DNA Extraction Methods
- Organic - uses organic chemicals, phenol, chloroform.
- Lysis with NaOH , Acidification with acetic acid and salt, extract with phenol or chloroform, DNA precipitate with ethanol
- Inorganic - uses inorganic chemicals, detergents, EDTA, acetic acid, salt (salting out, spooling)
-lysis with TRIS-edta-sds, precipitate with sodium acetate, DNA precipitate with isopropanol - Solid phase - DNA is immobilized on a solid support, beads or columns
-lysis with supplied reagents, acidification with reagents, wash DNA with buffer, and elute DNA - Solid-phase isolation media include silica spin columns and beads
- Nucleic acid binding to silica beads
Isolation of Mitochondrial DNA
- Isolate mitochondria by centrifugation
- Isolate total DNA
Methods to Assess DNA
*Gel electrophoresis with known standards
* Fluorescent dyes used to visualize the sample
- Spectrophotometry
- Nucleic acid absorbs light at 260nm
- Protein absorbs light at 280nm - purity
- Fluorometry
- Measures fluorescent related to DNA concentration using DNA-specific fluorescent dyes (Hoechst dye)
Nucleic Extraction Methods: RNA
- Lab preparation: designated RNase-free area
- Specimen collection:
- Bone marrow & peripheral blood – Acid citrate dextrose (ACD – yellow cap), liquid K3EDTA (lavender/pink cap)
- Tissue – fresh in saline, frozen
solation of mRNA (polyA-RNA)
*RNA extraction methods include organic and solid-phase methods
Methods to Assess RNA
- Gel electrophoresis (total RNA; quality)
- Spectrophotometry
- Fluorometry
- RiboGreen
Electrophoresis
- molecules will collect in gel
or form a band according to their speed of migration - The concentration of gel/buffer will affect the resolution (separation by size) of fragments
Pulsed-field gel electrophoresis
* large DNA molecules are separated by pulsed-field gel electrophoresis
(PFGE) systems
* Pulses of current applied in alternating dimensions
Polyacrylamide Gel Electrophoresis (PAGE)
* Polyacrylamide gels are chemically cross-linked gels by polymerization of acrylamide with a crosslinking agent & polymerization catalyst
* Used for very small DNA fragments, single-stranded DNA, RNA & proteins
Capillary Electrophoresis
* Separates solutes by charge/mass ratio
*Capillary gel electrophoresis is used to separate nucleic acids
Electrophoresis Buffers
- Carry current and protect samples during electrophoresis
- E.g. Tris-borate EDTA (TBE), Tris-acetate EDTA (TAE), Tris-phosphate EDTA (TPE) used most often for DNA
- E.g.10-millimolar sodium phosphate or MOPS buffer used for RNA
- Buffer additives can be used to modify sample molecules
- Formamide, urea (denaturing agents)
Visualization of Separated Bands
- Detect bands by staining during or after electrophoresis
- Ethidium bromide—for double-stranded DNA
- SYBR green or SYBR gold—for single- or double-stranded DNA or for RNA
- Silver stain—more sensitive for single- or double-stranded DNA or for RNA and proteins
Restriction Enzymes
- Recognize & cut specific sequences in DNA
Type 1
Methylation/cleavage (3 subunits
Type 2
Cleavage at specific recognition sites via:
* Sticky ends must match (be complementary) for optimal re-ligation
* Sticky ends can be converted to blunt ends with nuclease or polymerase
* Blunt ends can be re-ligated with less efficiency than sticky ends
* Blunt ends can be converted to sticky ends by ligating to synthetic adaptors
Type 3
Methylation/cleavage (2 subunits)
Restriction Enzyme Mapping
- Digest DNA with a restriction enzyme
- Resolve the fragments by gel electrophoresis
- The number of bands indicates the number of restriction sites
- The size of the bands indicates the distance between restriction sites
Southern Blot
- Detect for specific targets in the genomic DNA
1. Cleave DNA -
2. Gel electrophoresis – separates DNA based on size and charge
3. Gel is transferred to a solid support
4. DNA fragments are exposed to a labelled probe complementary to the gene of interest
5. The signal of the probe is detected to indicate presence of absence of the sequence of interest
target is DNA and probe is NA
Blotting Transfer
- Transfer of cut DNA from the gel to the membrane can be performed in three ways:
1. Capillary transfer – capillary movement of buffer from the soaked to dry paper
2. Electrophoretic transfer – uses electric current
3. Vacuum transfer – uses suction & buffer
Probes
Once the DNA is transferred to the membrane, the probe determines
what region is seen
- Probes are specific (complementary) to target gene
- Probes can be DNA, RNA, or protein
*Nonradioactive (digoxygenin, biotin, fluorescent) - Probes have covalently attached signal molecules
- Radioactive (32P, 33P, 35S)
Hybridization Conditions
the temperature at which 50% of a nucleic acid is hybridized to its complementary strand
MELTING TEMPERATURE
(Tm)
Stringency
Describes the conditions under which hybridization takes place
* Formamide concentration increases stringency
* Low salt increases stringency
* Heat increases stringency
Detection of Bound Probe
- Radioactive or chemiluminescent detection requires exposure of the
blot to autoradiography film - Chromogenic detection occurs directly on the blot
Northern Blot
- No restriction digestion required
- RNA is blotted to membranes
- Probes are labeled antisense RNA or DNA
- Internal/loading controls are required
target is RNA and probe is NA
Western Blot
- Proteins
- Serum, cell lysate, or protein extract is separated on SDS polyacrylamide gels (SDS-PAGE) or isoelectric focusing gels (IEF)
- Proteins are blotted to membranes by capillary or electrophoretic transfer
- Probes are specific binding proteins, polyclonal antibodies, or monoclonal antibodies
target and probe are proteins
Comparative Genomic Hybridization (CGH)
- CGH arrays are reverse dot blots.
- Test and reference DNA are labeled by nucleotides covalently attached to fluorescent dyes
- Test and reference DNA are hybridized to the probes on a chip
