Medical biotechnology introduction

Prof. Józef Dulak

Email: jozef.dulak@uj.edu.pl

Faculty of Biochemistry, Biophysics and Biotechnology

Department of Medical Biotechnology

Web: www.biotka.mol.uj.edu.pl/bmz

Lecture 1 – 05 March 2013

Rules

15 hours course – 2 ECTS

Final exam: 1. multiple choice test

2. open questions (eg. adding a missing

word or phrase or sentence)

Materials for the exam:

1. Lectures – slides will be available (not all) at the website of the department

- information provided during the lectures (hence attending them is adviced)

- additional materials to be distributed during the lectures

In the begining was…

Pre-History:

10,000 years ago - humans domesticate crops and livestock.

6,000 years ago - Biotechnology first used to leaven bread and ferment

beer, using yeast (Egypt).

6,000 years ago - Production of cheese and fermentation of wine (Sumeria,

China and Egypt).

2,500 years ago - First antibiotic: moldy soybean curds used to treat boils

(China).

Wall paintings from the Tomb of Kenamun

What is biotechnology?

Biotechnology:

bio - the use of biological processes;

technology - to solve problems or make useful

products.

Since thousands of years humans are trying to employ the natural biological

processes for their benefits:

1. Production of food

2. Treatment of diseases

Hence, genetically modified organisms(GMO) are not the results of

recent biotechnological development – all cultivated plants and

animals are the result of genetic modification

History of biotechnology

History of medical biotechnology

Edward Jenner's first vaccination

1797 - Jenner inoculates a child with a viral vaccine

to protect him from smallpox.

1919 - First use of the word biotechnology in print.

1928 - Penicillin discovered as an antibiotic: Alexander Fleming.

1938 - The term molecular biology is coined.

1941 - The term genetic engineering is first used, by Danish microbiologist

A. Jost in a lecture on reproduction in yeast at the technical institute in

Lwow, Poland.

1942 - Penicillin mass-produced in microbes.

1944 - Waksman isolates streptomycin, an effective antibiotic for

tuberculosis.

Medical biotechnology is the use of organisms and

organisms-derived materials for research

and to produce diagnostic and therapeutic products

that help

to treat and prevent human diseases

Medical biotechnology

T. Twardowski, S. Bielecki, European Biotechnology 2005

Divisions of biotechnology

The medical biotechnology field has helped bring to market microbial

pesticides, insect-resistant crops, and environmental clean-up techniques.

Strong interaction of medical biotechnology with

other branches of biotechnology

Medical biotechnology

= red biotechnology

Aims of medical biotechnology

1. Prevention of diseases

2. Diagnostic of diseases

3. Treatment of diseases

All those aspects are strongly related to basic research – investigation

on the mechanisms of diseases

Application of biotechnology for human health

„elucidation of the molecular structure of the genome including its nucleotide

sequence is fundamental to understanding the molecular pathogenesis of

human diseases”

A.J. Marian, John Belmont - Circ Res. 2011;108:1252-1269

Genomic and genetic determinants of phenotype (and diseases)

A.J. Marian, John Belmont - Circ Res. 2011;108:1252-1269

„Despite its apparent simplicity, the genome is a complex structure. The complexity is

far beyond the primary base sequence of the genome. DNA is a large macromolecule

that requires a complex system to orchestrate its compaction inside the nucleus in a

manner that selected genes are accessible to specific DNA processing enzymes, such

as polymerases, in an orderly and dynamic fashion, as demanded by the cell in

response to internal and external stimuli. Thus, understanding the functional content of

the genome necessitate knowledge beyond the complete genome sequence. Based on

today’s knowledge, only 1% of the human genome is transcribed into mRNA and

translated into proteins. An additional 0.5% serves as a template for noncoding

RNA and the regulatory regions that control gene expression. The functions of the

remaining 98.5% of the genome including functional conserved noncoding elements,

which comprise at least 6% of the genome, remain unknown. Hence, this large

segment of the genome is referred to as the dark matter of the genome. The

discoveries of noncoding RNA, microRNA, splice variants, and regulatory elements in

trans point to the complex mechanisms by which the genome governs various

biological processes, including phenotypic expression of diseases”

(see previous slide).

A.J. Marian, John Belmont - Circ Res. 2011;108:1252-1269

Complexity of human genome

„The estimated heritability of common complex diseases, defined as a proportion of the

phenotypic variance accounted for by genetic factors, varies from 20% to 80%,

depending on the phenotype and study characteristics”

„complex diseases result from the cumulative and interactive effects of a large

number of loci, each imparting a modest marginal effect on expression of the

phenotype”

Diseases

1. Monogenic diseases - inherited

2. Polygenic diseases – acquired

A.J. Marian, John Belmont - Circ Res. 2011;108:1252-1269

Genetic nature of diseases

Tools and products of medical biotechnology

1.Diagnostics at the nucleic acid level

2.Treatment

2.1. application of recombinant DNA technology

for drug development

2.2. treatment at the nucleic acid level and by

means of nucleic acids

2.2.1. genetic therapy

2.2.2. cell therapy

2.2.3. biomedical engineering

Genetic tests – detection of diseases

1. Cytogenetic analysis – chromosomes

1. Detection of mutations

- restriction enzymes & related techniques

- hybridisation: Southern blotting, Northern blotting

3. PCR technology

4. Sequencing

Here, six different DNA probes have been used to mark

the location of their respective nucleotide sequences on

human chromosome 5 at metaphase. The probes have

been chemically labeled and detected with fluorescent

antibodies. Both copies of chromosome 5 are shown,

aligned side by side. Each probe produces two dots on

each chromosome, since a metaphase chromosome

has replicated its DNA and therefore contains two

identical DNA helices. (Courtesy of David C. Ward.)

From: Isolating, Cloning, and Sequencing DNA

Molecular Biology of the Cell. 4th edition. Alberts B, Johnson A,

Lewis J, et al. New York: Garland Science; 2002.

Labeling of nucleic acids to detect mutations

www.hematogenix.com -

Detection of specific RNA or DNA molecules by gel-transfer hybridization

Molecular Biology of the Cell. 4th edition.Alberts B, Johnson A, Lewis J, et

al. New York: Garland Science; 2002.

Southern blot – detection of DNA

Western blot – detection of proteins

Northern blot – detection of RNA

Restriction enzymes for molecular diagnostics

Detection of the sickle-cell globin gene by

Southern blotting. The base change

(A → T) that causes sickle-cell anemia

destroys an MstII target site that is present

in the normal β-globin gene. This

difference can be detected by Southern

blotting. (Modified from Recombinant

DNA, 2d ed. Scientific American Books.

Copyright © 1992 by J. D. Watson, M.

Gilman, J. Witkowski, and M. Zoller.)

From: Using Recombinant DNA to Detect

Disease Alleles Directly

Copyright © 1999, W. H. Freeman and

Company.

Application of Southern blotting for disease detection

Polymerase chain reaction

Molecular Biology of the Cell. 4th edition.Alberts B, Johnson A, Lewis J, et

al. New York: Garland Science; 2002.

Detection of mutation by PCR

RJ Trent – Molecular medicine, 1997

The Cell: A Molecular Approach. 2nd edition.

Cooper GM.Sunderland (MA): Sinauer Associates; 2000.

Polymerase chain reaction

1. Classical PCR

2. real-time PCR

RFLP combined with PCR

RJ Trent – Molecular medicine, 1997

Techniques used for detection of mutations

From: Molecular medicine, 1997

Genetic tests

1.Preimplantation – after in vitro fertilisation

2.Prenatal diagnostics

3.Postnatal diagnostics

RJ Trent – Molecular medicine, 1997

Preimplantation diagnostics

From: From biology to biotechnology…

Prenatal diagnostics

Human genome project – HGP

Completed in 2003, the Human Genome Project (HGP) was

a 13-year project coordinated by the U.S. Department of Energy

and the National Institutes of Health. During the early years of the

HGP, the Wellcome Trust (U.K.) became a major partner; additional contributions

came from Japan, France, Germany, China, and others.

Project goals were to identify all the approximately 20,000-25,000 genes in human

DNA, determine the sequences of the 3 billion chemical base pairs that make up

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) that may arise from the project.

Though the HGP is finished, analyses of the data will continue for many years. An

important feature of the HGP project was the federal government's long-standing

dedication to the transfer of technology to the private sector. By licensing

technologies to private companies and awarding grants for innovative research, the

project catalyzed the multibillion-dollar U.S. biotechnology industry and fostered the

development of new medical applications.

History of Human Genome Project

History of Human Genome Project

The human genome

HGP and othe genome analysis was possible thanks to

the development of sequencing technology

Maxam-Gilbert sequencing

Allan Maxam and Walter Gilbert published a DNA sequencing method in 1977 based on chemical

modification of DNA and subsequent cleavage at specific bases.[7] Also known as chemical

sequencing, this method allowed purified samples of double-stranded DNA to be used without

further cloning. This method's use of radioactive labeling and its technical complexity discouraged

extensive use after refinements in the Sanger methods had been made.

Maxam-Gilbert sequencing requires radioactive labeling at one 5' end of the DNA and purification

of the DNA fragment to be sequenced. Chemical treatment then generates breaks at a small

proportion of one or two of the four nucleotide bases in each of four reactions (G, A+G, C, C+T).

The concentration of the modifying chemicals is controlled to introduce on average one

modification per DNA molecule. Thus a series of labeled fragments is generated, from the

radiolabeled end to the first "cut" site in each molecule. The fragments in the four reactions are

electrophoresed side by side in denaturing acrylamide gels for size separation. To visualize the

fragments, the gel is exposed to X-ray film for autoradiography, yielding a series of dark bands

each corresponding to a radiolabeled DNA fragment, from which the sequence may be inferred.[7]

Chain-termination methods

The chain-termination method developed by Frederick Sanger and coworkers in 1977 soon

became the method of choice, owing to its relative ease and reliability.[22][6] The chain-terminator

method uses fewer toxic chemicals and lower amounts of radioactivity than the Maxam and Gilbert

method. Because of its comparative ease, the Sanger method was soon automated and was the

method used in the first generation of DNA sequencers.

Principles of DNA sequencing

From: Wikipedia

The enzymatic—or dideoxy—method of sequencing DNA

Molecular Biology of the Cell. 4th edition.

Alberts B, Johnson A, Lewis J, et al.

New York: Garland Science; 2002.

RJ Trent – Molecular medicine, 1997

DNA sequencing

Automated sequencing

Next breakthrough in genomic medicine

Microarray technologies

Gene chips & gene arrays

Microarrays for disease diagnostics

RJ Trent – Molecular medicine 2012

The expression levels of thousands of genes can be

simultaneously analyzed using DNA microarrays (gene chips).

Here, analysis of 1733 genes in 84 breast tumor samples reveals

that the tumors can be divided into distinct classes based on their

gene expression patterns. Red corresponds to gene induction

and green corresponds to gene repression. [Adapted from C. M.

Perou et al., Nature 406(2000):747.]

New generation sequencing

The Human Genome Project, which was launched in 1990 with the

primary goal of deciphering sequence of the human genome, took

more than a decade to complete, even in a draft form, and cost close to

$3 billion.

DNA sequencing technology, however, has undergone a colossal shift

during the past 6 years. Various new techniques that sequence millions

of DNA strands in parallel have been developed. The new

technologies, which are collectively referred to as the next generation

sequencing (NGS) platforms, as opposed to the Sanger method,

which was used in the Human Genome

Project, have increased DNA sequencing output and have reduced the

cost of DNA sequencing by 500 000-fold. These advances in DNA

sequencing technologies along with the rapidly declining cost of

sequencing are changing the approach to genetic studies of not only

single gene disorders but also common complex disorders.

A.J. Marian, John Belmont - Circ Res. 2011;108:1252-1269

Direct DNA Sequencing

The cost of sequencing the entire human genome is expected to decrease to

$1000 by the end of 2011. This evolution has been made possible by switching to

massively parallel sequencing platforms wherein millions of DNA strands are

sequenced in parallel and simultaneously. The technologies have made it

feasible to sequence two or three genomes or a dozen of exoms in a week.

Application of the NGS extends beyond the DNA sequencing because the core

genome technology also affords the opportunity to sequence and analyze the

whole transcriptome (RNA-Seq), epigenetic modifications (Methyl-Seq), and

transcription factor binding sites (ChIP-Seq). The approach is quantitative and

enables relatively small amount of template.

New generation sequencing

A.J. Marian, John Belmont - Circ Res. 2011;108:1252-1269

Next-Generation Sequencing Platforms

Sydney Brenner, Nobel Laureate in Physiology and Medicine (2002), introduced the first

technique of sequencing of millions of copies of the DNA simultaneously, referred to as

MPSS in 2000. Soon, George Church et al described the technique of multiplex polony

sequencing. The first commercial NGS platform was based on pyrosequencing

technique. However, it was soon surpassed in output by reversible dye termination and

sequencing by ligation approaches. Sequencing platforms continue to evolve at a rapid

pace with enhanced capacity to generate bigger outputs and more accurate reads.

Accordingly, the newer instruments can generate up to 300 Gb of throughput per

sequencing run, which would be sufficient to cover two to three genomes and

approximately a dozen exomes and transcriptomes.

The two most commonly used platforms for whole exome and whole genome sequencing

are the SOLiD systems (Applied Biosystems), which are based on sequencing by

ligation-based chemistry and HiSeq systems (Illumina), which utilize reversible

terminator-based sequencing by synthesis chemistry. Both platforms generate short

reads that typically are 50 to 120 bases long and each can generate 20 to 30 Gb per day.

The accuracy of the sequence reads depends on various factors, including depth of

coverage. Overall, the systems have a high accuracy rate, typically 99.9%.

New generation sequencing

A.J. Marian, John Belmont - Circ Res. 2011;108:1252-1269

In contrast to short-read NGS platforms, pyrosequencing (Roche 454 sequencing

systems) can generate a read length of 400 bases and 1 million reads per run in 10

hours. However, the size of sequence output is much smaller and the cost per base

is much higher. Because of the length of the reads, the system is best suited for de

novo sequencing. The error rate is 0.1%. Therefore, for medical sequencing,

confirmation of the variants is essential.

Whole Genome Sequencing

Whole genome sequencing using NGS instruments only recently has become

feasible in individual laboratories. The existing platforms afford the opportunity to

sequence one to three genomes in a single run in 7 to 8 days. However, currently,

only few centers have the sequencing and bioinformatics capacity and financial

means to handle large-scale whole genome sequencing projects.

New generation sequencing

A.J. Marian, John Belmont - Circ Res. 2011;108:1252-1269

Whole Exome Sequencing

The whole exome sequencing approach is designed to capture, enrich, and sequence all

exons in the genome. Each genome is estimated to contain 300 Mbp representing 180

000 exons of 23 000 protein-coding genes. The focus on whole exome sequencing as

opposed to whole genome sequencing stems from the existing data, which indicate that

more than two-thirds of the known disease-causing genes in humans are located within

exons.

Traditional and next generation sequencing

P. Brown – Genomes 2002

From gene to genome and further….

Evolution of molecular medicine

RJ Trent – Molecular medicine 2012

Further reading and watching….

http://www.genome.gov/27539497

The Human Genome:

A Decade of Discovery, Creating a Healthy

Future

Agenda, Videos and Presentation Slides

Monday, June 7, 2010

Application of DNA recombination technology

Recombinant

proteins

Monoclonal

antibodies

Gene

localisation

and function

Gene modification

(mutations)

Forensic

medicine Molecular

diagnostics

Gene therapy

Transgenic

Animals

Creation of

new organisms

DNA recombination

technology

Next lectures:

12 March

9 April

16 April

23 April

14 May

21 May

(28 May)

Exam: planned on 18th June

Thank you