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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$
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
A.J. Marian, John Belmont - Circ Res. 2011;108:1252-1269
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
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 -
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
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
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
RJ Trent – Molecular medicine, 1997
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
Genetic tests and risks of diseases
RJ Trent – Molecular medicine, 2012
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)
Genetic tests
1.Preimplantation – after in vitro fertilisation
2.Prenatal diagnostics
3.Postnatal diagnostics
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.
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
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 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.
A.J. Marian, John Belmont - Circ Res. 2011;108:1252-1269
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.
New generation sequencing
A.J. Marian, John Belmont - Circ Res. 2011;108:1252-1269
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
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 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.
New generation sequencing
A.J. Marian, John Belmont - Circ Res. 2011;108:1252-1269
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.
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