Download Worksheet 1 (isolation)

Worksheet 1 (isolation)
Central Dogma: DNA-RNA-Protein
Transcription of DNA to RNA to protein: This dogma forms the backbone of
molecular biology and is represented by three major stages:
Replication, transcription and translation.
Draw a picture of these stages.
Worksheet 2 (isolation)
How does DNA replication work?
DNA helicases unwind the DNA. The unwound DNA strands are stabilised
by single strand-DNA binding protein. The new DNA is synthesised by DNA
polymerase in the 5'-3’ direction. All DNA polymerases synthesise in 5'-3’
direction. However, the two single strands have different directions. They are
antiparallel. The polymerase can synthesise the leading strand normally, but
at the lagging strand the polymerase and helicase will move away from one
other. The solution to this problem:
DNA polymerase synthesises a series of short DNA fragments (called
Okazaki-Fragments), the polymerase “jumps” back again on the lagging
strand. DNA ligase connects these fragments.
Draw a picture of this process.
Worksheet 3 (isolation)
How does RNA synthesis and processing
work?
Besides the coding information (exons), DNA contains a lot of non-coding
information (introns). During RNA processing these non-coding parts are removed.
Before the synthesis of a protein starts, the corresponding RNA molecule is formed
by RNA transcription. One strand of the DNA double helix is used as a template by
the RNA polymerase to synthesise a messenger RNA (mRNA). This mRNA
migrates from the nucleus to the cytoplasm. During this step, mRNA goes through
different types of maturation including one called splicing where the non-coding
sequences are eliminated. The coding mRNA sequence can be described in units
of three nucleotides called codons. In most mammalian cells, only 1% of the DNA
sequence is copied into a functional RNA (mRNA). Only one part of the DNA is
transcribed to produce nuclear RNA, and only a minor portion of the nuclear RNA
survives the RNA processing steps.
One of the most important stages in RNA processing is RNA splicing. In many
genes, the DNA sequence coding for proteins, or "exons", may be interrupted by
stretches of non-coding DNA, called "introns". In the cell nucleus, the DNA that
includes all the exons and introns of the gene is first transcribed into a
complementary RNA copy called "nuclear RNA," or nRNA. In a second step,
introns are removed from nRNA by a process called RNA splicing. The edited
sequence is called "messenger RNA" or mRNA.
The mRNA leaves the nucleus and travels to the cytoplasm, where it encounters
cellular bodies called ribosomes. The mRNA, which carries the gene's instructions,
controls the production of proteins by the ribosomes.
In eukaryotic cells, the mRNA is processed (essentially by splicing) and migrates
from the nucleus to the cytoplasm.
Draw a picture of this process.
Worksheet 4 (isolation)
How does protein synthesis work?
Messenger RNA carries coded information to the ribosomes. The ribosomes
"read" this information and use it for protein synthesis. This process is called
translation.
The ribosome binds to the mRNA at the start codon (AUG) that is
recognised only by an initiator tRNA. The ribosome proceeds to the
elongation phase of protein synthesis. During this stage, complexes,
composed of an amino acid linked to tRNA, sequentially bind to the
appropriate codon in mRNA by forming complementary base pairs with the
tRNA anticodon. The ribosome moves from codon to codon along the
mRNA. Amino acids are added one by one and translated into the
polypeptidic sequences encoded by DNA and represented by mRNA.
Finally, a release factor binds to the stop codon, terminating translation and
releasing the complete polypeptide from the ribosome.
One specific amino acid can correspond to more than one codon. The
genetic code is thus said to be degenerate.
(optional: find a table on the internet with the genetic code and try to
translate a genetic sequence into an amino acid sequence or translate an
amino acid sequence in a DNA chain)
Draw a picture of this process.
Worksheet 5 (isolation)
Structure of ribosomes
Ribosomes consist of two large subunits of different sizes and structures, which
are conglomerates of about 80 proteins and ribosomal RNA (rRNA). Usually,
naming of the components is by their sedimentation coefficients instead of their
mass. The whole ribosome has a sedimentation coefficient of 80 Svedberg (80
S), the subunits 40 S and 60 S, respectively. S-values are not additive. The
smaller 40 S-subunits consist of a molecule of 18S-rRNA and 32 protein
molecules.
The larger 60 S-subunit contains three kinds of rRNA, with
sedimentation coefficients of 5 S, 5.8 S and 28 S, as well as 46 proteins. In the
presence of mRNA, the subunits combine to form the complete ribosome,
whose molecular mass is about 650 times larger than a haemoglobin molecule.
Prokaryotic ribosomes are similarly constructed, but are slightly smaller than
those of eukaryotes; 70 S for the complete ribosome with values of 30 S and
50 S for the respective subunits.
Worksheet 6 (isolation)
Lac operon
Figure 1: E. coli encodes a -galactosidase that hydrolyses lactose into glucose
and galactose.
Normally, E. coli prefer glucose as carbon source. Under these conditions lactose
metabolising enzymes are not required and the coding genes are only weakly
expressed (Figure 2, A). The lacZ gene (the coding gene for -galactosidase) is
part of the lac operon. With lactose as the only carbon source, the synthesis of
the enzymes involved in metabolism of this nutrient increases more than 1000 fold
(Fig 2, B).
-galactosidase also hydrolyses chromogenic substrates as X-gal (5-bromo-4chloro-3-indolyl--D-galactoside) to a non toxic, insoluble, dense blue-coloured
product ( Fig.3).
Figure 2: (A): The lac operon of
E. coli with glucose as a carbon
source. The repressor binds to the
promotor of the lac operon and
expression of the upstream coding
genes is inhibited.
(B): Lactose in the culture medium
forms an inducer that binds to and
inactivates the repressor. As the
result, the coding genes located
upstream, ie. those for lactose
metabolism, are expressed and
lactose is metabolised.
Figure 3: -galactosidase also converts
the invisible, artificial-substrate X-gal
into a deep blue-coloured oxidation
product within cells.
Worksheet 7 (isolation)
What are the essential steps
to isolate DNA from an organism?
Cells are separated from their environment by a membrane. Bacteria have an
additional a cell wall of murein, plant cells one of cellulose.
To extract the DNA, the first step is to dissolve these barriers. Cell walls are
usually broken with enzymes that specifically attack the cell wall. The membrane
can be dissolved very effectively with detergents.
The next step is to separate the DNA from the remaining cell components.
Proteins can be denatured and precipitated either by phenol extraction or by the
effect of chaotrophic salts.
Either centrifugation or filtration separates the insoluble components from the
dissolved DNA, which can then be precipitated by addition of ethanol.
The
precipitated DNA can be washed with ethanol and then dissolved in a suitable
buffer solution.
Another way to purify the DNA is to bind it to an ion-exchange matrix and remove
all other components by washing. Then the pure DNA is eluted with a suitable
buffer solution from the matrix.
Worksheet 8 (isolation)
Classical Plasmid DNA preparation
The classical DNA preparation method was introduced by Birnboim and Doly 1979. This
method utilises an alkaline lysis in combination with the detergent SDS. The strongly
anionic detergent opens the cell wall of bacteria at high pH, denatures chromosomal
DNA and proteins, and releases plasmid DNA into the supernatant liquid. Due to the
highly alkaline conditions, the DNA base pairs are denatured, but the circular plasmid
DNA is intertwined so that the two strands are not separated. This way the two strands of
plasmid DNA will realign, if the alkaline stress is not too high. After the lysis of the
bacterial proteins, the broken cell walls and denatured chromosomal DNA are
precipitated by SDS in the presence of potassium ions (Ish-Horowicz and Burke, 1981).
After centrifugation, the plasmid DNA can be isolated from the supernatent liquid.
For purer DNA, an additional phenol/chloroform purification step may be performed to
remove residual protein contaminants. After adding phenol/chloroform, mixing, and
centrifugation, the upper aqueous phase contains the DNA, whereas the interface
contains denatured proteins. Remaining phenol can be removed by chloroform
extraction.
After addition of two volumes of ethanol and gentle shaking, the plasmid DNA is
precipitated by centrifugation. The DNA pellet should be washed with 70% ethanol and
dried for 10-15 minutes at room temperature to evaporate the remaining ethanol. Finally,
the DNA may be dissolved in a suitable buffer solution.
Worksheet 9 (isolation)
Commercial DNA isolating-kits
In the laboratory, DNA isolation is usually performed with commercial isolation kits.
This is a summary of some DNA kits from Quiagen:
Worksheet 10 (isolation)
Classical chromosomal DNA preparation
To obtain chromosomal DNA from organisms the following procedure is used.
The method was originally presented by Darly Stafford and colleagues in 1976.
First pellet and wash the cells by centrifugation in TE-buffer. The cells are lysed
by SDS and, in the same step, RNA is digested by RNAse (at 37°C).
Proteinase K
then
digests
remaining
protein
at
50 °C,
followed
by
phenol/chloroform extraction to remove all traces of both proteins and lipids. The
aqueous phase is separated by centrifugation and the extraction is repeated
several times. Lastly, the DNA in the aqueous phase is precipitated by addition
of ethanol and ammonium acetate. The DNA is pelleted by centrifugation,
washed with 70% ethanol, dried to remove the ethanol and finally dissolved in TE
buffer.
Worksheet 11 (isolation)
How can I quantify DNA?
In order to work with isolated DNA, you have to determine its concentration. DNA
absorbs light at a wavelength of 260 nm, as a result of the conjugated structure of the
purine and pyrimidine DNA bases. If the absorbance of a DNA solution is measured at
260 nm, then 1 OD (optical density) unit corresponds to a dsDNA concentration of
50 g/ml with a layer thickness of 1 cm. However, as proteins and RNA also absorb at
this wavelength, they interfere with the result. The quotient (A260/A280) of pure DNA is
between 1.8 and 1.9.