MIDTERM LEC: DNA & RNA ISOLATION Flashcards
Removal of nucleic acids (DNA and/or RNA) from the cells in which they normally resides
NUCLEIC ACID EXTRACTION
PURPOSE IS TO RELEASE NUCLEIC ACID FROM THE CELL FOR:
- Detecting a specific pathogen (bacteria and viruses)
- Diagnosing disease & genetic disorders
- Other applications: forensics, paternity tests, ancestry tracking, genetic engineering (vaccines, hormones), etc.
Free from contamination with macromolecules (proteins, carbohydrates, lipids, or other nucleic acids)
TARGET NUCLEIC ACID
Isolated the nuclein (DNA) from the WBCs he obtained from the pus on collected surgical bandages in a nearby hospital
FRIEDRICH MIESCHER in 1869
demonstrate the semiconservative replication of DNA
Meselson & Stahl (1958):
Developed from densitygradient centrifugation strategies
EARLY ROUTINE LABORATORY PROCEDURES OF DNA ISOLATION
Solubility differences among chromosomal DNA, plasmids, and proteins in alkaline buffers
LATER ROUTINE LABORATORY PROCEDURES OF DNA ISOLATION
Preparing the Sample for DNA ISOLATION
• Bacteria & fungi • Viruses • Nucleated cells in suspension (blood & bone marrow aspirates)
• Plasma • Tissue samples
Chemistries in DNA ISOLATION
• Organic isolation method
• Inorganic isolation method
• Solid-phase isolation
other chemistries for DNA ISOLATION
• Proteolytic lysis of fixed materials
• Rapid extraction methods
• Isolation of mitochondrial DNA (mtDNA)
sample with biggest yield
plasmid dna (300 ug-1g)
sample with smallest yield
hair follicles (0.1-0.2 ug)
Can be lysed by high pH and detergents
Gram-negative bacteria
Some bacteria & fungi with tough cell walls. what method is used???
• Enzymatic digestion
• Mechanical method
• Chemical lysis
Commercial reagents designed for isolation of DNA in amplification procedures (PCR)
Yeast, filamentous fungi, & gram-positive bacteria:
(lysozyme)
Enzymatic digestion
grinding/vigorously mixing with glass beads
Mechanical method
detergent (1% sodium dodecyl sulfate) & strong base (0.2 M NaOH) in the presence of Tris base, ethylenediaminetetraacetic acid (EDTA), & glucose
Chemical lysis
Held within free viruses/ integrated into the host genome along with host DNA
Viral DNA
Cell-free specimens (plasma) will be used for viral detection
Viral DNA
May require concentration of viroids by centrifugation or other methods
Viral DNA
what are the Nucleated Cells in Suspension???
Blood & Bone Marrow Aspirates
Anticoagulants (EDTA/citrate) will be added
▪ Obtained from the blood plasma
WBCS
▪ Purified of RBCs & other components by either differential density-gradient centrifugation or differential lysis
▪ Differential osmotic fragility of RBCs & WBCs
WBCS
Small vesicles (30-100 nm in diameter), which form by invagination & budding from the inside of cellular endosome vesicles & are secreted by living cells
Exosomes
▪ Contain nucleic acid & can be collected by centrifugation ▪ Diagnostic & prognostic analyses purposes (liquid biopsy)
Exosomes
what are 3 type of tissue samples??
Fresh Tissue
Frozen tissue
fixed, embedded tissue
Dissociated by mincing the tissue
Fresh Tissue