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Applied Molecular Microbiology summary reader+lectures
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Summary Applied molecular Microbiology (MIB-30806) Reader and lectures.
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Applied Molecular MicrobiologyChapter 1-Biotechnology, Engineering & Microorganisms
In biotechnology several areas are distinguished on the basis of the area: agro-good (green), medical
(red), marine (blue), and general industrial application (white).
Chapter 2- Central Dogma & Codon Bias
2.1 The Central Dogma of Molecular Biology
DNA is a linear polymer with repeating units of nucleotides, that consists of an invariant sugar-part
(deoxyribose) and a variable base-part (A, T, G, C). RNA has a similar structure, but the sugar is a
ribose and (apart from A, G, C) the fourth base is U. DNA is generally composed of two very long,
helical polynucleotide chains forming a double helix. A phosphate group covalently links between the
3’ and the 5’ carbon atom of the two adjacent deoxyriboses via a phospho-diester bond. The sugar-
phosphate backbone of each strand is on the outside of the double helix, whereas the purine and
pyrimidine bases are on the inside. The two chains are held together by two (A,T) or three (G,C)
hydrogen bonds. A requirement for base-pairing is that both strands are anti-parallel, they run in
different directions (5’-3’/3’-5’).
The central dogma of molecular biology:
DNA synthesis- theory & applications
The sequential addition of deoxyribonucleotide triphosphate (dNTPs) building blocks during DNA
synthesis is catalyzed by an enzyme called DNA polymerase. This enzyme uses single stranded DNA as
a template and generally starts with an oligonucleotide primer (DNA or RNA) with a free 3’-hydroxyl
group. Then the replication starts by incorporation of dNTPs (from 5’ to 3’) according to the anti-
parallel template sequence. Elongation happens via a phosphor-diester linkage between the 3’ and 5’
carbons and release of pyrophosphate. The complementarity of shape between correctly matched
nucleotide bases is crucial for the fidelity of base incorporations; same DNA polymerases have
proofreading activity allowing removal of mis-incorporated nucleotides, some do not possess this
activity (conformational change upon dNTP binding generates a tight pocket, that only allows
correctly matching nucleotide bases).
,Applications:
1. Oligonucleotide (=primer) synthesis; PCR and sequencing
3’ end is linked to a solid support. In each cycle, the 3’phosphorus of the incoming monomer is linked
to the 5’ oxygen atom of the growing chain. The DMT protective groups allows only 1 base to be
added each cycle.
,2. Polymerase Chain Reaction (PCR)
Needed: thermostable DNA polymerase, DNA template, primer and dNTP nucleotides.
3. DNA sequence analysis
Sanger sequencing uses ddATP’s. Due to the absence of the hydroxyl group at the 3’ carbon, no
phosphor-diester bond can be formed and thus the chain elongation by the DNA polymerase stops.
The result of DNA polymerization with a mix of dNTPs/ddNTPs will be a mix of fragments with
different sizes, that can be separated by polyacrylamide gel electrophoresis. This can be done with 4
separate reactions with all 4 dNTPs and 1 of the ddNTPs and loading them in separate lanes of the gel
or by adding a fluorescent dye to the ddNTPs, mixing the 4 reactions and loading it in a single lane.
Pyrosequencing is a DNA sequencing technique that is based on the detection of released
pyrophosphate (PPi) during DNA synthesis. In a cascade of enxymatic reactions, visible light is
generated that is proportional to the number of incorporated nucleotides.
, Apyrase is a nucleotide-degrading enzyme, to eliminate
the need for washing between the addition of different nucleotides.
RNA synthesis- theory & applications
The direction of RNA synthesis is 5’ to 3’. RNA polymerases do not need a primer and do not possess
proofreading nuclease activity.
Needed: RNA polymerase, DNA template, NTP nucleotides
Protein synthesis- theory & applications
Protein synthesis takes place on ribosomes (ribonucleoprotein particles, about two-thirds RNA and
one-third protein) consisting of a large and a small subunit. The mRNA is translated in the 5’ to 3’
directions, and proteins are synthesized in the amino to carboxyl direction. The start signal on
prokaryotic mRNA is usually AUG preceded by a purine-rich sequence, ribosome binding site or
Shine-Dalgarno box. During translation, the codons of the mRNA interact with the anti-codons of the
tRNA, at the 3’ end of which a specific amino acid has been coupled by a specific aminoacyl-tRNA
synthetase. There are 61 distinct amino acid-coding triplets and generally only 45-50 tRNA. Part of
this problem is solved by the so called codon bias (organisms with low GC genomes have preference
for A and T at the third position of the codon, and vice versa). In addition, some tRNAs can interact
with more than one codon, because of pairing of the third base of a codon is less crucial that that of
the other two (the wobble mechanism).
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