MIDTERM: DNA ISOLATION Flashcards
- Key to the molecular biology
- Removal of nucleic acids (DNA and/or RNA) from the cells in which they normally reside.
Nucleic Acid Extraction
APPLICATIONS OF NUCLEIC ACID EXTRACTION
DIAGNOSING DISEASE AND GENETIC DISORDERS
- FORENSICS
- PATERNITY TESTS
- ANCESTRY TRACKING
- GENETIC ENGINEERING
Target Nucleic Acids: free from contamination with macromolecules such as:
PROTEINS, CARBOHYDRATES, LIPIDS OR OTHER NUCLEIC ACIDS
isolated the nuclein (DNA) from the WBCs he obtained from the pus on collected surgical bandages in a nearby hospital.
Friedrich Miescher (1869)
process where the substance settle at the bottom of tube (the DNA)
PRECIPITATION
Developed from density-gradient centrifugation strategies
Early Routine Laboratory Procedures of DNA Isolation
demonstrate the semi-conservative replication of DNA
Messelson & Stahl (1958)
daughter DNA consists is consist of old and new strand, preserve the integrity of the parent strand
Semiconservative
- Can be lysed by high pH (alkaline) and detergents
- Break/lyse the cell wall by adding detergent or high pH
A. Bacteria and Fungi
thin cell layer
Gram +
thick cell layer of peptidoglycan (
Gram -
Some bacteria with tough cell walls:
- uses the enzyme “lysozyme”
ENZYMATIC DIGESTION
grinding/vigorously mixing with glass beads
- Grind the thick cell wall, physically break open the bacteria cell)
MECHANICAL METHOD
detergent (1% sodium dodecyl sulfate/SDS) and a strong base (0.2M NaOH) in the presence of Tris base, ethylenediaminetetraacetic acid (EDTA; anticoagulant) and glucose.
CHEMICAL LYSIS
commercial reagents designed for isolation of DNA in amplification procedures (PCR)
YEAST FILAMENTOUS FUNGI & GRAM + BACTERIA
o Held within free viruses or integrated into the host genome along with host DNA.
o Cell-free specimens (plasma) will be used for viral detection
o May require concentration of viroids by centrifugation or other methods
Viral DNA
naked virus, no protein capsule basic structure of virus
VIROIDS
o Obtained from the blood plasma
o Purified of red blood cells (RBCs) & other components by either differential density-gradient centrifugation or differential lysis
Nucleated Cells in Suspension (Blood & Bone Marrow Aspirates)
Exosomes (release by cancer cells)
o Small vesicles (30 – 100 nm in diameter), which form by imagination & budding from the inside of cellular endosome vesicles and are secreted by living cells
o Contain nucleic acid and can be collected through centrifugation
o Diagnostic & prognostic analyses purposes (liquid biopsy)
o Solid-phase collection of nucleic acid
Plasma
Dissolved by mincing the tissue
FRESH TISSUE
Dissociated by grinding and homogenizing the tissue
FROZEN TISSUE
- preserve tissue morphology
- May be deparaffinized by soaking in XYLENE and tissue is rehydrated by soaking it in decreasing concentrations of ethanol
- May be used directly without dewaxing (high contamination if not dewaxed)
- tissue fixatives affect the quality of DNA
FIXED EMBEDDED TISSUE
mercury based fixatives that can damage DNA
B-5 and BOUIN’S
DNA ISOLATION CHEMISTRIES
* uses a combination of HIGH SALT, and an ORGANIC MIXTURE of PHENOL & CHLOROFORM
* Readily dissolves hydrophobic contaminants (lipids & lipoproteins)
* collect cell debris & strips away most DNA – associated proteins
ORGANIC ISOLATION METHODS
- Does not include the organic extraction step
- Sometimes called “SALTING OUT”, makes use of LOW pH and high salt conditions to selectively precipitate proteins, leaving the DNA in the solution
- Excess salt is removed by rinsing the pelted nucleic acid in 70% ethanol, centrifuging & discarding the supernatant, then dissolving the DNA pellet in rehydration buffer (10 mM, Tris, ImM EDTA, or water)
INORGANIC ISOLATION METHOD
- Used SOLID MATRICES (silica-based products) in the form of columns or beads to bind & hold the DNA for washing
C. SOLID PHASE ISOLATION
NON – INVASIVE HUMAN DNA ISOLATION
- GENOMIC DNA PURIFICATION FROM HAIR
- GENOMIC DNA PURIFICATION FROM SALIVA
- GENOMIC DNA EXTRACTION FROM URINE SAMPLE
→ Hair sample is incubated (95°C) for 10 mins in NaOH (strong base/alkali) buffer & the supernatant is subjected to DNA purification after centrifugation
Simple Alkaline Lysis Methods (root of hair)
→ Hair sample is incubated (56°C) for 2 hours with buffer containing Tris= HCL, EDTA, NaCl, SDS, DTT and proteinase K, followed by gentle mixing and incubation (60 °C) for 2 hours or until the hair dissolved completely and DNA can be extracted from the solution
Smooth Chemical Digestion Method using Dithioreitol (DTT)
cells found in the saliva, exfoliated buccal epithelial cells and other cells
GENOMIC DNA PURIFICATION FROM SALIVA
→ Suspended in Lysis buffer which include Tris, EDTA, SDS & proteinase K
→ Incubated (56°C) for 1-3 hours until the tissue is totally dissolved and DNA
Buccal Swabs
→ Samples from saline rinse need to be processed/frozen immediately after collection
→ To inhibit growth of bacteria
→ Alcohol containing mouthwash must be used (kills microorganism and reduce bacterial growth)
Mouthwash Method
- Inverted and swirled in a specimen cup to create a homogenous suspension of cells followed by the centrifugation
- Supernatant is removed and a dry pellet containing cells is chilled (20°C) for 15 mins. Followed by the addition of Lysis buffer (Tris, EDTA, SDS, Proteinase K)
- Sample is incubated (56°C) for 2 hours and then DNA is extracted from solution
GENOMIC DNA EXTRACTION FROM URINE SAMPLE
PROTEOLYTIC LYSIS OF FIXED MATERIAL
- PARAFFIN – EMBEDDED SPECIMENS
- BEFORE LYSIS
- REAGENTS FOR CELL LYSIS
o Dewaxed w/ XYLENE specimens
o Rehydrated before nucleic acid isolation
PARAFFIN – EMBEDDED SPECIMENS
o Cells may be washed by suspension and centrifugation in saline/other isotonic buffers
BEFORE LYSIS
o Simple screens: detergents (SS or triton)
o PCR amplification: mixture of this buffer and proteinase K (capable of breaking down proteins)
REAGENTS FOR CELL LYSIS
