Techniques in molecular biology
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TECHNIQUES IN MOLECULAR BIOLOGY. CENTRIFUGATION- Separation of molecules/macromolecules/organelles according to the size, shape, density & gradient ELECTROPHORESIS- Separation of molecules/macromolecules according to charge

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TECHNIQUES IN MOLECULAR BIOLOGY

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TECHNIQUES IN MOLECULAR BIOLOGY

  • CENTRIFUGATION- Separation of molecules/macromolecules/organelles according to the size, shape, density & gradient

  • ELECTROPHORESIS- Separation of molecules/macromolecules according to charge

  • MICROSCOPY- Structural examination of minute molecule/macromolecule/organelle


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.html


ANALYTICAL CENTRIFUGATION

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


PREPARATIVE CENTRIFUGATION

  • TO SEPARATE SPECIFIC PARTICLES THAT IS REUSABLE

  • TYPES:

    - RATE ZONAL

    - DIFFERENTIAL

    - ISOPYCNIC CENTRIFUGATION


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


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


DIFFERENTIAL CENTRIFUGATION

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


DIFFERENTIAL CENTRIFUGATION


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)


RATE-ZONAL CENTRIFUGATION


ISOPYCNIC-ZONAL CENTRIFUGATION


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


SEDIMENTATION COEFFICIENTVALUES

PARTICLE OR SEDIMENTATION

MOLECULE COEFFICIENT

LYSOSOME9400S

TOBACCO MOSAIC VIRUS198S

RIBOSOME80S

RIBOSOMAL RNA MOLECULE 28S

tRNA MOLECULE4S

HEMOGLOBIN MOLECULE 4.5S


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)


SPEED OF CENTRIFUGATION

  • 2 WAYS OF EXPRESSING THE PULLING FORCE:

    a) RELATIVE CENTRIFUGATIONAL FORCE-RCF (g)

    b) ROTATION PER MINUTE (rpm)


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


RELATIVE CENTRIFUGATIONAL FORCE

  • EQUATION

    R.C.F. = 1.119 x 10 -5 (rpm2) r rpm=rotation per minute

    r=radius (in cm)

    UNIT g


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


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


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


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


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


+BASIC

-ACIDIC

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.


+

-

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


2-D PROTEIN ELECTROPHORESIS


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 PRESENTDURINGSAMPLE PREPARATION

  • SIMULATION OF 1-D ELECTROPHORESIS

    http://www.rit.edu/~pac8612/electro/

    Electro_Sim.html


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)


SDS-PAGE

  • PROTEIN IS STAINED USING COOMASIE BLUE OR SILVER

  • NON-DIRECTIONAL STAINING CAN BE DONE:

    -ANTIBODY BOUND WITH RADIOISOTOPE OR ENZYME, FLUORESENCE DYE


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


SDS-PAGE

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


NUCLEIC ACID ELECTROPHORESIS

  • AGAROSE OR POLIACRYLAMIDE IS THEMATRIX 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


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


NUCLEIC ACID ELECTROPHORESIS


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)


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


LIGHT MICROSCOPE

  • TYPES OF LIGHT MICROSCOPE :

    BRIGHT-FIELD MICROSCOPE DARK-FIELD MICROSCOPE

    PHASE-CONTRAST MICROSCOPE

    FLUORESENCE MICROSCOPE (UV)

    (FLUORESCIN/RHODAMIN)


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


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


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


ELECTRON MICROSCOPE

SCANNING ELECTRON

MICROSCOPE

MOSQUITO IMAGES

BY SCANNING ELECTRON

MICROSCOPE


OTHER TECHNIQUES

  • CHROMATOGRAPHY

    -PAPER: PROTEIN SEPARATION BY USING FILTER PAPER AS THE MATRIX

    -ION-EXCHANGE

    -GEL FILTRATION

    -AFFINITY

    -HIGH PRESSURE LIQUID CHROMATOGRAPHY (HPLC)


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


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