Biologist

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Biologist
Chemist
Physician
Basic Biology
Physicist
Biotechnology
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What is Biotechnology ?
Any technique that uses living organisms or
substances from those organisms, to make or
modify a product, to improve plants or animals.
Animal - Gene Transfer Methods - Transgenic
Animals - Their Application
Human - Human Genomic project - Gene Therapy
Technology - Medical Biotechnology - New
Pharmaceutical Production
Research (Recombinant Technology)
Plant - Plant Tissue Culture - Plant Genetic
Engineering
Microorganisms - Commercial Production - Food,
Agriculture, Energy
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Recombinant DNA Technology
DNA Structure
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Recombinant DNA Technology
RNA
DNA
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Bidirectional Semiconservation
Semidiscontinuous
Semiconservation
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ß
DNA
ß
RNA
Secondary channel
RNA exit channel
Active site
Using the lagging strand as template
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Three different modification occur in RNA that
becomes mature mRNA in eukaryotic cells.
Capping Capping of the pre-mRNA involves the
addition of 7-methylguanosine (m7G) to the 5'
end. In order to achieve this, the terminal 5'
phosphate requires removal, which is done by the
aid of a phosphatase enzyme. The enzyme guanosyl
transferase then catalyses the reaction which
produces the diphosphate 5' end. The diphosphate
5' prime end then attacks the a phosphorus atom
of a GTP molecule in order to add the guanine
residue in a 5'5' triphosphate link. The enzyme
S-adenosyl methionine then methylates the guanine
ring at the N-7 position. This type of cap, with
just the (m7G) in position is called a cap 0
structure. The ribose of the adjacent nucleotide
may also be methylated to give a cap 1.
Methylation of nucleotides downstream of the RNA
molecule produce cap 2, cap 3 structures and so
on. In these cases the methyl groups are added to
the 2' OH groups of the ribose sugar. The cap
protects the 5' end of the primary RNA transcript
from attack by ribonucleases that have
specificity to the 3'5' phosphodiester bonds.
Polyadenylaion A specificity factor -- called
Cleavage and Polyadenylation Specificity Factor
(CPSF). CPSF consists of three subunits, each of
which is an RNA-binding protein. Each of the
individual subunits will bind RNA
non-specifically. However, collectively, they
recognize and bind specifically to the AAUAAA
polyadenylation sequence. An endonuclease,
which consists of two components -- CFI and CFII,
cleaves the RNA. The poly(A) polymerase which
catalyses the addition of up to 250 adenine
nucleotides to the 3' end of the cleaved mRNA.
This enzyme, which is a monomeric protein, can
act either distributively or processively. An
additional protein, called Cleavage Stimulation
factor (CStF) is required to stabilize the
complex. CStF is an RNA-binding protein
Pre-mRNA hnRNA (heterogeneous nuclear RNA)
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Why does mRNA need the 5 and 3 modification ?
Modifications facilitate transport out of the
nucleus.
Modifications protect the mRNA from degradation
in cytoplasm. The cap protects the 5' end of
the primary RNA transcript from attack by
ribonucleases that have specificity to the 3'5'
phosphodiester bonds.
Modifications maintain stability for translation.
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3-Poly-A tail
Splicing
5-Cap
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Elongation/Termination
Initiation
Ribosome structure
Before translation begins, the ribosomes are
dissociated into small and large subunits
Met
5
3
tRNA
3
5
mRNA
Initiation codon
tRNA structure
An initiation complex forms, consisting of the
small ribosomal subunit, mRNA and the initiator
tRNA.
The large ribosomal subunit binds to the
initiation complex. The process of peptide
elongation begins with the addition of the
second tRNA with its amino acid.
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Wobble Hypothesis
Genetic Codon 64
Anti-Codon 61
61 Charged tRNA
31 40 Charged tRNA
G C, U UA, G
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• Transcriptional control
• transcription factors, promoter, RNA
  polymeras

2. Regulation of RNA processing
alternative splicing, stability of mRNA
3. Translational control/ Posttranslational
control protein folding, modification
(cutting, phosphorylation, SUMO-,
Ub-conjugation)
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mRNA goes to ribosome as direct copy of the gene
Constituitive gene expression (Promoter)
Regulating metabolism (Promoters and Operators)
A single promoter control several structural
genes (Operon, Polycistron)
An mRNA molecule is said to be monocistronic when
it contains the genetic information to translate
only a single protein. This is the case for most
of the eukaryotic mRNAs. On the other hand,
polycistronic mRNA carries the information of
several proteins, which are translated into
several proteins. Most of the mRNA found in
bacteria and archea are polycistronic.
Dicistronic is the term used to describe a mRNA
that encodes only two proteins.
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Recombinant DNA Technology
The Tools
Recombinant DNA is a set of tools that allows
scientists to move between genetics,
biochemistry and molecular biology.
What does it mean to clone a gene?
Cloning DNA means to isolate a gene or fragment
of DNA away from the other DNA of an organism
and be able to propagate this piece.
What is a vector (plasmids)?
Vector are naturally occurring small pieces of
circular DNA that replicated in various
organisms.
What do the restriction enzymes do?
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Two Major Enzymes Endonuclease and DNA ligase
Restriction endonucleases . Act as
scissors to cut DNA sugar-phosphate backbone at
specific sites. . The recognized sequences
are usually four to six base pairs. . The
recognized sequences are Palindromic
Sticky end
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DNA ligase . Join two DNA molecules
. Ligase is to create final phosphodiester bond
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Vector (Plasmid) Plasmid used in genetic
engineering are called vector.
What three features must vectors (Plamid)? .
Origins of replication that allows for
replication in organism. . A restriction site
recognized by a restriction enzyme in MCS. .
Selectable marker to allow us to select or screen.
Hfr bacteria possess F factor integrated into the
bacterial genome. F bacteria possess F factor
as a plasmid independent of the bacterial genome.
The F plasmid contains only F factor DNA and no
DNA from the bacterial genome. F' (F-prime)
bacteria possess an F plasmid that also includes
some DNA taken from the bacterial genome. F-
bacteria do not contain F factor.
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Cloning vectors
Bacterial vector Plasmid Phage vector
Bacteriophage Cosmid Hybrid of Phage DNA and
Plasmid Yeast artificial chromosomes
(YAC) Bacterial artificial chromosomes (BAC)
Vector
Insert size Plasmid
lt10 kb Bacteriophage
5-20 kb Cosmids
35-45 kb YAC
200 kb BAC
100-300 kb
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To introduce new DNA into organisms
Calcium Chloride Manganase Chloride
Hexamminecobalt Chloride Dimethyl Sulfoxide
(DMSO)
Exposing bacteria to salt solution
(Producing protoplast)
Bacteria
Electroporation (Transient openings in the cell
membrane)
Electroporation (Transient openings in the cell
membrane)
Yeast
Microprojectile (shot gun)
Calcium complex Liposome Electroporation Viral
infectiion
Transfection
Animals
Microprojectile (shot gun)
Microinjection
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Antibiotic resistance (Kanr, Ampr, Terr)
a-complementation
Bacteria
Colony PCR (Polymerase Chain Reaction)
Auxotrophic Gene (His, Trp, Leu)
Yeast
Antibiotic resistance (Neo, Hygro.)
Animals
PCR
Southern, Northern, Western blotting, Microarry
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PCR (Polymerase Chain Reaction)
Sequencing
Mutagenesis
Isolation of DNA fragment Sequencing Mutagenesis G
ene selection
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RT-PCR (Reverse transcriptase Polymerase Chain
Reaction)
Producing complementary DNA (cDNA)
cDNA library
Species cDNA library
Tissues specific cDNA library
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Southern/Northern
Western blotting
Probe
Template
Southern
DNA
DNA
Northern
DNA
RNA
Protein
Western
Protein
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DNA microarray Technology
To study gene expression To study changes in
genomic DNA (SNP)
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siRNA (Small interfering RNA)
To analysis the loss of function in cells
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Application of Recombinant DNA technology
Animal biotechnology
Embryonic stem cells (ES cells) pluripotent
stem cells
Animal clones
Research animal
Humanizing tissues and organs
Plant biotechnology
Insect/Virus/Herbicide resistance
GMO (Genetically Modified Organism) Food
Nutritionally Enhanced plant
Human genome project
Establishment of DNA sequence landmark
Generation of high-resolution genetic and
physical map
Identification of all the gene in the genome and
their function
Establishment of polymorphism database
Proteomics
Development of genome-tailored drugs
Pharmacogenomics
Bioinformatics
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