TECHNIQUES IN MOLECULAR BIOLOGY

GENETIC ENGINEERING

TOOLS OF MOLECULAR BIOLOGY

POLYMERASE CHAIN REACTION

Sequence to be copied is heated Primers are added and bind to ends of

single strands DNA polymerase uses free nucleotides to create complementary strands Doubles number of copies of DNA

GEL ELECTROPHORESIS
DNA is placed at one end of a gel A current is applied to the gel DNA molecules are negatively charged and

move toward positive end of gel

Smaller molecules move faster than larger

ones

DNA SEQUENCING
Developed by Frederick

Sanger
Reaction mixture:
Copies of DNA to be

sequenced
Primer DNA polymerase Standard nucleotides Modified nucleotides

DNA PROBE HYBRIDIZATION AND SOUTHERN BLOT

PROTEIN ANALYSIS

WESTERN BLOT

ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)

LIQUID CHROMATOGRAPHY

LIQUID COLUMN CHROMATOGRAPHY

A sample mixture is passed through a

column packed with solid particles which may or may not be coated with another liquid.
With the proper solvents, packing

conditions, some components in the sample will travel the column more slowly than others resulting in the desired separation.

 The 4 basic liquid chromatography modes are named according to the

FOUR BASIC LIQUID CHROMATOGRAPHY

mechanism involved:

1.

Liquid/Solid Chromatography (adsorption chromatography) A. B. Normal Phase LSC Reverse Phase LSC

2.

Liquid/Liquid Chromatography (partition chromatography)

A.
B. 3. 4.

Normal Phase LLC

Reverse Phase LLC

Ion Exchange Chromatography Gel Permeation Chromatography (exclusion chromatography)

TYPES OF CHROMATOGRAPHY
MOBILE PHASE LIQUID

FORMAT

Liquid-Liquid Chromatography (Partition)

Liquid-Solid Chromatography (Adsorption)

STATIONARY PHASE

Liquid

Solid

Normal Phase Mobile Phase Nonpolar Stationary phase Polar

Reverse Phase Mobile Phase Polar Stationary phase Nonpolar

Normal Phase

Reverse Phase

ION-EXCHANGE CHROMATOGRAPHY

GEL PERMEATION CHROMATOGRAPHY

MASS SPECTROMETRY

MASS SPECTROMETRY

analytical tool used for measuring

the molecular weight of a sample

for large samples such as

biomolecules, molecular weight can be measured within an accuracy of 0.01% of the total molecular weight of the sample

Structural information can be

generated using certain types of mass spectrometers, usually tandem mass spectrometers
achieved by fragmenting the sample and

analysing the products generated

useful for the structural elucidation of organic

compounds, for peptide or oligonucleotide sequencing, and for monitoring the existence of previously characterised compounds in complex mixtures

BASIC PRINCIPLES
An object that is moving and subjected to a

sideways force is deflected out of its original path

Amount of deflection depends on the mass of the object force and the curved path of deflection, mass can be calculated

Given the speed of the object, the size of the

STEPS IN MASS SPECTROMETRY

1. Ion source ionizes molecule of

interest
2.Mass analyzer differentiates the ions

according to their m/z

3.Detector measures the ion beam

current

IONIZATION METHOD
depends on:

type of sample under investigation the mass spectrometer available

MALDI TOF MS

SELDI-TOF MS