This summary includes all the lectures of the course Molecular Biology of the Cell of the master Biomedical Sciences - lectures were given by Maike Stam, Hans van der Spek and Stanley Brul.
Summary is only good for the specific course at the university. It is not a proper summary of the text book, and it does not summarize the chapters stated (Chapter 3, 4, 5, 6, 7, 8, 9 and 17), at least not in a way that is useful -no clear division of the chapters etc
Central Dogma
Gene to function - Information flow from DNA RNA protein metabolite phenotype
Direction and polarity - DNA has two antiparallel polarities, double stranded. Both strands are
synthesized 5’ 3’
Proteins also have polarity – N-terminal to C-terminal
DNA replication is semi-conservative - every new
double stranded molecule consists of one old and one
new strand. The old strand serves as a template for
the new strand – because synthesis of the new strand
goes 5’3’ the template strand should be read 3’5’
DNA synthesis is the process of the formation of a
phosphodiester bond while hydrolyzing the matching
dNTP molecule energy for the formation of the covalent phosphodiester bond comes from the
substrate itself – hydrolysis of the substrate (dNTP) results in the release of a pyrophosphate (PPi)
molecule and the base is incorporated into the sugar backbone.
Synthesis of the new strand has to go into the 5’3’ direction, because this allows for easy removal
of wrongly inserted bases (proofreading)– when the base is removed, a new base can easily be
attached to the free 3’ -OH group. The triphosphate group of the dNTP will provide the energy for
this incorporation. On the 5’ end of the DNA strand this energy (in the form of cleavage of a high-
energy bond) cannot be provided and the synthesis/elongation will stop.
DNA replication
Helicase unwinds the double-stranded DNA by
walking over one strand from 5’3’. Thus the
other strand, the leading strand, will be opened
from 3’5’. The leading strand can be replicated by
DNA polymerase continuously, since the new DNA
strand needs to be synthesized from 5’3’.
The strand the helicase ‘walks’ on is the lagging strand. The DNA from
this strand can be replicated starting from the replication fork – during the
replication more DNA will be unwound by helicase and the next fragment can be synthesized starting
from the new position of the replication fork.
These fragments are called Okazaki fragments and for each of the fragments an RNA primer (made
by RNA primase) is needed. The fragments need to be connected to each other by ligase.
Topoisomerase relieves the tension on the DNA in front of the replication fork by making a nick in
one or both of the strands so that the DNA in front of the replication fork is not overly coiled
,Mistakes in DNA replication
During DNA replication about 1 in 105 bases is wrongly inserted. These mistakes can be restored by
the proofreading activity of the DNA polymerase complex 1 in 107 errors per added nucleotide
The DNA polymerase complex has a polymerization active site and an editing site – the complex can
‘eat back’ like a pacman (3’5’ editing/exonuclease activity)
The complex removes one entire nucleotide (sugar backbone included) and adds the correct
nucleotide – after that polymerization in the 5’3’ direction resumes
Mismatch repair
MutS and MutL scan the DNA and detect a mismatch/nick
in the new DNA strand a whole stretch of nucleotides
around the incorrect base is removed (±12 nucleotides)
DNA polymerase is recruited again to make a correct
strand 1 in 1010 errors
The new strand can be distinguished from the old strand
because the old strand already contains DNA methylation
and/or other modifications
Chemical changes in DNA bases can cause mutations in the
DNA
Depurination a whole base (A or G) is removed – if not
recovered this results in deletion of a nucleotide pair and
frameshift
Deamination a cytosine is deaminated (hydrolysis of C
resulting in removal of NH3) into a uracil
One of the daughter cells will have the correct C-G pair the other will have a A-T (the U will be
replaced by a T, since U is not a DNA base)
DNA sequencing
Dideoxy sequencing (Sanger sequencing) when ddNTPs are incorporated this results in chain
termination - ddNTPs lack the free 3’OH needed for chain extension
The reaction mixes include a high concentration of dNTPs and a low concentration of ddNTPs.
Occasionally/stochastically synthesis will stop because a ddNTP gets incorporated.
The primer determines the start of the reaction.
In the beginning 4 different reactions (each with a different ddNTP) had to be performed per DNA
sequence – now it is possible to pool all ddNTPs into one reaction, because they are labeled with
different fluorescent/radioactive labels
The different fragments that are obtained with dideoxy sequencing only differ 1 nucleotide in length
– higher in the gel (bigger fragments) it gets more difficult to distinguish which sequence is the longer
one (e.g. fragments of 20 or 21 nucleotides
differ more from each other than fragments
of 400 and 401 nucleotides)
innovation – automated dideoxy
sequencing - instead of reading the fragments
yourself from a gel the fluorescently labeled
fragments are loaded onto a capillary and
fluorescence is detected by a laser/detector
,The sequence data/peaks are not always clear
For example CG rich regions are difficult to reach by DNA polymerase which might result in double
peaks. Repetitive DNA sequences with long stretches of the same nucleotide are also difficult for
DNA polymerase – nucleotides might be skipped or synthesized twice (insertion/deletion)
Whole genome sequencing (WGS)
Hierarchical shotgun sequencing - Genomic DNA is cut up
into smaller fragments BAC library – DNA fragments are
amplified, inserted into BACs (vectors/plasmids) and
sequenced genome is assembled by comparison of
overlapping sequences
Contigs – the assembly of smaller DNA sequences into one
continuous strand
This method works well for small genomes that lack
repetitive DNA repeats are a problem in WGS – you have
to fill in the gaps between contigs to be able to figure out how
big the repeat is
To circumvent this problem the human genome was first cut
up into big DNA fragments that were cloned into BACs
The order of the BAC clones was then determined by the
comparing the pattern of restriction enzyme sites within a clone with that of the whole genome
Massive parallel sequencing
Pyrosequencing separation of different DNA sequences is not necessary – each
sequence/fragment is fixed onto a grid – individual spots on the grid all contain a specific DNA
sequence attached to a bead
When a dNTP is incorporated a pyrophosphate (PPi) is released and converted into ATP – with
pyrosequencing each ATP molecule is consumed by the luciferase enzyme, yielding one flash of light
a camera is needed to detect a light-signal/incorporation of a dNTP
because light is used as a signal all four dNTPs need to be added individually, because otherwise
you cannot distinguish which dNTP was incorporated
A total of 4 enzymes are used in pyrosequencing, while dideoxy sequencing requires only 1 enzyme
(DNA polymerase) and chain termination ddNTPs
1. DNA polymerase incorporates the dNTP, resulting in the release of PPi
2. Sulfurylase converts PPi into ATP
3. Luciferase uses ATP to produce light
4. Apyrase degrades unincorporated dNTPs so that the reaction can restart with a new dNTP
Other differences between pyrosequencing and dideoxy sequencing include:
o Pyrosequencing generally produces smaller reads
o Imaging is done with microscopy and a camera, while in dideoxy sequencing imaging is done
with gel- or capillary electrophoresis or laser-based detection of fluorescent signals
o In pyrosequencing nucleotides keep getting incorporated until the end of the sequencing
process while incorporation of a ddNTP in dideoxy sequencing results in chain termination
o Fragment sequencing on small spheres vs gels/tubes/capillaries
, In pyrosequencing incorporation of for example 2 A’s after each other results in a stronger signal – so
this can be distinguished from incorporation of just 1 A however it does remain difficult to identify
how many nucleotides exactly are incorporated
difficulty with identifying polymer tracts
Illumina sequencing
Instead of luciferase the dNTPs are labeled with different fluorescent groups the 4 dNTPs can be
added at the same time, since the fluorescent labels are detected at different wavelengths
Photos are taken after each incorporation to establish which dNTP was incorporated.
ION TORRENT sequencing
When a nucleotide gets incorporated during DNA synthesis
not only a PPi is released but also a proton (H+) this
results in a change in pH.
The resulting pH change can be detected on a chip
Only one dNTP can be added at a time to establish
incorporation of which dNTP resulted in the pH change.
Every one of these methods has its advantages and
disadvantages – fast/slower, expensive/cheaper, more/less
bases per read, more/less reads per run, different error
rates etc.
Quick and dirty genome sequencing
Many short but overlapping reads assemble contigs and
whole genome data
MinION/Nanopore
USB with a nanopore DNA strands are pulled through the
nanopore by immobilized/fixed motor proteins the ion
flow is disturbed when the DNA is pulled through and the
disturbance/current is different for each nucleotide
determine sequence of the DNA that passes through
About 100.000 bp per read (a lot)
BioNano
Rough genome scan – parts of the DNA are labeled, such as repetitive sequences or restriction sites
you can find where these regions are present in the genome but there is no zooming into the
actual sequence
Polymerase chain reaction (PCR)
With PCR you can decide yourself which sequence you want to amplify because you have to design
the primers yourself exact amplification of a desired fragment (so the sequence has to be known
already)
Denaturation of the double stranded DNA (± 95°C) annealing of the primers to the single DNA
strands (±50-55°C) DNA synthesis/elongation (±72°C) starting from the primer
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