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

Others 6 withdraw1980-2005

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 drug-induced 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 QT-prolongation 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 DNATwo-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

G - 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

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

Maxam-Gilbert DNA Sequencing

Fred Sanger (dideoxy) DNA SequencingSanger 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

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)

Advantages of dideoxy DNA Sequencing

Automated Fluorescence SequencingIn 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 ProjectBegun 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

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.

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

3. Incubate your hybridization mixture containing fluorescently labeled cDNAswith your DNA chip.

4. Detect bound cDNA using laser technology and store data in a computer. 5. Analyze data

using computational methods.

2. Generate a hybridization solution containing a mixture of fluorescently labeled cDNAs.

A DNA Microarray Experiment : step by step

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 Mg ionB. cDNA piecesC. dNTPD. ddNTPE. 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 muchC. mRNA can not hybridize with DNA tagD. cDNA is cheaper