Prof. Józef Dulak Email: [email protected]

Medical biotechnologyintroduction

Prof. Józef Dulak

Email: [email protected]

Faculty of Biochemistry, Biophysics and Biotechnology Department of Medical Biotechnology

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

Lecture 1 – 07 March 2016

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 at the website of the department

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

- additional materials may be distributed during the lectures

- Lectures at the conference - „Perspectives in Medical Biotechnology” – 22-23 May

Book is available in the library – several copies

Used copies at Amazon – from 12.5 $

New and Kindle versions – for 40$

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 fermentbeer, 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 biologicalprocesses for their benefits:

1. Production of food

2. Prevention, diagnosis and treatment of diseases

Hence, genetically modified organisms(GMO) are not only the results of recent biotechnological development – all cultivated plants and

animals are the result of genetic modification

History of biotechnology

History of medical biotechnology – some milestones

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 microbiologistA. Jost in a lecture on reproduction in yeast at the technical institute inLwow, Poland.

1942 - Penicillin mass-produced in microbes.

1944 - Waksman isolates streptomycin, an effective antibiotic fortuberculosis.

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 – investigationon 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

„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 thephenotype”

Diseases

1.Monogenic diseases - inherited

2. Polygenic diseases – acquired


Genetic nature of diseases

Tools and products of medical biotechnology

1. Prevention

2. Diagnostics at the nucleic acid level

3. Treatment

3.1. application of recombinant DNA technologyfor drug development3.2. treatment at the nucleic acid level and by

means of nucleic acids3.2.1. genetic therapy3.2.2. cell therapy3.2.3. biomedical engineering

Prevention measures can be implemented at different stages of disease

1. Primary - promoting health prior to development of disease or injury- immunization; health promotion campaings

2. Secondary – detecting disease in its early (asymptomatic) stage- screening case finding, detection

3. Tertiary – reversing, arresting or delaying the progression of the disease- preventing complication of chronic diseases such as diabetes

including rehabilitation

4. Quaternary – avoiding consequences related to overmedication,overdiagnosis or incidental findings, eg. imaging - availability of medical based information from the internet - direct-to-consumer DNA genetic testing

From: RJ Trent – Molecular Medicine, Academic Press 2012

Genetic tests – detection of diseases

1. Cytogenetic analysis – chromosomes

2. Detection of mutations

- restriction enzymes & related techniques

- hybridisation: Southern blotting, Northern blotting

3. PCR technology

4. Sequencing

Cytogenetic diagnosis - chronic myeloid leukemia

AJ Trent – Molecular medicine, 2012

Cytogenetic diagnosis - chronic myeloid leukemia

First cancer for which the genetical mutation causing the disease has been identified (1960, Philadelphia)

- Mutated chromosome Philadelphia forms after translocation fragment of long arm of chromosome 9 (coding for Abl kinase) to long arm of chromosome 22 (coding for Bcr protein) (9 22)

- This mutation is present in 95% patients with CML; it can also be found in patients with other leukemias (e.g. in 15-30% cases of acute lymphoid leukemia)

- Translocation leads to formation of hybrid geneBcr/Abl and fusion protein of constitutive, unregulated kinase activity

AJ Trent – Molecular medicine, 2012

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 -

Methods to detect variations/changes in the genes/gene expression

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

Sir Edwin Southern

Restriction enzymes for molecular diagnostics

Detection of the sickle-cell globin gene by Southern blotting. The base change (A → T) that causes sickle-cell anemiadestroys an MstII target site that is present in the normal β-globin gene. This difference can be detected by Southernblotting. (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 DirectlyCopyright © 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.

Kary Mullis Nobel Prize 1993

Science, 19 June 2009

History of research on cystic fibrosis (1)

24

25

History of research on cystic fibrosis (2)

Cystic fibrosis transmembrane conductance regulator

168 kDa protein; 1480 aa residues

CFTR codes for a chloride ion channel

Over 1500 mutations produce CF; deltaF508 (D508) is the most common (in exon 10, interferes with ATP binding)

Cystic fibrosis – mutations in CFTR gene

deltaF508 mutation – deletion of one codon leading to loss of phenylalanine in amino acidposition 508

Detection of mutation by PCR

RJ Trent – Molecular medicine, 1997

The Cell: A Molecular Approach. 2nd edition.Cooper GM.Sunderland (MA): Sinauer Associates; 2000.

Heteroduplex formation in PCR

RFLP combined with PCR


When it is not possible to detect mutations using only enzyme digestion and Southern blotting

Polymerase chain reaction

1. Classical PCR

2. real-time PCR- quite expensive- cannot be used for a genome wide survey

Techniques used for detection of mutations

From: Molecular medicine, 1997

Genetic tests and risks of diseases


Life time risk of breast cancer:BRCA1 mutations – 50-80%BRCA2 mutations – 40-70%

Risk of ovarian cancerBRCA1 mutations – 40%BRCA2 mutations – 20%

Mutations in BRCA1 & BRCA2account only for 5-10% of allbreast and ovarian cances

HbS – one mutation; but some affectedheterozygotes will have a milder phenotypebecause of other genetic factors, such ascoexisting thalasemia, a raised HbF (boththese will reduce the level of HBS in the blood)

The purposes of DNA tests


Genetic tests

1.Preimplantation – after in vitro fertilisation

2.Prenatal diagnostics

3.Postnatal diagnostics


Preimplantation diagnostics

From: From biology to biotechnology…

Prenatal diagnostics

The breakthrough in basic research, genetic diagnostics and (potentially) treatment

Human genome project – HGP

Completed in 2003, the Human Genome Project (HGP) wasa 13-year project coordinated by the U.S. Department of Energyand 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

Sequencing of human genome and genomes of other organisms was possible thanks to the developement of DNA sequencing technology

Combination of automatic sequencing with PCR allowed the rapid analysisof the large number of sequences in a relatively short time

How this happened?

Maxam-Gilbert sequencingAllan 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 methodsThe 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 – Sanger technique

Molecular Biology of the Cell. 4th edition.Alberts B, Johnson A, Lewis J, et al.New York: Garland Science; 2002.

Frederick Sanger


DNA sequencing- Sanger method

Automated sequencing

Next breakthrough in genomic medicine

Microarray technologies

Source: Wikipedia

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.]

Microarray technologies

New generation sequencing

The Human Genome Project, which was launched in 1990 with theprimary 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 newtechnologies, which are collectively referred to as the next generationsequencing (NGS) platforms, as opposed to the Sanger method,which was used in the Human GenomeProject, 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.


Direct DNA SequencingThe cost of sequencing the entire human genome is expected to decrease to $1000 by the end of 2011 (not fullfilled – 2014). 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 technologieshave 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 sequencingbecause 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.



Next-Generation Sequencing PlatformsSydney Brenner, Nobel Laureate in Physiology and Medicine (2002), introduced the first technique of sequencing of millions of copies of the DNA simultaneously, referred to asMPSS 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 andapproximately 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 reversibleterminator-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 – various platforms


Next generation seqeuencing methodology

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 SequencingWhole 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.



Whole Exome SequencingThe 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 thatmore than two-thirds of the known disease-causing genes in humans are located within exons.

Traditional and next generation sequencing

Next generation seqeuencing

Next generation seqeuencing

The evolution of DNA sequencing

Progress in sequencing technology

Illumina

P. Brown – Genomes 2002

From gene to genome and further….

Next generation sequencing applications

KS Frese, Biology 2013,

Evolution of molecular medicine

RJ Trent – Molecular medicine 2012

Application of DNA recombination technology

Recombinant proteins

Monoclonal antibodies

Genelocalisation and function

Gene modification(mutations)

Forensic medicineMolecular

diagnosticsGene therapy

Transgenic Animals

Creation of new organisms

DNA recombinationtechnology

Next lectures:

15 March

22 March

5 April

12 April

19 April

26 April

Exam: planned on 24th May or 14th June

Thank you

Documents

Prof. Józef Dulak Email: [email protected]