cDNA synthesis

PCR (Polymerase Chain Reaction)

Designing primers for PCR

Chemical synthesis of genes

Shotgun experiment

Gene library

A Recombinant DNA molecule is produced by joining together two or more DNA segments usually originating from different organisms.

GENE LIBRARY

A Gene library is a collection of cloning vectors which contains genes of interest, representing entire set of foreign DNA.

TWO TYPES:COMPLEMENTARY DNA (cDNA) LIBRARYGENOMIC LIBRARY

WHAT IS cDNA library?It is a combination of cloned cDNA fragments inserted into a collection of host cells, which together constitute some portion of the transcriptome of the organism.

cDNA library is a more specialized and exclusive DNA library .

5’ 3’mRNA

CAP

POLY A TAILPROCESSING

SPLICING

INTRON DELETED

MATURE mRNA

cDNA library preparation

cDNA is created from a mature mRNA from eukaryotic cell with the use of an enzyme known as reverse transcriptase.

In eukaryotes, poly (A) tail distinguishes mRNA from tRNA and rRNA and can therefore used as a primer site for reverse transcription.

c D N A

CONSTRUCTION

cDNA LIBRARY

GENOMIC LIBRARY

Collection of plasmid clones containing recombinant DNA molecules so that the sum total of DNA inserts in this collection, ideally represents the entire genome of the concerned organism.

Preparation:- DNA Partial Digestion Gel Electrophoresis Inserted into Vector.

This constitutes the shotgun approach to gene cloning.

SHOTGUN METHOD- In this method, 1-2µm tungsten or gold particles coated with DNA to be used for transformation, are accelerated to velocities which enable their entry into plant cell/nuclei.

Components Pressurised He gasGas acceleration tubeRupture discStopping screenMicro-carrier

DNA AMPLIFICATION BY PCR

PCR is a method for producing an extremely large number of copies of a specific DNA sequence from DNA mixture without having to clone it, a process called amplification.

Majors steps are:DenaturationAnnealingPrimer extension

EVENTS IN PCR

PCR PRIMERS

PCR uses a primer that pair to the region of DNA molecule located just outside the sequence to be amplified.

Length of primer

Self complementary regions

GC content

Melting temperature

CHEMICAL SYNTHESIS OF GENEGene synthesis refers to chemical synthesis of a strand of DNA, base by base.An automated machine which synthesizes the desired gene, chemically from the free nucleotide is known as the GENE MACHINE.The gene machine contains the 10 containers, synthesizer column, valves, spectrophotometer, programmed computer.

PHOSPHOTRIESTER OR PHOSPHORAMIDITE APPROACH

To prevent the side reaction the amino group of the nitrogenous base is protected. This chemically protected nucleotide is called the phosphoramidite. Hence this method is called Phosphoramidite Method.

Detritylation ProcessThe First Nucleotide is linked with the Spacer Molecule, which when pumped into synthesizer column, binds with the glass beads, to form the ‘starting complex’, which acts as solid support for chemical synthesis followed by addition of acetonitrile. TCA pumped to release DMT from nucleotide, this is called detritylation process.

As programmed, the second nucleotide is pumped into synthesizer column, and simultaneously TETRAZOLE is pumped into it. The Tetrazole activates the Phosphoramidite.

This is called Activation and Coupling!

Capping:Acetic anhydride and Dimethyl amino pyridine pumped into the column which in turn prevents the further reactions.

Oxidation:Iodine mixture is pumped into column.

REFERENCESBIOTECHNOLOGY BY B D SINGHGENETIC ENGINEERING BY SANDHYA MITRA

PRESENTED BY JYOTI DEVENDRA ADALA MSc PART II

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