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Amino Acids are chiral molecules

(well, most of them are)

Rule of CORN

An example that small conformational


kink in helix makes a difference of
function

Toxicity and Drug Withdraw


Heart

7 withdraw

Liver

4 withdraw

Eye

3 withdraw
1980-2005

Others

6 withdraw

All due to similar symptom with heart condition


development and eventually one protein

Adapted from presentation from Organon Pharmaceutical

Borrowed from P.G Poveda

hERG is especially interesting for


us
Most important mechanism leading to druginduced torsades de pointes (TdP).
1:100000, but sudden death.
QT prolongation (with/out TdP) responsible for
60% withdraw for last 16 years.
On market, a percentage of drugs still has QTprolongation potential.
60% of drug in development has inhibitory effect
on hERG.

Borrowed from P.G Poveda

The side effects happen rarely and can


hardly be detected in clinical trials.
in vivo model, abnormal ventricular
function (long QT-syndrome)
in vitro model, inhibition of hERG channel
(abnormal K+ flux).

Proteins are the molecules to get


things done
But they are very fragile and high
maintenance. They are inactive in a lot of
conditions and they unfold very easily.
Nature uses DNA instead. DNA contains
linear information to be transcribed into
RNA and then eventually protein.
With DNA, life goes on.

The Birth of Molecular Biology: DNA Structure

In science, we made a hypothesis


and demonstrate it is right or wrong
A lot of time, even if it is wrong, the testing
gives us information.
That is the basic model of scientific
research.
Before the double helix model was
demonstrated to be right by x-ray
crystallography, other model exists.
What other models would you propose?

Alternative model for DNA


Amount of C=G
Amount of A=T
It is alpha helical
Why this model doesnt fit the
scientific data?

Helix pitch 34
Diameter 20
10.4 base per turn
In contrast, helix has a
pitch of 5.4 and radius
of 10 . 3.6 residues
per turn.

Let us talk about sequencing


With the development of PCR techniques,
DNA sequencing can be done fast and
cheap with the much longer reading.

Sequencing DNA
Prior to the mid-1970s no method existed by
which DNA could be directly sequenced.
Knowledge about gene and genome organization
was based upon studies of prokaryotic organisms
and the primary means of obtaining DNA sequence
was so-called reverse genetics in which the amino
acid sequence of the gene product of interest is
back-translated into a nucleotide sequence based
upon the appropriate codons.

Protein sequencing strategy

Maxam-Gilbert DNA Sequencing

Allan Maxam / Walter Gilbert DNA Sequencing


Sequencing single-stranded DNA
Two-step catalytic process:
1) Break glycoside bond between the ribose sugar and the base /
displace base
Purines react with dimethyl sulfate
Pyrimidines react with hydrazine
2) Piperidine catalyzes phosphodiester bond cleavage where base
displaced

- dimethyl sulfate and piperidine

A + G - dimethyl sulfate and piperidine in formic acid


C

- hydrazine and piperidine in 1.5M NaCl

C + T - hydrazine and piperidine

Maxam-Gilbert DNA Sequencing

200-300 bases of DNA sequence every few days


Use large amounts of radioactive material, 35S or 32P
Constantly pouring large, thin polyacrylamide gels
Hydrazine is a neurotoxin

Fred Sanger (dideoxy) DNA Sequencing


Sanger knew that,
whenever a
dideoxynucleotide was
incorporated into a
polynucleotide, the chain
would irreversibly stop,
or terminate. Thus, the
incorporation of specific
dideoxynucleotides in
vitro would result in
selective chain
termination.

Sanger (dideoxy) DNA Sequencing

Advantages of dideoxy DNA Sequencing


Elimination of dangerous chemicals (hydrazine)
Greater efficiency (>3x)
Taq polymerase makes DNA strands off of a template at
rate of about 500 bases per minute
Chemical synthesis of a 25-mer oligonucleotide takes
more than two hours.
High Throughput Methods (Human Genome Project)

Automated Fluorescence Sequencing


In 1986, Leroy Hood and colleagues reported on a DNA sequencing method in
which the radioactive labels, autoradiography, and manual base calling were all
replaced by fluorescent labels, laser induced fluorescence detection, and
computerized base calling.

Human Genome Project


Begun formally in 1990, the U.S. Human Genome Project was a 13-year effort
coordinated by the U.S. Department of Energy and the National Institutes of
Health. The project originally was planned to last 15 years, but rapid
technological advances accelerated the completion date to 2003.
Project goals:
identify all the approximately 20,000-25,000 genes in human DNA,
determine the sequences of ~3 billion chemical base pairs of human DNA,
store this information in databases,
improve tools for data analysis,
transfer related technologies to the private sector, and
address the ethical, legal, and social issues (ELSI) from the project.
sequence 500 Mb/year at < $0.25 per finished base
(Sequenced >1,400 Mb/year at <$0.09 per finished base)
complete genome sequences of E. coli, S. cerevisiae, C. elegans, D. melanogaster
develop genomic-scale technologies (oligo syn, DNA microarrays, 2-hybrid sys)

HGP Hero - Jim Kent (research


scientist at UC Santa Cruz)
The human genome project was ultimately
a race between Celera Genomics and the
public effort, with the final push being a
bioinformatics problem to put all of the
sequence reads together into a draft
genome sequence. Jim Kent was a grad
student at UCSC, who worked for weeks
developing the algorithm to put all of this
together, beating Celera by 3 days to an
assembled human genome sequence.
genomics and Parasol.

Microarray
The relative concentration of any given
mRNA in a population of transcripts can
be determined by hybridization to its
specific complementary sequences.
The relative abundance of each of the
mRNAs encoded in a given genome can
therefore be assessed by their
simultaneous hybridization to the complete
set of corresponding sequence.

A DNA Microarray Experiment : step by step

1. Prepare your
DNA chip using
your chosen target
DNAs.

3. Incubate your
hybridization
mixture containing
fluorescently
labeled cDNAs
2. Generate a
with your DNA
hybridization
solution containing chip.
a mixture of
fluorescently
labeled cDNAs.

4. Detect bound
cDNA using laser
technology and
store data in a
computer.

5. Analyze data
using
computational
methods.

http://www.dnalc.org/ddnalc/resources/dnachip.html

Note: Thanks to Prof. Vishy Iyer for many of these slides on microarrrays.

To conduct microarray
hybridization, which of this
ingredient is needed?
A
B.
C.
D.
E.

Mg ion
cDNA pieces
dNTP
ddNTP
DNA polymerase

The application of microarray


1. Expression profiling.

2. Gene loss or gain.

3. Mutation
4. Function assignment for unknown proteins

Hierarchical clustering of gene expression data (as ratios).

In reality, we use cDNA rather


than mRNA for microarray
experiments, why?
A. mRNA has too much secondary
structure.
B. mRNA degrades too much
C. mRNA can not hybridize with DNA
tag
D. cDNA is cheaper