Recombinant DNA technology •
Biotechnology •
And •
Recombinant DNA (rDNA) •
**Since World War I microbes have been used to produce a
variety of chemicals=== (ethanol, acetone, citric acid …)
**Since World War II microbes used to produce certain
antimicrobial agents=== antibiotics
•
**Now microbes are being used as factories to produce
chemicals that the organism do not naturally make
•
Biotechnology •
•
Biotechnology == is the use of m.o, cell or cell •
component to make a product
by
•
inserting genes into cells by rDNA technology == •
genetic engineering
**Gene from vertebrate animal (man) can be inserted •
into the DNA of a bacterium
**the recipient can then be made to express the •
gene, which may code for a commercially useful
product
**Human insulin ------ treating diabetes •
**Vaccine for hepatitis B virus •
**Human growth hormone •
**Make thousands of copies of the same DNA •
molecule to amplify DNA ----experiments &
analysis for identifying microbes (viruses)
Bacterial genetic material
Bacterial chromosome
Single,long,D.S DNA = contain
genes carry all genetic
information required for
bacterial structure and function
Plasmids=5-100 genes not needed for
growth & reproduction == (survival in
stressful situation)
Small,circular,D.S. DNA
Extra chromosomal genetic material
replicate independently of
chromosomal DNA
DNA structure
DNA molecule is composed •
of repeating units of
nucleotides===are
composed of deoxyribose
sugar molecule linked to a
phosphate group and to a
nitrogenous base (adininethymidine, cytosineguanine)
Nucleotides are twisted •
together in pairs to from a
double helix ---the 2 strands
are held together by
hydrogen bonds
The 2 strands are •
complementary
Duplication of
DNA during
division
•
==replication •
DNA polymerase •
Genetic code •
Set of rules that determines how a •
nucleotide sequence is converted
into the a.a. sequence of a protein
that control cell activities
Transcription==synthesis of a •
complementary strand of RNA(carry
all information) from DNA template
by RNA polymerase
Translation==protein •
synthesis==is the process in which
the information in the nucleotide
base sequence of mRNA is used to
dictate the a.a. sequence of a
protein (in 3 base segments)
---this associated with ribosome •
which consist of rRNA and protein
Genetic transfer & Recombination •
Genetic transfer & Recombination
Genetic recombination==exchange of genes between •
two DNA molecules to form new combinations of genes on
a chromosome by crossing
over
the chromosome break & then rejoin •
The original chromosomes carry a portion of the others gene •
(carry a new function) •
2 different chromosomes
Recombination/ Crossing over
breakage
Cross over
rejoin
Chromosomes== different
than either parents
Genetic transfer
Involves a •
donor cell (give portion of its DNA) •
and •
a recipient cell •
Part of donors DNA is usually incorporated •
into the recipient DNA
The recipient === recombinant •
How can gene transfer from one bacterial
cell to other?
Transformation
recombination
How can gene transfer from one bacterial
cell to other?
Transformation===genes transferred as naked DNA in •
solution==occurs naturally
***after bacterial death or lyses •
***some bacteria release their DNA into the environment •
***other bacteria of the same species can come in contact with
these fragments
***take them up and incorporate them into their DNA by
recombination
#### Recombinant cell ###
•
Has different genetic makeup than the donor or the recipient
•
Transduction
How can gene transfer from one bacterial
cell to other?
Transduction=== involve •
bacteriophage==virus that infect bacteria
***transfer DNA from one bacterium •
to another •
•
How can gene transfer from one bacterial
cell to other?
Conjugation==require contact between living cells •
and the presence of a special plasmid
F plasmid ==facilitates conjugation •
==can give a bac. new genes that may •
help it survive in a changing •
environment •
==25 genes that code for production of pilli •
F+ == have F plasmid •
F- == have no F plasmid •
This event occurs when •
bacteria extend sex pilli
to attached the
cytoplasmic bridge and F
plasmid transferred to
the recipient cell and
integrated within the
chromosome (crossing
over)
Recombinant genome •
can be passed on to
future generations
Other plasmid of equal importance
R plasmid== resistance plasmid =transferred by •
conjugation and pass to future generation
== carry genes that confer •
resistance to certain antibiotics •
code for•enzymes that inactivate certain drugs
Bacteria with R plasmid •
make the drug inactive
•
Bacteria without R plasmid •
• to that antibiotic and destroyed by it
sensitive
Recombinant DNA technologies
### A desired gene is inserted into a DNA •
vector (plasmid or viral genome)
### The vector inserts the DNA into a new cell •
which is grown to form a clone
### Large quantities of the gene product can •
be harvested from the clone (protein)
Molecular cloning
**is a set of experimental methods in molecular biology that
are used to assemble recombinant DNA molecules and to
direct their replication within host organisms
Cloning== replication of a single DNA molecule starting •
from a single living cell to generate a large population of cells
containing identical DNA molecules.
Molecular cloning generally uses DNA sequences from two •
different organisms: the species that is the source of the DNA
to be cloned, and the species that will serve as the living host
for replication of the recombinant DNA.
•
Molecular cloning
Molecular cloning
**is a set of experimental methods in molecular biology that are •
used to assemble recombinant DNA molecules and to direct their
replication within host organisms
Cloning== replication of a single •
DNA molecule starting from a
single living cell to generate a large
population of cells containing
identical DNA molecules.
Molecular cloning generally uses •
DNA sequences from two different
organisms: the species that is the
source of the DNA to be cloned,
and the species that will serve as
the living host for replication of
the recombinant DNA.
Molecular cloning experiment
DNA to be cloned is obtained from an organism of interest
Tools of rDNA technology
Microbes & vectors •
==Lab.strain of bacteria (E.coli) •
==plasmid cloning vector •
**widely available •
**versatile •
**offer rapid growth •
**smaller size are more easily manipulated •
**circular (destruction) •
Tools of rDNA technology
Enzymes •
**restriction enzyme •
==recognizes & cuts only one particular nucleotide •
sequence in DNA-----some produce sticky ends of the
DNA fragments
**DNA ligase •
==covalently link the DNA back bones by base pairing-- •
-rejoin DNA pieces
Endonuclease •
Alkaline phosphatase •
Reverse transcriptase •
•
Tools of rDNA technology
DNA to be cloned •
==extracted from the organism of interest •
*any tissue source can be used (extinct animals)
*DNA also can be obtained from RNA using •
reverse transcriptase
==The extracted DNA is then purified to •
remove contaminating proteins
==Amplified •
Method
DND ligase
cloning
Application of rDNA technology
provides an essentially unlimited quantity of any •
DNA segments derived from any genome. This
material can be used for a wide range of purposes,
including those in both basic and applied biological
science.
recombinant protein ===Obtaining the molecular •
clone of a gene can lead to the development of
organisms that produce the protein product of the
cloned genes.
1-medically useful proteins ==whose administration can correct a
defective or poorly expressed gene
**factor VIII a blood-clotting factor deficient in •
some forms of hemophilia •
**insulin used to treat some forms of diabetes •
•
2-proteins ==that can be administered to assist in a life threatening
emergency
**tissue plasminogen activator used to treat strokes •
•
3-recombinant subunit vaccines == purified protein can be used to
immunize patients against infectious diseases, without exposing
them to the infectious agent itself
**hepatitis B vaccine •
4- recombinant proteins == standard material •
** diagnostic laboratory tests •
•
Other application •
Probes= DNA with known genetic sequence •
**nucleic acid hybridization •
**antibody probes •
**polymerase chain reaction (PCR) •
**DNA sequencing •
Transgenic organisms==genetically modified •
organisms (GMO)
**transgenic mouse •
**commercial use (pharmaceu.,plants, •
pharming) •
Gene therapy
*** supplying a functional gene to cells •
lacking that function----with the aim of
correcting a genetic disorder
fix a faulty gene by replacing it with a healthy gene •
=== the use of DNA as a drug to treat disease by •
delivering therapeutic DNA into a patient's cells
SCID=Sever Combined Immunodeficiency Disease
due to a defective gene for Adenosine Deaminase
(ADA)
First Term Exam.
Good Luck