MOLECULARMETHODS

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ELECTROPHORESIS

Gel electrophoresis is a method that separates biomolecules-either nucleic acids or proteins-on the basis of size, electric charge, and other physical properties.

The term electrophoresis describes the migration of charged particle under the influence of an electric field.

Electro refers to the energy of electricity.

Phoresis, from the Greek verb phoros, means "to carry across."

Many important biological molecules such as amino acids, peptides, proteins, nucleotides, and nucleic acids, posses ionisable groups and, therefore, at any given pH, exist in solution as electically charged species either as cations (+) or anions (-).

Depending on the nature of the net charge, the charged particles will migrate either to the cathode or to the anode.

COMMONLY USED ELECTROPHORETIC TECHNIQUE:

Agarose gel electrophoresis- Separation of low molecular weight DNA.

NativePAGE(PolyAcrylamide Gel Electrophoresis)- Separation of high molecular weight DNA

SDS-PAGE (Sodium Dodecyl Sulfate PAGE)- Separation of proteins.

HORIZONTAL AND VERTICAL GEL ELETROPHORESIS CHAMBER

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BLOTTING

Blotting is the process by which the separated biomolecules are immobilized onto a solid support.

There are 3 types of blotting:1. Southern blotting.2. Northern blotting.3. Western blotting.

Southern blotting:

Southern blotting was named after Edward M. Southern who developed this procedure at Edinburgh University in the 1970s.

DNA molecules are transferred from an agarose gel onto a membrane.

Northern blotting:

Developed by James Alwine and George Stark.

The Northern blot is a technique used to study gene expression.

RNA molecules are immobilized by this technique.

Western blotting:

The technique was developed by W. Neal Burnette.

Used to immobilize proteins in a given sample of tissue homogenate or extract.

Widely used confirmatory test for HIV-AIDS.

BLOTTING SET-UP

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HYBRIDIZATION

Hybridization is the process of combining complementary, single-stranded nucleic acid to a single nucleotide strand.

DNA strand will bind to their complement under normal conditions, so two perfectly complementary strands will bind to each other readily.

PROCEDURE:

The double-stranded DNA helix is heated in solution.

Due to external conditions, the hydrogen-bonded base pairing becomes thermodynamically unfavorable and the complementary strands separate.

Denatured DNA is then mixed with denatured probe.

There are two types of probes:

Radioactive probes. Non-radioactive probes

The combined sets are then cooled slowly to allow the DNA to reanneal and form a new "hybridized" DNA molecule.

Hybridization analysis can now be used for tracing defective genes in human DNA

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A – Denaturation B – Renaturation C - RNA probe D - Hybridization with the probe

INSITU HYBRIDIZATION The technology can provide rapid, quantitative, multiparameter analyses on

single living (or dead) cells based on the measurement of visible and fluorescent light emission.

This technique is based on the measure of fluorescence intensity produced by fluorescent-labled antibodies and ligands that bind specific cell-associated molecules.

FLUORESCENT INSITU HYBRIDIZATION:

Fluorescence in situ hybridization (FISH) uses fluorescent molecules to vividly paint genes or

chromosomes.

This technique is particularly useful for gene mapping and for identifying chromosomal abnormalities.

FISH involves the preparation of short sequences of single-stranded DNA, called probes, which are complementary to the DNA sequences.

These probes hybridize, or bind, to the complementary DNA.

Since they are labeled with fluorescent tags, the location of DNA of interest could be detected.

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FISH can also be performed on nondividing cells, making it a highly versatile procedure.

APPLICATIONS:

Aneuploid Screening.

Detection of microdeletion.

To detect specific chromosome rearrangements seen in certain cancers.

FLOW CYTOMETRYFlow cytometry is a technique for counting, examining and sorting microscopic particles suspended in a stream of fluid.

FACS [Fluorescent Activated Cell Sorter]:

A method for sorting a suspension of biological cells into two or more containers, one cell at a time, based upon specific light scattering and fluorescent characteristics of each cell.

APPLICATIONS:

The applications of flow cytometry include:

1. Can be used to measure the amount of DNA in cells.

2. Flow cytometry is a widely used method for characterizing and separating individual cells.

3. Lymphocyte subset enumeration for Immunodeficiency disease.

4. Analysis of DNA Ploidy, the Cell Cycle, and Cell Death.

5. Measurement of the efficacy of cancer chemotherapy.

6. Reticulocyte enumeration.

7. Detection of minimal residual disease.

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FLUORESCENCE ACTIVATED CELL SORTER

MICROARRAY DNA microarray (gene chip, DNA chip, or gene array) is a collection of

microscopic DNA spots attached to a solid surface, such as glass, plastic or silicone chip forming an array for the purpose of expression profiling.

Works on the principle of nucleic acid hybridization.

APPLICATIONS:

Expression level for thousands of genes can be monitored simultaneously.

Identification of gene mutations.

MICROARRAY

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EXPRESSION PROFILING OF NORMAL AND CANCER CELL

MICROARRAY SLIDE

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