This document discusses various techniques used in recombinant DNA technology including cDNA synthesis, PCR, primer design, chemical gene synthesis, gene libraries, and the shotgun method. It provides details on creating cDNA and genomic libraries, which are collections of cloned DNA fragments that represent all or part of the transcriptome or genome, respectively, of an organism. The document also describes the chemical phosphoramidite method for synthesizing genes base by base in an automated gene machine and the steps involved in the PCR process such as denaturation, annealing, and primer extension to amplify DNA.
sem4-cdna sythesis,pcr,designing primers for pcr, synthesis of genes, shotgun experiment and gene library.
1. 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.
2. 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) LIBRARY
GENOMIC LIBRARY
3. 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 TAIL
PROCESSING
SPLICING
INTRON DELETED
MATURE mRNA
4. 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.
7. 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.
8. 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 gas
Gas acceleration tube
Rupture disc
Stopping screen
Micro-carrier
9. 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:
Denaturation
Annealing
Primer extension
11. 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
12. CHEMICAL SYNTHESIS OF GENE
Gene 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.
13. 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.
14. Detritylation Process
The 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.
15. 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!
16. 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.
17. REFERENCES
BIOTECHNOLOGY BY B D SINGH
GENETIC ENGINEERING BY
SANDHYA MITRA
PRESENTED BY
JYOTI DEVENDRA ADALA
MSc PART II
