A full summary combining class notes, slides, and the reader. Filled up with images for a better understanding. The whole summary is 70 pages perfect for starting to study straightforward for the exam. With this summary i got myself an 8 in the exam.
1. THE CENTRAL DOGMA
1.1. DNA AND RNA:
DNA is the molecule of heredity in prokaryotic and eukaryotic → genetic info passed to coming
generations.
In viruses it can be either DNA or RNA.
1953→ Watson and crick revealed the DNA structure:
- They defined it as a linear polymer, with repeating units
of nucleotides.
Non-variant part → deoxyribose sugar backbone
Variant part→ bases: adenine(A), thymine (T), cytosine
(C) and guanine (G).
In nature DNA is a double very long helical polynucleotide
chains→ double helix.
- A phosphate group is linked between the 3´and the
5´carbon atom of the two adjacent deoxyriboses→
phosphor-diester bond.
- Sugar-phosphate backbone→ outside of the helix.
- Bases→ inside of the helix, attach to the 1´carbon.
- Chains held together by 3 hydrogen bonds between pair of
bases.
- One strand is complementary to the other and anti-parallel→ run
in different directions.
- The 2´carbon determines whether is RNA or DNA.
- The 3´carbon determines the strand orientation and the next nucleotide is added.
RNA has a similar structure → sugar is ribose and uracil (U) instead of thymine.
1.2. FLOW OF GENETIC INFORMATION:
+ How is the information stored in the form of nucleic acids and passed on the functional
molecules of the cell, the proteins?
“The central dogma” solves this problem.
DNA→ transcribed into mRNA→ translated to protein
Translation to protein is done via rRNAs→ catalytic
component responsible for peptide bond synthesis
in the frame of the ribosome.
Translation can only happen with tRNAS→ specific
in terms of mRNA codon recognition, and aa
delivery.
This code is conserved in all cellular organisms.
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, 1.3. DNA SYNTHESIS:
The synthesis of DNA (in nature: replication) can only happen with a template molecule of DNA.
DNA polymerase is the enzyme in charge of adding new dNTPs, however it requires an RNA primer,
synthesised by primase.
Elongation happens on the 5´-3´direction→ template on the leader strand is 3´-5´.
The complementarity of the correct matches bases is crucial for the fidelity of base incorporation.
DNA polymerase has proofreading activity; therefore, it can detect mismatch bases.
All synthesis processes can be reversed→ DNA by nucleases, RNA by ribonucleases, protein by
proteases.
RNA is less stable than DNA since it is usually single stranded, and nucleases have a better access to
ribose compared to deoxyribose.
To add dNTPs there is a conformational change in DNA pol→ generated a tight pocket that allows
only the correct nucleotide pairing addition.
After a phosphodiester bond has been form→ release of pyrophosphate (PPi, with H2O converts
back to organic phosphate).
1.4. DNA SYNTHESIS APPLICATION:
2. Oligonucleotide synthesis: primer
DNA needs a primer to initiate synthesis.
Oligonucleotide chain is linked to a 3´end to a solid support→ activated
dNTPS can be added.
In each cycle→ 3´-phosphorus atom of the incoming monomer is bound to
the 5´oxygen atom of the growing chain.
Elongation proceeds 3´-5´(opposite to synthesis).
5´end of a primer does not contain a phosphate group→ block by a chemical
protective group to render it inactive and prevent binding through the 5´end
instead of the 3´end.
3. Polymerase chain reaction (PCR):
DNA polymerase thermos stable→ Taq polymerase for instance
Generated DNA becomes template strand within progression of PCR.
Chain reaction in which DNA template is exponentially amplified (region
between the oligonucleotides primers)
Start with few copies and end up with millions of copies.
4. DNA sequencing analysis:
Process determining the nucleotide order.
Until recently → Sanger method→ in which uses a chain termination method by using
dideoxynucleoside triphosphates (ddA,G,C,TTPs), lack the OH on the 3´C→ cannot add more
dNTPs.
The radioactive fragments are removed from the templated and loaded into a gel with four
distinct columns→ one per nucleoside.
The DNA sequence is read directly from the gel.
The smallest fragments travel the furthest, the larger ones the least→ read from bottom to
top.
Read-out of 800-1000 nucleotides→ very cheap.
Automated sequencing → nucleotides labelled with fluorescent dyes→ each ddNTP with a
colour.
The products are mixed and electrophoresed together.
Detector can scan and rapidly determine the sequence from the order of colours.
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, Fully automated capillary electrophoresis→ nowadays is much faster ad allows up to 96
samples simultaneously.
5. Pyrosequencing:
DNA sequencing technique based on the detection of PPi during DNA synthesis.
Cascade of enzymatic reactions light is generated→ proportional to the number of
nucleotides added.
+process:
a) Annealing of primer to ssDNA template.
b) Nucleic acid polymerization and inorganic PPi released.
c) PPi is converted to ATP-by-ATP sulfurylase.
d) ATP is used by luciferase→ oxidize luciferin and generate light.
Only one dNTP is added at a time→ know whether is introduced or not via light.
Standard pyrosequencing uses a minimum of 3 enzymes→ DNA pol, luciferase and TAP
sulfurylase.
Detection takes between 3-4 seconds at room temperature→ by a charged-coupled device
(CCD).
Solid-phase pyrosequencing→ immobilized DNA with 3 enzyme system. Requires washing to
remove the excess of substrate dNTP after addition.
Liquid-phase pyrosequencing→ nucleotide degrading enzyme is introduced→ apyrase
eliminates the need for solid support and intermediate washing.
6. High-throughput pyrosequencing→ large scale parallel pyrosequencing.
Sequencing of 400-600 megabases per 10h run.
Unbiased sample preparation, long and highly accurate reads.
Relies on fixing adapter-ligated DNA fragments to small DNA-capture beads in a water-in-oil
emulsion.
DNA fixed in the beads is amplified by PCR.
Each DNA bound bead is placed in a PicoTiter plate in combination with a 3 enzyme
system→ sequenced.
1.5. RNA SYNTHESIS AND APPLICATIONS:
RNA synthesis by RNA polymerase→ does not require a primer and do not have proofreading
activity.
Transcription by the DNA template molecule→ create an RNA molecule by ribonucleotide
triphosphates (NTPs) in the 5´to 3´direction.
RNA pol are multi-subunit complexes.
Recognition of the transcription site → promoter regions in archaea and eukaryotes by TATA box
bound to the TATA-binding protein.
In bacteria via the recognition of the sigma subunit of the RNA pol.
Applications → Homo/heterologous gene expression.
1.6. PROTEIN SYNTHESIS AND APPLICATIONS:
Translation→ mediated by the interplay of more than a hundred macromolecules→mRNAs, tRNAs,
rRNAs, aminoacyl- tRNA synthesases and protein factors.
3
, Protein synthesis takes place in the ribosomes-ribonucleoprotein particles (2/3 RNA and 1/3
protein)→ large and small subunit.
mRNA is translated in the 5´- 3´direction, and proteins are synthesised in the amino-carboxyl
direction.
Prokaryotes→ AUG start signal preceded by a purine rich sequence that base pairs with 16S rRNA.
Codon interacts with anti-codon of tRNA at the 3´end→ a specific amino acid has been coupled by
aminoacyl tRNA synthetase.
61 amino acid coding triplets + 3 codon stops and only 45-50 tRNA. Why? Codon bias.
Some tRNAs can interact with more than one codon → pairing of the third base codon is less crucial.
Applications→ Taxonomy ( by the conserved protein machinery, comparative analysis for
classification purposes) and protein production (homologous and heterologous).
QUESTIONS LECTURE:
• during DNA replication, RNA primers are used – why?
Because DNA polymerase cannot start synthesis of dNTPs without a 3´OH with a triphosphate group.
• in gel electrophoresis, DNA migrates to the positive electrode – why ?
Because it has a negative charge due to the phosphate groups in the DNA chain.
• draw the ribose-derived part of NTP (RNA), dNTP (DNA), and ddNTP; indicate the 2’, the 3’, and the
5’ carbon atom of the ribose ring
Look at the picture in page 2.
• describe DNA sequencing by the Sanger (dideoxy-)method
It uses chain stop sequencing molecules, since they add nucleotides which are missing the OH group
in the 3´carbon. This prevents any nucleotides for binding. Different fragments which each of the
four nucleotides is obtained. They are loaded in an electrophoresis gel. Analysis reading the gel from
bottom to top ( more info on page 2).
• describe the relevant details of the pyrosequencing method
3 enzymes is the minimum requirement. Light is detected upon nucleotide addition and PPi release.
• define ORF, gene, genome
ORF→ Open Reading frame→ is it a region of the genome that has potential to be translated into
protein. It usually has a start codon such as AUG. It is a region in the DNA where is it free of
nucleosomes and where usually the transcription machinery can bind to it to start the DNA
transcription process, followed by translation into protein.
Gene→ fragment of the DNA which codes for a specific protein or synthesis of other functional
molecules. Contains relevant information for the organism. Is the unity of heredity and influences
the organism structure, function and behaviour.
Genome→ Is what we define ad the total DNA within a cell. Is the set of genetic material in an
organism. Contains all information necessary for the structure, function, growth and development of
that organism. Contains all genes and non-coding sequences.
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