TECHNIQUES IN MOLECULAR BIOLOGY

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TECHNIQUES IN MOLECULAR BIOLOGY

• CENTRIFUGATION- Separation of molecules/macromolec
  ules/organelles according to the size, shape,
  density gradient
• ELECTROPHORESIS- Separation of molecules/macromole
  cules according to charge
• MICROSCOPY- Structural examination of minute
  molecule/macromolecule/organelle

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CENTRIFUGATION

• MATERIALS OR PARTICLES IN A SOLUTION CAN BE
  SEPARATED BY A CENTRIFUGE THAT USES THE PRINCIPLE
  OF CENTRIFUGATION
• CLASSES
• -ANALYTICAL/PREPARATIVE
• -ULTRACENTRIFUGATION AND LOW SPEED
• -DIFFERENTIAL/ZONAL CENTRIFUGATION
• http//ntri.tamuk.edu/centrifuge/centrifugation.ht
  ml

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ANALYTICAL CENTRIFUGATION

• IS USED TO MEASURE THE SEDIMENTED PARTICLE
  PHYSICAL CHARACTERISTICS SUCH AS SEDIMENTATION
  COEFFICIENT AND MOLECULAR WEIGHT

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PREPARATIVE CENTRIFUGATION

• TO SEPARATE SPECIFIC PARTICLES THAT IS REUSABLE
• TYPES
• - RATE ZONAL
• - DIFFERENTIAL
• - ISOPYCNIC CENTRIFUGATION

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ULTRACENTRIFUGATION AND LOW SPEED

• DEPENDS ON SPEED
• ULTRACENTRIFUGATION - THE SPEED EXCEEDS 20,000
  RPM
• SUPER SPEED ULTRACENTRIFUGATION- THE SPEED IS
  BETWEEN 10,000 RPM-20,000 RPM
• LOW SPEED CENTRIFUGATION- THE SPEED IS BELOW
  10,000 RPM

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DIFFERENTIAL CENTRIFUGATION

• PARTICLES IN SAMPLE WILL SEPARATE INTO
  SUPERNATANT AND PELLET OR IN BOTH DEPENDING ON
  THEIR SIZE, SHAPE, DENSITY AND CENTRIFUGATION
  CONDITION
• THE PELLET CONTAINS ALL THE SEDIMENTED COMPONENT
  MIXTURE AND CAN CONTAIN MATERIALS THAT WAS NOT
  SEDIMENTED EARLIER

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DIFFERENTIAL CENTRIFUGATION

• SUPERNATANT CONTAINS MATERIALS THAT ARE NOT
  SEDIMENTED BUT CAN BE SEDIMENTED WHEN
  CENTRIFUGATION IS DONE AT A HIGHER SPEED

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DIFFERENTIAL CENTRIFUGATION
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ZONAL CENTRIFUGATION

• SAMPLE IS APPLIED ON TOP OF SUCROSE OR CESIUM
  CLORIDE SOLUTION
• PARTICLE CAN BE SEPARATED ACCORDING TO SIZE
  SHAPE (TIME-RATE ZONE) OR DENSITY (ISOPYCNIC)

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RATE-ZONAL CENTRIFUGATION
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ISOPYCNIC-ZONAL CENTRIFUGATION
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SEDIMENTATION COEFFICIENT

• WHEN CELL COMPONENTS ARE CENTRIFUFED THROUGH A
  GRADIENT SOLUTION, THEY WILL SEPARATE INTO THEIR
  OWN ZONE OR LINE/LAYER
• THE RATE WHEN THE COMPONENT SEPARATES IS CALLED
  AS SEDIMENTATION COEFFICIENT OR THE s VALUE
  (SVEDBERG UNIT )
• 1 S 1 X 10-13 SECONDS

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SEDIMENTATION COEFFICIENTVALUES

• PARTICLE OR SEDIMENTATION
• MOLECULE COEFFICIENT
• LYSOSOME 9400S
• TOBACCO MOSAIC VIRUS 198S
• RIBOSOME 80S
• RIBOSOMAL RNA MOLECULE 28S
• tRNA MOLECULE 4S
• HEMOGLOBIN MOLECULE 4.5S

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SPEED OF CENTRIFUGATION

• A PARTICLE THAT IS ROTATING WILL HAVE A PULLING
  FORCE IN A FORM OF MAGNITUDE TO SPEED FUNCTION AT
  DEFINED ANGLE (ROTATION SPEED) AND CENTRFUGATION
  RADIUS (THE DISTANCE BETWEEN THE SAMPLE CONTAINER
  AND THE ROTOR CENTRE)

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SPEED OF CENTRIFUGATION

• 2 WAYS OF EXPRESSING THE PULLING FORCE
• a) RELATIVE CENTRIFUGATIONAL FORCE-RCF (g)
• b) ROTATION PER MINUTE (rpm)

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RELATIVE CENTRIFUGATIONAL FORCE

• THE PULLING FORCE OF CENTRIFUGATION IS BASED ON
  OR RELATIVE TO THE STANDARD GRAVITATIONAL FORCE
• FOR EXAMPLE 500x g MEANS THAT THE PULLING FORCE
  IS 500 TIMES BIGGER THAN THE STANDARD
  GRAVITATIONAL FORCE

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RELATIVE CENTRIFUGATIONAL FORCE

• EQUATION
• R.C.F. 1.119 x 10 -5 (rpm2) r rpmrotation
  per minute
• rradius (in cm)
• UNIT g

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ELECTROPHORESIS

• THE MOVEMENT OF CHARGED PARTICLE IS INFLUENCED BY
  ELECTRICAL CURRENT
• ELECTROPHORESIS IS THE METHOD OF SEPARATING
  MACROMOLECULE SUCH AS NUCLEIC ACID AND PROTEIN
  ACCORDING TO SIZE, ELECTRICAL CHARGE AND PHYSICAL
  PROPERTIES SUCH AS DENSITY ETC
• SEPARATION IS AIDED BY A MATRIX SUCH AS
  POLIACRYLAMIDE OR AGAROSE

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ELECTROPHORESIS

• PRINCIPLE SEPARATION OF MACROMOLECULE DEPENDING
  ON TWO PROPERTIES WEIGHT AND CHARGE
• ELECTRICAL CURRENT FROM THE ELECTRODE WILL PUSH
  THE MOLECULE AND AT THE SAME TIME THE OTHER
  ELECTRODE WILL PUT IT
• MOLECULES WILL MOVE ALONG THE PORES THAT ARE
  FORMED BETWEEN THE INTER-WOVEN MATRIX THAT ACTS
  LIKE A SIEVE TO SEAPARATE THE MOLECULE ACCORDING
  TO THEIR SIZE

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ELECTROPHORESIS

• ELECTRICAL CURRENT WILL FORCE THE MACROMOLECULE
  TO MOVE ALONG THE PORES
• THE MACROMOLECULE MOVEMENT DEPENDS ON THE
  ELECTRICAL FIELD FORCE, THE MOLECULE SIZE AND
  SHAPE, THE SAMPLE RELATIVE HYDROPHOBIC PROPERTY,
  IONIC STRENGTH AND THE TEMPERATURE OF THE
  ELECTROPHORESIS BUFFER
• DYEING WILL AID THE VISUALISATION OF
  MACROMOLECULE IN THE FORM OF SEPARATED SERIES OF
  STRIPES

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PROTEIN ELECTROPHORESIS

• PROTEIN HAS A POSITIVE OR NEGATIVE NET CHARGE AS
  A RESULT OF THE COMBINATION OF CHARGED AMINO
  ACIDS CONTAINEDIN THEM
• THE MATRIX THAT IS USUALLY USED FOR PROTEIN
  SEPARATION IS POLIACRYLAMIDE
• TWO DIMENSIONAL GEL ELECTROPHORESIS- PROTEIN
  SEPARATION ACCORDING TO ISOELECTRICAL POINTS AND
  MOLECULAR WEIGHT

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2-D PROTEIN ELECTROPHORESIS

• FIRST STEP/DIMENSION
• PROTEIN SEPARATION ACCORDING TO ISOELECTRIC
  POINT (PROTEIN CONTAINS DIFFERENT POSITIVE AND
  NEGATIVE CHARGE RATIO)
• -ELECTROPHORESIS IS DONE ON THE GEL IN THE FORM
  OF TUBE PROTEIN WILL MOVE IN A SOLUTION WITH
  DIFFERENT pH GRADIENT

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2-D PROTEIN ELECTROPHORESIS

• FIRST STEP/DIMENSION
• -PROTEIN WILL STOP WHEN IT REACHES THE pH WHICH
  IS EQUAL TO ITS ISOELECTRIC POINT i.e WHEN THE
  PROTEIN DOES NOT HAVE A NET CHARGE.

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2-D PROTEIN ELECTROPHORESIS

• SECOND STEP/DIMENSION
• PROTEIN SEPARATION BY MOLECULAR WEIGHT
• ELECTROPHORESIS IS DONE IN AN ORTHOGONAL
  DIRECTION FROM
• THE FIRST STEP
• SODIUM DODECYL SULPHATE
• (SDS) IS ADDED

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2-D PROTEIN ELECTROPHORESIS
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1-D PROTEIN ELECTROPHORESIS

• PROTEIN IS SEPARATED BY ITS MOLECULAR WEIGHT ONLY
  
• THE TECHNIQUE IS ALSO KNOWN AS POLIACRYLAMIDE GEL
  ELECTROPHORESIS (PAGE) OR SDS-PAGE IF SDS IS
  PRESENT DURING SAMPLE PREPARATION
• SIMULATION OF 1-D ELECTROPHORESIS
• http//www.rit.edu/pac8612/electro/
• Electro_Sim.html

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SDS-PAGE

• TO SEPARATE PROTEIN WITH THE SIZE OF 5 - 2,000
  kDa
• PORES IN BETWEEN THE POLIACRYLAMIDE MATRIX CAN
  VARIES FROM 3-30
• THE PROTEIN SAMPLE IS IN THE FORM OF PRIMARY
  STRUCTURE (SAMPLE IS BOILED WITH SDS AND
  ?-MERCAPTOETHANOL PRIOR BEING LOADED ONTO GEL)

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SDS-PAGE

• PROTEIN IS STAINED USING COOMASIE BLUE OR SILVER
• NON-DIRECTIONAL STAINING CAN BE DONE
• -ANTIBODY BOUND WITH RADIOISOTOPE OR ENZYME,
  FLUORESENCE DYE

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SDS-PAGE

• SDS FUNCTION
• NEGATIVELY CHARGED DETERGENT THAT
• BINDS TO THE
• HYDROPHOBIC REGION
• OF THE PROTEIN
• MOLECULE AS A
• RESULT THE PROTEIN BECOMES A LONG POLIPEPTIDE
  CHAIN AND FREE FROM OTHER PROTEINS AND LIPIDS

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SDS-PAGE

• ?-MERCAPTOETHANOL FUNCTION TO BREAK DISULPHIDE
  BONDS SO THAT PROTEIN SUBUNIT CAN BE ANALYSED

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NUCLEIC ACID ELECTROPHORESIS

• AGAROSE OR POLIACRYLAMIDE IS THE MATRIX USUALLY
  USED TO SEPARATE NUCLEIC ACID IN A TECHNIQUE
  KNOWN AS AGAROSE GEL ELECTROPHORESIS
• SAMPLE CONTAINING DNA IS LOADED INTO WELLS
  LOCATED NEAR TO THE NEGATIVELY CHARGED ELECTRODE
• DNA THAT IS NEGATIVELY CHARGED WILL BE ATTRACTED
  TO THE POSITIVE ELECTRODE

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NUCLEIC ACID ELECTROPHORESIS

• DNA WITH A BIGGER SIZE WILL MOVE SLOWER THAN THE
  SMALLER SIZE WHICH MOVE FASTER
• STAINING IS DONE USING ETHIDIUM BROMIDE (EtBr)
  THAT ENABLES THE VISUALISATION OF NUCLEIC ACID
  EtBr IS INSERTED BETWEEN THE BASES ON THE NUCLEIC
  ACID
• EtBr IS ORANGE IN COLOUR WHEN LIT-UP BY
  ULTRA-VIOLET LIGHT

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NUCLEIC ACID ELECTROPHORESIS
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MICROSCOPY

• ONE OF THE EARLIEST TECHNIQUE TO STUDY
  MACROMOLECULE
• PRINCIPLE TO ENLARGE SMALL IMAGES
• TYPES OF MICROSCOPY ACCORDING TO THE SIZE OF
  IMAGE ENLARGEMENT
• - LIGHT MICROSCOPE (300nm-2mm)
• - ELECTRON MICROSCOPE
• (0.15nm-100?m)

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LIGHT MICROSCOPE

• IMAGE ENLARGEMENT PRINCIPLE
• LIGHT FROM BELOW OF THE MICROCOPE GOES THROUGH
• THE CONDENSOR TO FOCUS THE
• LIGHT TO THE SPECIMEN.
• LIGHT FROM THE SPECIMEN IS
• RECOLLECTED BY THE OBJECTIVE LENSE TO FORM AN
  IMAGE

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LIGHT MICROSCOPE

• TYPES OF LIGHT MICROSCOPE
• BRIGHT-FIELD MICROSCOPE DARK-FIELD
  MICROSCOPE
• PHASE-CONTRAST MICROSCOPE
• FLUORESENCE MICROSCOPE (UV)
• (FLUORESCIN/RHODAMIN)

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ELECTRON MICROSCOPE

• PRINCIPLE
• -ELECTRON IS USED (NOT LIGHT) TO ENLARGE IMAGE
• -SPECIMEN MUST UNDERGO A SERIES OF PREPARATION
  PROCESSES SUCH AS COATING WITH THIN LAYER OF GOLD
  TO ALLOW EMITTED ELECTRON TO COLLIDE TO AND THEN
  RECOLLECTED TO FORM IMAGE ON THE SCREEN

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ELECTRON MICROSCOPE

• TYPES
• 1) TRANSMISSION ELECTRON MICROSCOPE
• -ELECTRON GOES THROUGH THE SPECIMEN AND IMAGE IS
  RECOLLECTED ON A FLUORECENS SCREEN
• -THE INNER STRUCTURE OF THE SPECIMEN CAN BE SEEN

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ELECTRON MICROSCOPE

• TYPES
• 2) SCANNING ELECTRON MICROSCOPE
• -ELECTRON IS FOCUSSED TO THE SPECIMEN AND THEN
  REEMITTED (SCANNED) TO THE DETECTOR AND IMAGE IS
  SEND TO THE SCREEN FOR VIEWING
• -THE OUTER STRUCTURE CAN BE SEEN

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ELECTRON MICROSCOPE
SCANNING ELECTRON MICROSCOPE
MOSQUITO IMAGES BY SCANNING ELECTRON MICROSCOPE
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OTHER TECHNIQUES

• CHROMATOGRAPHY
• -PAPER PROTEIN SEPARATION BY USING FILTER
  PAPER AS THE MATRIX
• -ION-EXCHANGE
• -GEL FILTRATION
• -AFFINITY
• -HIGH PRESSURE LIQUID CHROMATOGRAPHY (HPLC)

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OTHER TECHNIQUES

• RADIOISOTOPES FOR MOLECULE TAGGING 32P, 131I,
  35S, 14C, 45Ca, 3H
• - RIA, PULSE-CHASE EXPERIMENT,
  AUTORADIOGRAPHY
• ANTIBODY (MONOCLONE/POLYCLONE) FOR TAGGING
  MOLECULE EIA, IF, ELISA
• X-RAY DIFFRACTION ANALYSIS PROTEIN STRUCTURE
  DETERMINATION
• DNA RECOMBINANT TECNOLOGY