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Seminar System Immunology
Experimental techniques to acquire
high‐throughput quantitative data
Author
Christoph Schwörer
Betreuer
Sven Nahnsen
13.11.2008
1. Introduction
In the past few years System Biology has emerged from the field of computational biology. The
processing power of new computers and the development of new techniques led to new
approaches in the understanding the complete picture of what happens inside a single cell or
an entire organism. Instead of looking at one particular reaction, interaction of between single
proteins or even a whole pathway we now want to look at the status of a whole cell at once.
Thus we can come to understand the interaction of whole Pathways or the complete cellular
reaction to a certain stimulus.
But to build these new models we need reliable statistics. In order to get to these reliable
statistics we need many sets of data from different sources. One of the reasons is why there
have been several new techniques developed to acquire data in huge amounts. Which is why
they are called high throughput methods. Because they process whole experiments at once,
like screening the genome for a certain sequence. This report will now give an introduction to
the basic techniques used to prepare these high throughput methods as well as an introduction
to the most important high throughput methods.
2. Basic techniques
In order to conduct high throughput experiments we have to prepare them carefully. This
means we have to separate cells from one and another if we want to test only certain cells with
specific properties. Or we have to separate certain compartments within a cell if we want to
test them alone. On the other hand we have to provide certain cells with these wanted
properties in order to do comparison tests. In this chapter we will now discuss the basic
techniques used to prepare high throughput experiments.
2.1 Restriction Enzymes / Gel Electrophoresis
Gel electrophoresis can be used for
two different purposes. On the one
hand it can be used to identify the
relationship between different cell
lines on the other it can be used to
break down the isolate short strands
of DNA for further use.
The first step in this procedure is to
break down the very large strands of
cellular DNA into short fragments.
This is accomplished by restriction
enzymes.
enzymes
Restriction
recognize short sequences of double
stranded DNA, which are typically
about 10 to 12 basepairs long, and
these specific
cut
sequences. There exist about several hundred different restriction enzymes which all have
different recognition sites.
Figure 1: Agarose Gel with luminescent DNA strands
the DNA at
After the DNA is completely digested by a restriction enzyme the solution is put on an agarose
gel. The gel is then applied with an electrical field so DNA strands are pulled to the electrodes.
In dependency of their length and charge the different DNA strands will travel at different
speed so that after a given time they separate and reach different points in the gel. With the
addition of luminescent chemicals the strands can be made visible so that they form a pattern
of strands on the agarose gel (see figure 1)
2.2 1D/2D Protein Gels
Gel electrophoresis can not only be used to
separate DNA strands but it can also be used
to separate proteins. The problem is that
there are so many proteins within a cell with
approximately the same size that it is almost
impossible to separate them by size only.
That is why one has to use another criterion
to separate the proteins further. In this case
2D electrophoresis uses
the different
isoelectric points of the proteins which they
reach at different ph‐values (O’Farrel 1975).
In the procedure the first step is to linearize
the proteins because in their natural tertiary
structure they won’t fit through the pores of
the gel. So all the intramolecular bonds which
give the protein its form have to be broken.
(E.g. H‐H bonds or sulfuric bonds) The next step is to separate the proteins by size as it is done
with the DNA on a polyacrylamid gel which is applied with an electrical field. After the second
step another gel with a ph gradient is put on the first and because of their charge the proteins
begin to travel to their isoelectric point. Afterwards the gel with the previously luminescent
marked proteins is visualized.
Figure 2: 2D Protein gel. Each dot represents one protein.
2.3 Cloning Vectors an DNA Libraries
Cloning Vectors are short DNA fragments (up to 19 kbp), as for example the ones we have
retrieved with the restriction enzyme/gel electrophoresis technique. To analyze these DNA
fragments and the genes on them we have to bring them into a living environment. Because
DNA is the same in all living beings they can be inserted into bacteria which then express the
proteins encoded on the DNA strands.
This is achieved by transformation where the DNA fragments, which are called cloning vectors,
are added to a solution of bacteria cells. The cloning vectors can now penetrate the cells
surface and get into the cell. There the original bacterial DNA plasmid is cut with the same
restriction enzyme used to obtain the cloning vectors. Now there is a chance that the cloning
vector is inserted into the plasmid by recombining the cut locations called sticky ends.
After the cloning vector is inserted the cells proliferate and are later separated by the newly
resistances)
new
obtained
properties
antibiotic
through
DNA.
(e.g.
the
2.4 Hybridization and Blotting
Another basic problem is to identify whether a specific DNA sequences or protein is present in a
given DNA/protein sample.
For DNA the technique at hand is the so called Southern Blotting (Southern 1975). A given DNA
sample is first put through a gel electrophoresis to separate the DNA strands by size and is then
washed on a nylon patch to fixate the strands. Afterwards the nylon patch is incubated at up to
80°C to break the hydrogen bonds so that the DNA gets single stranded. Now the nylon patch is
washed again with a solution of hybridization probes, which are short fragments of the
complementary DNA we want to test for. These probes are radioactively marked and will
hybridize with the single stranded target DNA. Now the nylon patch is pressed against a X‐ray
film where the hybridized probes will be visualized.
To test for the existence of specific proteins a similar technique is used which is named Western
Blotting. Like Southern Blotting first the given protein sample is separated using 2D
electrophoresis and then washed onto a carrier patch. In order to test for the targeted protein
this technique uses marked antibodies as probes. Those marked probes can then again be
visualized with an X‐ray film.
2.5 Centrifugation
One of the oldest techniques used for the separation of cell compartments is centrifugation.
There the centrifugal force is used for the separation. More exactly the fact that molecules with
different density will have different sedimentation rates. So that after a given time the
compartments will be separated. Hereby the Sedimentation rate is measured in Svedenberg
m
units:
1(
r
r
)
/
=
S
V
²
w
r
=
par
sol
f
Where m is the mass of the particle, f the friction of the medium and r sol/ r par the density of
the medium/particle
2.6 Column Chromatography
In column chromatography the molecules one wants to separate are washed through a solid carrier
material. Because of the different size and shape of the different molecules they arrive at different
times at the bottom of the column. A more sophisticated method is also available where the carrier
material is spiked with antibodies for a target protein. The antibodies will bind to target protein and
hold it back while everything else is washed through. Then a solution is washed through which will
loosen the protein form the antibodies and the protein can be retrieved.
-
3. Advanced Techniques
After having prepared the proteins or DNA we want to test we now need to have methods so
that we can retrieve data from a large number of parallel experiments. To get confirmation or
even more data to create statistics we need to do several of the same experiment at once. The
techniques used for this purpose are called high throughput experiments because of the sheer
amount of parallel processing and data we get.
3.1 PCR (Polymerase Chain Reaction)
PCR is not an experiment to retrieve data
but more a method to amplificate DNA we
already have prepared to an amount where
it can be used in later high throughput
techniques. (Saiki et al. 1985) Simply put
PCR duplicates the amount of DNA per
cycle. The first step is to heat the DNA
solution so that the hydrogen bonds
between the two DNA strands is broken an
the DNA gets single stranded. Then primers
are added to the solution which will
hybridize with the single stranded DNA
while the solution is cooling down. Now the
DNA‐polymerase kicks in and extends the
single stranded DNA with primer to a new
double stranded DNA strand. This leads to the duplication of DNA with each cycle so that after
a few cycles there is sufficient DNA to use in a high throughput experiment.
Figure 3: PCR
3.2 DNA-/Protein Chips (Microarrays)
Microarrays are a newly developed method to test the expressions of thousands of genes at
once (Cahill and Nordhoff 2003). There are two different types of microarrays, DNA‐chips and
protein‐chips. While DNA chips test for the occurrence of mRNA in a cell, protein‐chips test for
the occurrence of proteins. Both methods applied to the same cell will lead to different
expression patterns because there are several factors influencing the translation from mRNA to
proteins. Both methods work in a similar way.
DNA chips are carrier spotted with cDNA primers
from exons which one can get from a DNA library.
Those chips are then incubated with DNA reversely
transcribed from the target cells mRNA. This DNA is
also marked with fluorescing dye so that the
coloring of the chip reveals the expression of the
correspondent genes. As you can see in fig.4 with the
use of different dyes one can also do comparrison
expereriments on one microarray.
Protein‐chips on the other hand are carriers spotted
with binding partners for proteins which can be
other proteins, antibodies, DNA or drugs. But
protein‐chips are not that easy to apply because
different proteins have different optimal conditions
(e.g ph‐value) so that one has to find a sufficient
compromise to acquire usable data.
3.3 Yeast Two-hybridization
Figure 4: Heatplot of a comparative microarray with two
sources
The yeast‐two‐hybrid system is a technique used to test if two proteins, prey and bait, interact.
(Uetz et al.2000) It uses the fact that the Gala4 Transcription factor consists of two parts. Those
two parts are fused to either of the proteins one wants to test. If bait and prey do interact they
come close together. When this happens the two parts of Gala4 TF also come close enough
together so that it can
the
promote
expression of a given
reporter gene which is
promoted by Gala4.
For screening purposes
this technique can be
Figure 5: Yeast-two-hybrid system
extended to a high
throughput technique
by adding multiple prey proteins or even multiple bait proteins.
3.4 Mass Spectrometry
Mass spectrometry allows the identification of proteins through their mass/charge ratio
(Abersold & Mann 2003). In a mass spectrometer basically the digested protein is ionized by an
ion source and the fragments are accelerated through a magnet onto a mass analyzer. The
detector then delivers a fingerprint of the
containing fragments. This fingerprint is now
compared to the precomputed theoretical
fingerprints from a protein database.
There are different methods available for
the ionization or the mass analysis. The two
methods for ionization are ESI (Electrospray
ionization) which is used to ionize proteins
out of solutions and MALD (matrix assisted
laser desorption/ionization) which is used
on proteins in dry crystals.
Figure 6: Mass spectrometer
For the mass analysis there exist four basic
types. The first is the sector field analyzer
which is depicted in fig.6. It measures the
deviation of a fragment from its trajectory
according to the fact that heavier fragments
won’t be deviated so much then lighter
fragments. The second type of analyzer is the TOF (time of flight) analyzer which measures the
time between entrance in the magnetic field and impact on the analyzer. This type also bases
on the fact that heavier fragments won’t accelerate so fast then lighter ones because of their
inertia. The third type is the quadrupole which allows only fragments to pass that have a
specific mass/charge ratio. The quadrupole is used to measure the quantity of the targeted
fragment. The last type is the Fourier transform ion cyclotron. Here the ions are accelerated in
circular magnetic field. It measures the radius and the frequency of the flying fragments and
computes from that the mass fingerprint. This is also by far the most accurate and sensitive
type of analyzer.
3.5 Transgenic Animals
Transgenic animals are animals who’s DNA have been altered. Either by inserting foreign DNA
or by willingly cutting out specific genes. Either of both happens with the firs stem cell before it
begins to proliferate. There are two ways of getting the foreign DNA into the cell. The first is to
directly inject it into the cell, which is called DNA microinjection. The second is to use an altered
retrovirus which infects the cell.
Transgenic animals are mostly used as knockout animals where one specific gene is cut out to
identify its function.
3.6 RNA Interference
RNA interference is mechanism inhibiting DNA expression where a double stranded RNA has
been inserted into a cell (Fire et al. 1998). It is part of the cells defense system against viruses or
other genomic material. The double stranded RNA is recognized by an endoribonuclease called
DICER. DICER cuts the dsRNA into short strings (~20bps) which are then assembled to RISC
(RNA‐induced silencing complex). The RISC complex then recognizes the correspondent mRNA
and cuts it into short pieces which are then digested thus inhibiting the translation of this
mRNA.
In opposition to transgenic animals this method is usable in high throughput experiments
where many cells and/or genes can be inhibited at once. The only problem with RNA
interference studies is that longer dsRNA strands lead to an interferon response in mammalian
cells. This is why in these cases synthetically produced siRNA strands are used.(Dykxoorn et al.
2003)
4. Discussion and Conclusion
As shown in the chapters above there are several techniques available to acquire high
throughput data. The most upcoming are surely the microarray and the DNA interference
techniques. What all techniques have in common is that they are very expensive to conduct
either in the individual experiment like microarrays or in the needed infrastructure and
machinery like a mass spectrometer. What they also have in common is that every one of them
needs a lot of processing power to analyze the results. Not only to fit the data into models but
simply to handle the sheer amount of data. This processing power is only available to everyone
since the last few years. As research goes on and the field of system biology will surely grow it
stands to hope that in mass production the techniques will be more affordable.
5. References
5.1 Literature
Cahill, D.J. and Nordhoff, E. Protein arrays and their role in proteomics (2003) Adv. Biochem.
Eng. Biotechnol. 83, 177‐87.
Dykxoorn, D.M., Nivina, C.D. and Sharp, P.A. Killing the messenger: short RNAs that silence gene
expression.(2003) Nat. Rec. Mol. Cell. Biol. 4, 457-67
E.Klipp, R.Herwig, A.Kowald, C.Wierling, H.Lehrbach
System Biology in Practice. Concepts,Implementation and Application, (2005)Wiley-VCH 109-
133
Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E., and Mello, C.C. Potent and specific
genetic interference by double stranded RNA in Caenorhabditis elgeans (1998) Nature 391, 806‐
11
O’Farrel. P.H. High resolution two-dimensional electrophpresis of proteins(1975) J. Biol. Chem
250, 4007-4021
Ruedi Aebersold & Matthias Mann
Mass spectrometry-based proteomics (2003) Nature 422, 198-207
Saiki, R.K., Scharf, S., Faloona, F., Mullis,K.B., Horn, G.T., Erlich, H.A. and Arnheim, N. Enzamtic
amplification of beta globin genomic sequences and restriction site analysis for diagnosis of
sickle cell anemia.(1985) Science 230, 1350-1354
Southern, E.M. Detection of specific sequences among DNA fragments separated by gel
electrophpresis (1975) J. Mol. Biol. 98, 503-517
Uetz, P., Giot, L., Cagney, G. Mansfield, T.A., Judson, R.S., Knight, J.R., Lockshon, D., Narayan, V.,
Srinivasan, M., Pochart, P., Qureshi‐Emili, A., Li, Y., Goodwin, B., Conover, D., Kalbfleisch, T.,
Vijayadamo‐Dar, G., Yang, M. Johnston, M., Fields, S., and Rothenberg J.M. A comprehensive
analysis of protein-protein interaction in Saccharomyces cerivisiae (2000) Nature 403, 623‐7
5.2 Figures
Fig. 1: http://upload.wikimedia.org/wikipedia/commons/6/60/Gel_electrophoresis_2.jpg
Fig. 2: http://upload.wikimedia.org/wikipedia/de/b/b2/2D‐Gel.jpg
Fig. 3: http://www.obgynacademy.com/basicsciences/fetology/genetics/images/pcr.png
Fig. 4: http://www.bio.davidson.edu/COURSES/genomics/2005/Durnbaugh/microarray.jpg
Fig. 5: http://upload.wikimedia.org/wikipedia/en/e/e4/Three‐hybrid‐system.svg
Fig. 6:
http://upload.wikimedia.org/wikipedia/commons/b/b8/Mass_spectrometer_schematics.png