Extraction of DNA Extraction of DNA
/RNA /RNA /RNA /RNA
Professor Dr. Md. Akram Hossain Professor Dr. Md. Akram Hossain
MMC, 2012 MMC, 2012
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PURPOSE PURPOSE
INTELLECTUAL INTELLECTUAL
TO UNDERSTAND TO UNDERSTAND GENE EXPRESSION GENE EXPRESSION
TO UNDERSTAND TO UNDERSTAND
GENOME STRUCTURE GENOME STRUCTURE
APPLIED APPLIED
TO ADVANCE TO ADVANCE MEDICAL
MEDICAL
KNOWLEDGE
KNOWLEDGE--GENE GENE THERAPY &
THERAPY &
GENOME STRUCTURE GENOME STRUCTURE
TO UNDERSTAND TO UNDERSTAND THE PROCESSES OF THE PROCESSES OF MOLECULAR
MOLECULAR EVOLUTION EVOLUTION
THERAPY &
THERAPY &
DIAGNOSTICS DIAGNOSTICS
TO PRODUCE TO PRODUCE
COMMERCIALLY COMMERCIALLY USEFUL PRODUCTS USEFUL PRODUCTS
PRINCIPAL STEPS IN PRINCIPAL STEPS IN
CLONING A GENE CLONING A GENE
MAKE A GENE LIBRARYMAKE A GENE LIBRARY
OBTAIN A SOURCE OF DNA FRAGMENTSOBTAIN A SOURCE OF DNA FRAGMENTS
PREPARE A SUITABLE VECTORPREPARE A SUITABLE VECTOR
JOIN FRAGMENTS TO VECTOR JOIN FRAGMENTS TO VECTOR -- LIGATIONLIGATION
SCREENING THE LIBRARYSCREENING THE LIBRARY-- CLONE SELECTIONCLONE SELECTION
NUCLEIC ACID HYBRIDISATIONNUCLEIC ACID HYBRIDISATION
EXPRESSION SCREENINGEXPRESSION SCREENING
COMPLEMENTATIONCOMPLEMENTATION
CHARACTERISATION OF CLONED GENECHARACTERISATION OF CLONED GENE
RESTRICTION MAPPINGRESTRICTION MAPPING
SEQUENCESEQUENCE
THIS SLIDE SUMMARISES THE MAIN TOPICS I WILL COVER
DNA FRAGMENTS DNA FRAGMENTS
GENOMIC DNA GENOMIC DNA
RESTRICTION FRAGMENTS RESTRICTION FRAGMENTS
MECHANICALLY SHEARED MECHANICALLY SHEARED
cDNA cDNA
cDNA cDNA
REVERSE TRANSCRIPTION OF mRNA REVERSE TRANSCRIPTION OF mRNA
CHEMICAL SYNTHESIS CHEMICAL SYNTHESIS
POLYMERASE CHAIN REACTION POLYMERASE CHAIN REACTION
EXTRACTION OF GENOMIC EXTRACTION OF GENOMIC
DNA DNA
1.
1.
OBTAIN TISSUE OBTAIN TISSUE – – FROM RELEVANT FROM RELEVANT
SOURCE eg ORGAN FROM ORGANISM OR SOURCE eg ORGAN FROM ORGANISM OR CELLS FROM TISSUE CULTURE
CELLS FROM TISSUE CULTURE
2.
2.
IF NECESSARY IF NECESSARY DISPERSE IF NECESSARY IF NECESSARY DISPERSE DISPERSE DISPERSE THE CELLS THE CELLS THE CELLS THE CELLS
3.
3.
GENTLY GENTLY LYSE LYSE THE CELLS IN AN THE CELLS IN AN IONIC IONIC DETERGENT
DETERGENT SUCH AS SUCH AS SARKOSYL SARKOSYL OR OR SDS SDS IN THE PRESENCE OF
IN THE PRESENCE OF EDTA EDTA – – WHAT IS WHAT IS THE FUNCTION OF THE DETERGENT THE FUNCTION OF THE DETERGENT AND THE EDTA?
AND THE EDTA?
EXTRACTION OF GENOMIC EXTRACTION OF GENOMIC
DNA DNA
4. REMOVE PROTEIN USING 4. REMOVE PROTEIN USING
PROTEINASE K
PROTEINASE K AND AND PHENOL PHENOL
Aqueous = DNA KEEP THIS
Interface = PROTEIN (DISCARD) Phenol = LIPIDS (DISCARD)
EXTRACTION OF EXTRACTION OF
GENOMIC DNA GENOMIC DNA
5.
5.
REMOVE REMOVE AQUEOUS PHASE AQUEOUS PHASE TO A FRESH TO A FRESH TUBE AND ADD
TUBE AND ADD SALT SALT (SODIUM (SODIUM ACETATE TO 0.3 M)
ACETATE TO 0.3 M)
6.
6.
ADD 2.5 VOLUMES ADD 2.5 VOLUMES – –20 20
OOC C ETHANOL ETHANOL – – CAUSING DNA TO
CAUSING DNA TO PRECIPITATE PRECIPITATE CAUSING DNA TO
CAUSING DNA TO PRECIPITATE PRECIPITATE
7.
7.
HARVEST DNA BY CENTRIFUGATION HARVEST DNA BY CENTRIFUGATION
8.
8.
WASH THE PRECIPITATE WITH WASH THE PRECIPITATE WITH 70% 70%
ETHANOL
ETHANOL – – WHY? WHY?
9.
9.
DRY AND DISSOLVE IN AQUEOUS DRY AND DISSOLVE IN AQUEOUS
BUFFER SUCH AS 10 mM TRIS pH 7.5, 1 BUFFER SUCH AS 10 mM TRIS pH 7.5, 1 mM EDTA (TE) AND MEASURE
mM EDTA (TE) AND MEASURE OD OD 260/280
260/280
Theory of Electrophoresis
The movement of a charged molecule subjected to an electric field is represented by the following equation:
V = Eq V = f
f
V: the velocity of the molecule E: the electric field in volts/cm
q: the net charge on the molecule
f: frictional coefficient, which depend on the mass and shape of the molecule
DNA Structure Influences DNA Structure Influences
Migration Through Gels
Migration Through Gels
Gel Electrophoresis of DNA
Wells for sample loading
Direction for DNA
migration Anode (+)
Agarose slab gel submerged in buffer
Cathode (-)
Agarose is a polysaccharide derived from seaweed, which forms a solid gel when dissolved in aqueous solution. When an electric field is applied to an agarose gel in the presence of a buffer solution which will conduct electricity, DNA fragments move through the gel towards the positive electrode (DNA is highly negatively charged) at a rate which is dependent on its size and shape.
Gel Electrophoresis of DNA
For linear DNA molecules, they have uniform shape and charge to mass ratio. The shape and charge to mass ratio. The electrophoretic mobility of the DNA molecule is influenced primarily by the molecular size:
The larger molecules are retarded by the
molecular sieving effect of the gel, and the
small molecules have greater mobility.
Gel Electrophoresis of DNA
• The DNA can be stained by the inclusion of ethidium bromide in the gel, or by soaking the gel in a solution of ethidium bromide after electrophoresis. The DNA shows up as an orange band on illumination by UV light. Alternatively, methylene blue can be used to light. Alternatively, methylene blue can be used to stain DNA.
• Gels composed of polyacrylamide can separate DNA molecules that differ in length by only one nucleotide and are used to determine the base sequence of DNA.
Agarose gels are used to separate DNA fragments that have larger size differences.
Digestion of DNA by Restriction Enzymes Digestion of DNA by Restriction Enzymes
GAATTC CTTAAG
EcoRI
Before
Before thethe electrophoresis,electrophoresis, DNADNA isis digesteddigested byby restriction
restriction enzymesenzymes intointo smallsmall fragmentsfragments ofof DNADNA..
G AATTC CTTAA G
EcoRI
• In order to detect specific sequences, DNA is usually transferred to a solid support, such as a sheet of
nitrocellulose or nylon paper.
• The paper is treated with an alkaline solution to
Procedures of DNA Fingerprinting
• The paper is treated with an alkaline solution to denature DNA, that is, separate the two strands of each double helix.
• The single-stranded DNA can be hybridized with a probe, and the regions on the nitrocellulose blot
containing DNA that base-pairs with the probe can be identified.
Pulsed Field Gel Electrophoresis Pulsed Field Gel Electrophoresis
Ordinary gel has a constant current in one Ordinary gel has a constant current in one direction
direction
Only small fragments can enter gel and be Only small fragments can enter gel and be separated
separated separated separated
PFGE PFGE -- direction of current changes regularly direction of current changes regularly (pulsed)
(pulsed)
large fragments twist and move slowly through large fragments twist and move slowly through gel
gel
Pulsed Field Gel Electrophoresis
Molten Agarose Cultured bacteria
Incubate with Proteinase K
Trapped HMW DNA
Embedded Cells DNA extraction &
wash to remove cell debris
Pulsed Field Gel Electrophoresis
Digest with Rare-cutting restriction nuclease
+ +
Period Switching (pulsing) between electrode pairs
Net migration
- -
Applications of Electrophoresis Applications of Electrophoresis
Detection of unknown DNA Detection of unknown DNA – – by Southern by Southern blotting
blotting
Detection of unknown RNA Detection of unknown RNA – – Northern Northern blotting
blotting blotting blotting
Detection of proteins Detection of proteins – – by western blotting by western blotting
