2. What is gene?
• A gene is a stretch of DNA
that codes for a type of
protein that has a function in
the organism.
• It is a unit of heredity in a
living organism.. All living
things depend on genes
• Genes hold the information to
build and maintain an
organism's cells and pass
genetic traits to offspring.
3. What are gene components?
• Genes contain:
EXONS: a set of coding regions…
INTRONS: Non-coding regions removed sequence
and are therefore labeled split genes (splicing).
4. What is the genome?
• The genetic
complement of an
organism, including
all of its GENES, as
represented in its
DNA
5. What is the gene expression?
• Is the process by which
information from a gene is
used in the synthesis of a
functional gene product
(proteins)
• The process of gene
expression is used by all
known life - eukaryotes ,
prokaryotes , and viruses - to
generate the macromolecular
machinery for life.
6. Steps of gene expression
• (1) Transcription (mRNA
synthesis),
• (2) Post-transcriptional
process (RNA splicing),
• (3) Translation (protein
synthesis)
• (4)post-translational
modification of a protein.
7. What are the genetic changes?
• An alteration in a segment of
DNA, which can disturb a
gene's behavior and
sometimes leads to disease.
• It may be:
• (1) Small genetic change,
genetic drift (mutation)
• (2) large genetic change,
genetic shift (recombination)
8. What is mutation?
• Are changes in the
DNA sequence of a
cell's genome
caused by
radiation, viruses,
transposons and
mutagenic
chemicals,
9. What is recombination?
• The exchange of
corresponding DNA
segments between
adjacent chromosomes
during the special type
of cell division that
results in the
production of new
genetic make up...
11. In genetic engineering,
recombination can also
refer to artificial and
deliberate recombination
of pieces of DNA, from
different organisms,
creating what is called
recombinant DNA.
12. • Cloning technology - generation of many
copies of DNA template (e.g., recombinant
DNA molecule) that is replicated in a host.
13. Steps of rDNA
• DNA Cloning
Goal is to generate large amounts of pure DNA that can be manipulated and
studied.
DNA is cloned by the following steps:
1. Isolate DNA from organism (e.g., extract DNA)
2. Cut DNA with restriction enzymes to a desired size.
3. Splice (or ligate) each piece of DNA into a cloning vector to create a
recombinant DNA molecule.
4. Cloning vector = artificial DNA molecule capable of replicating in a host
organism (e.g., bacteria).
5. Transform recombinant DNA (cloning vector + DNA fragment) into a host
(e.g., bacteria) that will replicate and make copies.
6. Expression and Multiplication of DNA insert in the host
E. coli is the most common host.
14. (i) Isolation of gene of intrest
First step in rec DNA technology is the
selection of a DNA segment of interest
which is to be cloned. This desired DNA
segment is then isolated enzymatically.
This DNA segment of interest is termed as
DNA insert or foreign DNA or target DNA
or cloned DNA
15. Cut DNA with restriction enzymes
to a desired size
Restriction enzymes recognize specific bases pair sequences in DNA called
restriction sites and cleave the DNA by hydrolyzing the phosphodiester
bond.
Cut occurs between the 3’ carbon of the first nucleotide and the
phosphate of the next nucleotide.
Restriction fragment ends have 5’ phosphates & 3’ hydroxyls
Restriction enzyme
16. ii) Selection of suitable cloning
vector:
A cloning vector is a self replicating DNA
molecule, into which the DNA insert is to be
integrated. A suitable cloning vector is
selected in the next step of rec DNA
technology. Most commonly used vectors are
plasmids and bacteriophages
17. (iii) Introduction of DNA insert
into vector to form recDNA
molecule:
The target DNA or the DNA insert which has
been extracted and cleaved enzymatically by
the selective restriction endonuclease
enzymes [in step (i)] are now ligated (joined)
by the enzyme ligase to vector DNA to form a
rec DNA molecule which is often called as
cloning vector insert DNA construct.
19. (iv) rec DNA molecule is
introduced into a suitable host:
Suitable host cells are selected and the rec
DNA molecule so formed [in step (iii)] is
introduced into these host cells. This process
of entry of rec DNA into the host cell is called
transformation. Usually selected hosts are
bacterial cells like E. coli, however yeast, fungi
may also be utilized.
20. (v) Selection of transformed host
cells:
Transformed cells (or recombinant cells) are
those host cells which have taken up the rec
DNA molecule. In this step the transformed
cells are separated from the non transformed
cells by using various methods making use of
marker genes.
21. (vi) Expression and Multiplication
of DNA insert in the host:
Finally, it is to be ensured that the foreign
DNA inserted into the vector DNA is
expressing the desired character in the host
cells. Also, the transformed host cells are
multiplied to obtain sufficient number of
copies. If needed, such genes may also be
transferred and expressed into another
organism
