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Transcript genetic et.al - UniMAP Portal

Lect Dec 2013 Genetic
Review -->Additional enzyme app: essential oil extraction
1. Nucleotides
2. Central Dogma :
3. PCR,
4. ELECTROPHORESIS
5. SOUTHERN BLOT
Gene extrc
A nucleotide is
base + sugar + phosphate covalently bonded together
The BASES
The purines
NH
O
N
H
uracil
O
pyrimidines
DNA= deoxy ribo nucleic acid
RNA = Ribo nucleic acid
Sugar
Exp : sugar + base (adenosine)
3,5 phosphodiester bonds formed between
The double helix
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Each base is hydrogen bonded to a
base in the opposite strand to form
a planar base pair.
Each adenine residue must pair
with a thymine residue and vice
versa, and each guanine residue
must pair with a cytosine residue
and vice versa.
These hydrogenbonding
interactions, a phenomenon known
as complementary base pairing,
result in the specific association of
the two chains of the double helix.
Build the dna
Comparison between DNA & RNA
The base composition of DNA
•
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Chargaff’s rules.
DNA has equal numbers of adenine and thymine residues (A =
T) and equal numbers of guanine and cytosine (G = C).
4/11/2016
Central dogma of molecular biology
replication
transcription
DNA
RNA
translation
PROTEIN
DNA directs its own replication to produce new DNA molecule;
DNA is transcribes into RNA;
RNA is translated into protein.
gene1
DNA REPLICATION
The two strands separate, then each serves as a template for the synthesis of
a complementary strand
Each of the two new DNA molecules contains one old strand and one new
strand.) Hence it is called semiconservative
TRANSCRIPTION
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messenger RNA (mRNA) carry genetic
information from chromosomes to ribosomes
ribosomal RNA (rRNA) combine with
ribosomal polypeptides to form ribosomes-the
organelles that synthesize polypeptides
transfer RNA (tRNA) deliver amino acids to
the ribosomes
TRANSLATION
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Translation is the process whereby ribosomes
use the genetic information of nucleotide
sequences to synthesize protein.
quiz
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Code Quiz
Click for the answer.
1. CCU codes for: ?
2. CGA codes for: ?
3. UCA codes for: ?
1. pro
2. arg
3. ser
Multiplying DNA in vitro:
The Polymerase Chain Reaction (PCR)
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The polymerase chain reaction (PCR) is a technique
to produce a large number of identical molecules of
DNA in vitro.
Using PCR, we start with a single molecule of DNA
and generate billions of exact replicate within hours.
PCR
PCR steps
•
Denaturation.
•
Priming.
Extension
Exposure to heat (about 94°C)
Separates the double strands DNA
A mixture containing an excess of DNA primers, DNA
polymerase, and the four deoxyribonucleotide
triphosphates (A, T, G, and C) is added to the target DNA
This mixture is then cooled to about 65°C, enabling
double-stranded DNA to reform.
Raising the temperature to about 72°C increases the rate
at which DNA polymerase replicates each strand to produce
more DNA
These steps are repeated over and over, so the number of DNA molecules
increases exponentially
Separating DNA Molecules:
Gel Electrophoresis
Southern Blot
Electrophoresis
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Gel electrophoresis is a
technique for separating
molecules by size, shape, and
electrical charge.
It involves drawing DNA
molecules (negative charge),
through a gel by an electric
current toward the positive
electrode
Smaller DNA fragments move
faster and farther than larger ones.
Size of a fragments is determined
by comparing the distance it
travels to the distances traveled by
standard DNA fragment of known
size.
gelelectroanim
Southernblot
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The Southern blot technique is the extension of gel
electrophoresis to stabilize specific DNA sequences and then
localize them using DNA dyes or probes.
Once the DNA fragments have been separated by size, the
liquid in the electrophoresis gel is blotted out, the DNA is
denatured with NaOH, and its single strands are transferred and
bonded to anitrocellulose membrane.
If the gel separates DNA and the DNA is detected with a DNA
probe, it is called a Southern blot.
Southern
blot
technique
CITRIC ACID CYCLE
Electron transport Chain (ETC)
PHOTOSYNTHESIS
Types of Cell Respirations:
Obligate anaerobes,
organisms that grow only in the absence of oxygen, avoid the gas by living in
highly reduced environments such as soil. They use fermentative processes to
satisfy their energy requirements.
Aerotolerant anaerobes,
also depend on fermentation for their energy needs, possess detoxifying
enzymes and antioxidant molecules that protect against oxygen's toxic
products.
Facul- tative anaerobes
not only possess the mechanisms needed for detoxifying oxygen metabolites,
they can also generate energy by using oxygen as an electron acceptor when
the gas is present.
Obligate aerobes
highly dependent on oxygen for energy production. They protect themselves
from the potentially dangerous consequences of exposure to oxygen with
elaborate mechanisms composed of enzymes and antioxidant molecules.
Respiration Main steps:
1. Oxidation of organic fuels (fatty acids, glucose, and some
amino acids) yields acetyl-CoA.
2. Oxidation of acetyl groups in the citric acid cycle includes
four steps in which electrons are abstracted.
3. Electrons carried by NADH and FADH2 are funneled into
a respiratory chain, ultimately reducing O2 to H2O. This
electron flow drives the production of ATP.
citric acid
kreb
Explanation:
The citric acid cycle is a metabolic pathway where 2 carbon
fragments derived from organic fuel molecules are oxidized to form
CO2 and the coenzymes NAD+ and FAD are reduced to form NADH
and FADH2, which act as electron carriers.
The electron transport Chain (ETC) is a mechanism where
electrons are transferred from reduced coenzymes to an acceptor
(usually O2).
mido e transport.swf
Oxidative phosphorylation, synthesis of ATP from ADP ( the
energy released by electron transport is captured in the form of a
proton gradient that drives the synthesis of ATP, the energy
currency of living organisms.
Main Reactions of the Citric Acid Cycle.
Oxaloacetate (4 C ) + acetyl-CoA (2C) ---->citrate (6 C)
Also formed :
2 molecules of CO2
3 molecules of NADH,
1 molecule of FADH2,
In each turn of the citric acid cycle:
2 carbon atoms enter as the acetyl group of acetyl-CoA and
2 molecules of CO2 are released.
OVERALL REACTION
glycolysis
After its transport into the mitochondrial matrix, pyruvate is converted to
acetyl- CoA in a series of reactions catalyzed by the enzymes in the pyruvate
dehydrogenase complex. The net reaction, an oxidative decarboxylation, is as
follows:
The net reaction for the citric acid cycle is as follows:
Electron Transport Chain.
Complexes I and II transfer electrons from NADH and succinate, respectively, to
UQ. Complex III transfers electrons from UQH2 to cytochrome c. Complex IV
transfers electrons from cytochrome c to O2.
Complex I, called NADH dehydrogenase complex, catalyzes
the transfer of electrons from NADH to UQ. The major sources of NADH include
several reactions of the citric acid
complex II ( The succinate dehydrogenase complex ) mediates the transfer of
electrons from succinate to UQ.
Complex III (cytochrome c complex).
transfers electrons from reduced coenzyme Q (UQH2) to cytochrome c.
Complex IV (Cytochrome oxidase)
catalyzes the 4-electron reduction of O2 to form H2O.
Summary of the flow of electrons and protons through the four
complexes of the respiratory chain. Electrons reach Q through Complexes I
and II. QH2 serves as a mobile carrier of electrons and protons. It passes
electrons to Complex III, which passes them to another mobile connecting link,
cytochrome c. Complex IV then transfers electrons from reduced cytochrome c
to O2.
etc
The Chemi osmotic Theory
Electrons from NADH and other oxidizable substrates
pass through the inner membrane. Electron flow is
accompanied by proton transfer across the membrane,
producing pH and charge gradient.
The inner mitochondrial membrane is impermeable to
protons; protons can re-enter the matrix only through
proton-specific channels (Fo). The proton-motive force
that drives protons back into the matrix provides the
energy for ATP synthesis
Photosynthesis : the trapping of light energy and its conversion to chemical energy,
which then reduces carbon dioxide and incorporates it into organic molecules
Photorespiration: a light-dependent process occurring in plant cells actively
engaged in photosynthesis that consumes oxygen and liberates carbon dioxide
The essential feature of photosynthesis is the absorption of light energy
by pigment molecules.
The chlorophylls are green pigment molecules that absorp light energy
to drive photochemical events.
PHOTOSYNTHESIS
•
Photosynthesis Reaction
• 12H20 + 6CO2 ----- light -----> 6O2+ C6H12O6 +
6H20
Photosynthesis
Photorespiration
• It is a light-dependent process
• consuming oxygen and releasing CO2.
EXERCISES : SOME EXAMPLE OF QUESTIONS
Example Question : citric acid
1.
Define citric acid cycle. Write the different types of
biochemical reactions involved in this cycle.
2.
Acetyl-CoA is an important compound in citric acid cycle.
Explain how this compound is formed and explain its role in
this cycle.
Answer:
1. The citric acid cycle is a central pathway for
recovering energy from the three major metabolic
fuels: carbohydrates, fatty acids, and amino acid.
These fuels are broken down to yield acetyl-CoA,
which enters the citric acid cycle by condensing
with the C4 compound oxaloacetate. The citric acid
cycle is a series of reactions in which 2 CO2 are
released for every acetyl-CoA that enters the cycle,
so that oxaloacetate is always reformed.
QUESTION EXAMPLES : PHOTOSYNTHESIS:
1. Differentiate between the light reactions and
dark (light independent)
reactions in
photosynthesis
2. Differentiate between
photorespiration
photosynthesis
and
Answer :
1. In the light reactions, organisms capture light energy to synthesize
ATP and generate reducing equivalents in the form of NADH.
In the dark reactions, carbon dioxide is converted to
carbohydrates using the ATP and NADPH generated in the light
reactions.
2. Photosynthesis & Photorespiration
Question Example . ETC
1. The electron transport chain (ETC) components are
organized into four complexes. Name the complexes
and explain general activities occuring within each
of the four complexes.
Answer
• Complex I, called NADH dehydrogenase complex, catalyzes the transfer of
electrons from NADH to UQ. The major sources of NADH include several
reactions of the citric acid
• Complex II ( The succinate dehydrogenase complex ) mediates the transfer
of electrons from succinate to UQ.
• Complex III (cytochrome c complex). transfers electrons from reduced
coenzyme Q (UQH2) to cytochrome c.
• Complex IV (Cytochrome oxidase) catalyzes the 4-electron reduction of O2
to form H2O.