Post on 04-Jun-2018
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
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
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.
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) 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.
The human genome
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.
Automated sequencing
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.
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