Masterarbeit/StilVorlagen/Seminar System Imunology - Ausarbeitung.md

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