air dry briefly by inverting tubes in paper towel at room temperature
ETOH
- Remove RNA and Elute
a. Add 100uL high salt TE and 1 uL RNAse. Incubate 30 minutes
at 37C.
b. Dilute sample with:
i. 200uL 1XTE
ii. 10uL 3M sodium acetate (5.2)
iii. 250uL cold 70% ETOH then gently mix to precipitate DNA
- Resuspend DNA
• Resuspend in 20uL TE buffer (10mM Tris-HCl, 1mM
EDTA, pH8) to dissolve precipitate
• Store at -20C to -40C until further use
- DNA Measurement
• Measure DNA yield using______ at _____
UV-Visible Spectrophotometer at 260 nm
Centrifugation is performed intermittently and the upper____ phase is transferred to a new tube while avoiding the____
aqueous
interphase
• The contaminants are denatured and accumulate in the_____ phase and the nucleic acids are preserved in the_____ phase
organic
aqueous
Anotherway of removing proteins is by using the enzyme…
proteinase K
