4. DNA Extraction, Detection and Quantification
DNA Extraction, Detection, and Quantification
Overview of Nucleic Acids
Nucleic Acids: Biological macromolecules essential for all forms of life.
DNA (Deoxyribonucleic Acid): Carries genetic information.
RNA (Ribonucleic Acid): Plays various roles including acting as a messenger carrying instructions from DNA for controlling the synthesis of proteins.
Transcription: Process of copying a segment of DNA into RNA.
Translation: Process where ribosomes synthesize proteins using the messenger RNA transcript.
Proteins: Polymers made from amino acids and perform various functions within organisms.
Genotype vs Phenotype
Genotype:
Definition: The sequence of nucleotides in an organism's DNA inherited from its parents.
Phenotype:
Definition: Observable characteristics or traits of an organism.
Factors influencing phenotype include:
Genotype
Epigenome: Modifications on DNA that affect gene expression without altering the underlying sequence.
Environmental influences: Factors from the organism's surroundings that can affect both genotype and phenotype.
DNA Extraction
Definition: The first step in any molecular test that involves isolating DNA or RNA from a sample and purifying it by removing other cellular components.
Common Steps in DNA Extraction:
Collection of the sample
Lysis of the cell membrane: Breaking open the cell and nuclear membranes to release DNA.
Removal of contaminants: Removing membrane fragments, proteins, and RNA.
Concentration of purified DNA: Isolating and concentrating the DNA for further use.
Methods of DNA Extraction:
Phenol/Chloroform-Organic Extraction
Salt (Salting Out)-Inorganic Extraction
Column Methodology
Magnetic Bead Method
Specimen Collection and Handling for DNA Extraction
Anticoagulants used in blood samples:
EDTA (Ethylenediaminetetraacetic acid): Prevents clotting and inhibits enzyme activity by chelating ions like Mg$^{2+}$.
Note: Preferred over Heparin, as Heparin can interfere with downstream molecular methods, inhibiting Taq polymerase during PCR, and inhibiting DNases and RNases.
Types of Specimens for DNA extraction include:
Whole blood
Bone marrow
Buffy coat
Fresh or frozen tissue
Tumor samples
Skin samples
Fetal chorionic villi
Umbilical cord
Formalin-fixed paraffin-embedded tissue (FFPE)
Saliva
Amniotic fluid
Cultured cells
Specimen Storage and Stability
Storage Conditions:
Whole blood and Bone marrow: Can be stored at room temperature for 4 days.
Buffy coat: Room temperature for 4 days or frozen at -80 °C for many years.
Fresh tissue: Good at 4 °C for 2 days.
Frozen tissue: -80 °C for years.
FFPE: Indefinitely.
Saliva: Can be stored in preserving agent for years.
Amniotic fluid: Must be centrifuged to obtain the cell pellet, which can be frozen at -80 °C for years.
Cultured cells: Viable for up to 8 weeks with proper maintenance.
DNA Extraction Methods
Requirements for all extraction methods:
Lysis of cell and nuclear membranes.
Removal of protein and cellular debris.
Isolation of DNA.
Common Extraction Methods:
Phenol/Chloroform Method: Organic extraction method that dissolves proteins and isolates DNA.
Column Method: Uses silica membranes in spin columns to adsorb DNA under specific conditions.
Salt Method: Uses anionic detergents, followed by alcohol precipitation for DNA recovery.
Magnetic Bead Method: Isolates DNA via binding to magnetic beads under chaotropic salt presence.
Specific Methods of Sample Preparation
Whole Blood Preparation:
Process:
Remove red blood cells (RBCs) using NH$_4$Cl and Tris-HCl solution.
Incubate at 37 °C for 25 min.
Centrifuge, resuspend nuclear pellet in 0.85% NaCl.
Further purify with TE buffer and SDS.
Digest with proteinase K at 50 °C.
Buffy Coat Preparation:
Process:
Centrifuge whole blood.
Use a transfer pipette to recover the buffy coat.
Add protease and lysis buffer; incubate at 56 °C for 10 min.
Tissue Preparation (Fresh/Frozen):
Process:
Move tissue to biosafety cabinet.
Cut with a sterile scalpel and resuspend in cell lysis buffer with proteinase K.
Incubate overnight at 56 °C.
FFPE Preparation:
Process:
Cut sections from FFPE blocks.
Remove paraffin wax and wash in EtOH.
Resuspend in lysis buffer with proteinase K, incubate overnight at 65 °C.
Saliva Preparation:
Process:
Collect saliva and centrifuge.
Resuspend in PBS and add lysis buffer with proteinase K.
Amniotic Fluid Preparation:
Process:
Centrifuge the fluid, leaving a small volume.
Resuspend pellet in lysis solution, add proteinase K, incubate overnight at 55 °C.
Precipitation Techniques
Ethanol Precipitation:
Principle: DNA, being a polar molecule with a negatively charged phosphate backbone, is typically prevented from forming ionic bonds with positive ions by water. Ethanol, being less polar, allows these bonds to form, leading to DNA precipitation.
Principles of Extraction Techniques
Phenol/Chloroform Extraction:
Conducted under fume hood. Involves repeated layers of phenol and chloroform to separate DNA from proteins and contaminants.
Essential steps include mixing lysate with phenol, centrifugation to separate the aqueous layer (containing DNA), and successive washes until DNA is precipitated with cold ethanol.
Column Method:
The principle is that DNA binds to silica membranes under specific conditions, followed by washing and elution through a change in buffer conditions.
Steps:
Combine lysate with EtOH, apply to spin column.
Centrifuge to remove lysate and wash with buffers AW1 and AW2.
Elute DNA with Buffer AE.
Salt Method:
Cells are lysed with detergent in presence of stabilizers. Precipitation of proteins followed by extraction of DNA using alcohol.
Magnetic Bead:
Principle:
• DNA binds silica‑coated magnetic beads
• Magnet separates DNA from contaminants
Pros: Automation‑friendly, clean
Cons: Cost of reagents/instruments
RNA Extraction
Principles: RNA extraction requires careful techniques to avoid contamination from RNase enzymes, which can degrade RNA.
Contamination Control Measures:
Use of RNase-destroying solutions for cleaning surfaces and equipment.
Employment of nuclease-free reagents and consumables.
Adding reducing agents such as β-mercaptoethanol to lysis buffers to disrupt RNase activity.
Storage Conditions for Nucleic Acids
Short Term Storage: DNA at 4 °C, RNA at -80 °C.
Long Term Storage: Both DNA and RNA should be stored at -80 °C to ensure stability.
Quantification of Nucleic Acids
Spectrophotometry:
Estimates nucleic acid quantity and purity by absorbance readings at 260 nm and 280 nm.
260/280 Ratio:
A ratio of 1.8 indicates pure DNA; 2.0 indicates pure RNA.
Reading at 260 nm for estimating concentration:
DNA yield = Absorbance at 260 nm × 50 (0)
RNA yield = Absorbance at 260 nm × 40 (0)
260/230 Ratio: To identify organic contaminants, a ratio of <1.8 suggests contamination from organic materials
Troubleshooting Purity
260/280 = DNA <1.7
• Protein contamination
• Low DNA concentration
• Phenol contamination
260/280 =DNA > 2.0
• RNA contamination
260/230 =DNA < 1.8
• Organic or carbohydrate contamination
Advantages and Disadvantages of Spectrophotometry
Advantages:
Simple and direct measurement of nucleic acid purity.
No sample prep required.
Disadvantages:
Not selective (cannot differentiate between DNA and RNA), limited sensitivity, and cannot detect fragmentation.
Other Methods for DNA Quantitation
Fluorescence: Utilizes specific fluorogenic dyes for selective binding to nucleic acids, offering high sensitivity and accuracy despite contamination.
Quantitative Real-Time PCR (qPCR): Evaluates amplifiable DNA by measuring the quantity of amplified housekeeping genes, useful for assessing fragmented DNA in samples such as FFPE.
Assessing Quality of Nucleic Acids
Integrity Gels for Quality Assurance: Loading DNA or RNA onto a gel; intact DNA shows a high molecular weight band while intact total RNA will display distinct bands corresponding to 28S and 18S rRNA on a denaturing gel.