THE CABINET of MINISTRI of UKRAINE NATIONAL UNIVERSITY of LIFE and ENVIROMENTAL SCIENCES of UKRAINE Faculty of biotechnology Department of molecular biology, microbiology and biosafety "APPROVED" The dean of faculty of biotechnology ______________ J. Kolomiets “___” _______________ 2011 SCIENTIFIC-METHODOLOGY COMPLEX Discipline "BIOSAFETY (use of biotechnologies)" For grounding of specialists in direction 0514 "Biotechnology" speciality 6.051401 – “Biotechnology” in agrarian higher educational institutions of III – IV levels of accreditation KYIV - 2011
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THE CABINET of MINISTRI of UKRAINE NATIONAL UNIVERSITY of LIFE and ENVIROMENTAL SCIENCES
of UKRAINE Faculty of biotechnology
Department of molecular biology, microbiology and biosafety
"APPROVED"
The dean of faculty of biotechnology
______________ J. Kolomiets
“___” _______________ 2011
SCIENTIFIC-METHODOLOGY COMPLEX
Discipline
"BIOSAFETY (use of biotechnologies)"
For grounding of specialists in direction 0514 "Biotechnology" speciality 6.051401 – “Biotechnology”
in agrarian higher educational institutions of III – IV levels of accreditation
KYIV - 2011
2
THE CABINET of MINISTRI of UKRAINE NATIONAL UNIVERSITY of LIFE and ENVIROMENTAL SCIENCES
of UKRAINE Faculty of biotechnology
Department of molecular biology, microbiology and biosafety
"APPROVED"
The dean of faculty of biotechnology
______________ J. V. Kolomiets
“___” _______________ 2011
WORKING EDUCATIONAL PROGRAM Discipline
"BIOSAFETY (use of biotechnologies)"
For grounding of specialists in direction 0514 "Biotechnology" speciality 6.051401 – “Biotechnology”
in agrarian higher educational institutions of III – IV levels of accreditation
Educational-qualification level "Bachelor's of Science Degree " Semester - 5 Number of weeks - 16 Number of ECTS credits – 4 Lectures - 16 hours Practice works – 32 hours Independent work under the supervision of lecturer - 16 hours. Independent work – 30 hours. Term paper – 75 hours. Final form of control: test – 7 hours.
KYIV - 2011
3
Working educational program was completed by Professor of department of molecular biology, microbiology and biosafety Starodub N.F.
Working educational program was discussed at the department of molecular biology, microbiology and biosafety Report № _____ from _____ .______________.2011. Head of department Patyka M.V. Decreed by methodological commission of Faculty of Biotechnology Report № ____ from . ___. _____________. 2011 Head of Educational-methodological council PhD, assistant of professor Kolomiets J. V. Secretary of Educational-methodological council Marchenko O.A. Author: professor, PhD Starodub M.F.
ANNOTATION of discipline
«Biosafety (use of biotechnologies)» Speciality 6.051401 – “Biotechnology”
In course of «Biosafety» it is studied the heredity and variability of organisms
with the new formed characteristics and their dispersion and possible role for biocenose. This course contents from two principal modules which include 8 theoretical
themes, 8 seminar-practical and laboratory training themes and 8 themes for the independent work. It allows to be acquainted in:
Modern conception about heredity and variability, their origin and molecular substance;
Understanding consequence of effect of scientific-technical progress on the planet gene pool, distinguishing positive and negative aspects of interaction of living organisms with the environment changing in result of climatologic, technical and informational reorganization;
Main methodological approaches for the control of genetic status of organisms; Modern analytical methods for the control of food and feed quality; Ethical aspects and problems of biosafety; Main rules and agreements in the field of biosafety which are accepted in Ukraine
and in other countries; Principles and mechanisms for manipulation with genome, achievements of
genetic engineering and therapy as well as modern biotechnologies, their advantage and risk for planet biosystem.
5
1. Memorandum
1.1. Place and role of discipline in the system of proficient training
Biosafety includes studying of the heredity and variability of organisms with the new formed characteristics and their dispersion and possible role for biocenose. Protection of the heredity of living organisms – it is preservation of life on the Earth in all its aspects and evolution. Because of activation of interaction of human and nature, increasing effect of endogenous factors on the heredity of living organisms, creation of new genetically modified organisms the problem of ecological changes in environment is arisen due to its contamination as well as appearance and dispersion of different types of biotechnologies including construction of transgenic organisms. People should know what kind of consequence will be in the result of non-controlled effect of appeared in environment mutagenic factors on the heredity of living organisms. People should understand all problems which are connected with the intensive involving: a) the genetically modified organisms to solve problems of deficit of products in countries of third world; b) environment recultivation from different types of toxic substances; c) synthesis and obtaining of pharmacological substances; d) improving quality of the existing plant sorts and animal species; e) use plants as factories for directed chemical synthesis of any substances and so on.
1.2. The main goals and tasks of discipline
The main goals of discipline are: theoretical and practical training of students for providing safe environment. The main task of discipline is forming of specialists which are able: to provide an analysis of quality and background of different species of plants,
animals and microorganisms used for biotechnological production; to provide selection of methods staff safety during technological processes.
1.3. Requirements to the knowledge and abilities gotten in the process of study of
discipline
In result of discipline studying the student should know: Modern conception about heredity and variability, their origin and
molecular substance; Understanding consequence of effect of scientific-technical progress on
the planet gene pool, distinguishing positive and negative aspects of interaction of living organisms with the environment changing in result of climatologic, technical and informational reorganization;
Main methodological approaches for the control of genetic status of organisms;
Modern analytical methods for the control of food and feed quality;
6
Ethical aspects and problems of biosafety; Main rules and agreements in the field of biosafety which are accepted
in Ukraine and in other countries; Principles and mechanisms for manipulation with genome,
achievements of genetic engineering and therapy as well as modern biotechnologies, their advantage and risk for planet biosystem.
Student should be able:
To use scientific, educational and methodical literature which concerned biosafety;
To analyze possible consequences of active and wide involving of genetically modified organisms and number of modern biotechnologies on the state of environment;
To be aligned in the use of the separate achievements of scientific-technical progress which are most non-destructive for living organisms and how much these achievements may be used without effect on the genetic pool of living organisms;
To estimate advantages and risk for people, animal and plants; the application of genetic engineering and modern technologies.
1.4 Lists of disciplines which are needed for the study of discipline
Studying of discipline «Biosafety (use of biotechnologies)» is based on the
knowledge which students get while studying of normative part of EPP – cycle of preparation of humanitarian, social, economical and nature-study professional and selected disciplines.
Student has to know: 1) the main philosophical denominations, which explain interrelations in working
collective, common intercommunication of things, development as passage quantitative changes to qualitative which have place in course “Philosophy of science and innovative development”
2) labor, labor process, object of labor, product of labor that are studied in course “International standardization, technology certification, raw material and ready products in agriculture”.
3) chemical elements and their interaction, chemical compounds that are used for producing biotechnological products and which are studied in course “Chemistry”;
4) the main methods of creating of organisms which synthesize biotechnological raw-stuff and are considered in course “General biotechnology”;
5) heredity and variability of living organisms, influence of mutations from “Genetics”
6) an influence of ecological factors to the quality of biotechnological products, which are studied in course “General ecology”.
7
2. THE PROGRAM STRUCTURE OF EDUCATIONAL DISCIPLINE «Biosafety (use of biotechnologies)»
Course:
preparation of bachelors
Form of studies: daily
Direction speciality
educationally qualifying level Description of educational disciplines
Amount of credits, correspond to ЕСТS:
Amount of credits - 4 (1 credit = 30 hours.)
Modules: 2+ term paper1 The substantial moduls:
amount of the modules - 2
General amount of hours: amount of hours - 146
A week's hours:
amount of hours - 2
Code and name of direction
0514 «Biotechnology»
Code and name of speciality
6.051401 – “Biotechnology” Educationally qualifying level
bachelor
Obligatory Year of preparation: 3. Semester: 1. Lectures (theoretical preparation):
amount of hours - 16 Practical work:
amount of hours - 32 Independent work:
amount of hours - 16 Individual work:
term paper amount of hours - 75
Type of control: test
1 Примітка До залікового кредиту можуть включатися окремі або всі модулі навчальної діяльності студента.
8
3. REFERENCE STRUCTURE OF TEST COURSE of educational discipline
«Biosafety (use of biotechnologies)»
№
Theme title Lectures Practical (seminar,
laboratory) training
Independent work of students
Individual work
The substantial module I Theme title amount of
hours amount of
hours amount of
hours amount of
hours 1 Biosafety, its main points and tasks.
General characteristics of separate directions of scientific-technical progress and possible variants of its effect on the genome of living organisms
2 4 2
2 Heredity and variability – basic abilities of living organisms. Molecular basis of heredity and variety.
2 4
2
3 Horizontal and vertical genes transfer. 2 4 2
4 Practical achievements of modern biotechnology and genetic engineering.
2 4 2
The substantial module II 1 Modern methods of molecular
genetics. Characteristics of mutations.
2 4 2
2 Biotechnologies of manipulation with genes. Genetically modified organisms: their main points, directions of use.
2 4
2
3 Problems of possible ecological consequences from use of genetically modified organisms.
2 4 2
4 Main rules and agreements in the field of biosafety. 2 4 2
Altogether: 146 16 32 16 75
9
3. MODULAR STRUCTURE OF DISCIPLINE «Biosafety (use of biotechnologies)»
Modular structure of discipline Form of control
Т.1. Biosafety, its main points and tasks. General characteristics of separate directions of scientific-technical progress and possible variants of its effect on the genome of living organisms
Т.2. Heredity and variability – basic abilities of living organisms. Molecular basis of heredity and variety.
Т.3. Horizontal and vertical genes transfer.
The substantial module 1
Т.4. Practical achievements of modern biotechnology and genetic engineering.
Т.1. Modern methods of molecular genetics. Characteristics of mutations.
Т.2. Biotechnologies of manipulation with genes. Genetically modified organisms: their main points, directions of use.
Т.3. Problems of possible ecological consequences from use of genetically modified organisms.
MO
DU
LE
The substantial module 1I
Т.4. Main rules and agreements in the field of biosafety.
Test
Final control of knowledges Final test Test
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4. TABLE OF CONTENTS OF EDUCATIONAL DISCIPLINE «Biosafety (use of biotechnologies)»
SUBSTANTIAL MODULE I.
Theme 1. Biosafety, its main points and tasks. General characteristics of separate
directions of scientific-technical progress and possible variants of its effect on the genome of living organisms
Unit of heredity – gene. Gene localization. Molecular structure of genes. Genome. Genome of pro- and eukaryotes. Natural mobile genetic elements, retrotransposones. Problems of application of hereditary and non-hereditary transgenic characters. Changing of hereditary during natural and industrial hybridization. Changing hereditary by methods of genetic engineering. Problems of protection of hereditary of organisms.
Theme 2. Heredity and variability – basic abilities of living organisms. Molecular
basis of heredity and variety. Unit of heredity – gene. Gene localization. Molecular structure of genes. Genome.
Genome of pro- and eukaryotes. Natural mobile genetic elements, retrotransposones. Problems of application of hereditary and non-hereditary transgenic characters. Changing of hereditary during natural and industrial hybridization. Changing hereditary by methods of genetic engineering. Problems of protection of hereditary of organisms.
Theme 3. Horizontal and vertical genes transfer.
Traditional intraspecific and interspecific hybridization (transference of gene blocks with different dimensions) plants, animals, microorganisms as basis of evolutionary process. Theme 4. Practical achievements of modern biotechnology and genetic engineering.
Obtaining of new pharmacological preparations (insulin, vaccine to poliomyelitis). Expression of human somatotropine (grown hormone) in the tobacco chloroplasts. Genetically modified plants (transgenic rice sorts, potatoes, maize, tomatoes and others). Tasks, achievements and problems of genetic engineering. Compensation of inherent genetic defects of maturity and treatment of diseases aroused during ontogenesis.
SUBSTANTIAL MODULE II.
Theme 1. Modern methods of molecular genetics. Characteristics of mutations.
Ferments of restriction. Vectors for the molecular cloning. Plasmids, bacteriophage, cosmide, shuttle vectors, artificial chromosomes of yeast. Creation of genomic libraries. Construction of restrictive maps. Southern blot analysis.
Mutations connected with the destruction of genetic code. Theme 2. Biotechnologies of manipulation with genes. Genetically modified
organisms: their main points, directions of use.
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Strategy of genetic engineering works. Preparation of DNA of the needed gene from genome. Transfer genes in the cells of other organisms: microinjection, electroporation, transfection, packing in liposomes, bombardment by micro-particles.
Overcoming problems which are connected with the intensive involving: a) the genetically modified organisms to solve problems of deficit of products in countries of third world; b) environment recultivation from different types of toxic substances; c) synthesis and obtaining of pharmacological preparations; d) improving quality of the existing plant sorts and animal species; e) using plants as factories for directed chemical synthesis of any substances and so on. Theme 3. Problems of possible ecological consequences from use of genetically
modified organisms. Possibility of GMO effect on environment. Advantages and risk. Principles of
caution and sufficient equivalence. Marking genetically modified foods, feeds, seed and medical preparations. Theme 4. Main rules and agreements in the field of biosafety.
Cartagena protocol and Orchuskaja convencion. Codex of Alimentariusa. Bilbao and Inujama Declarations. General declaration of JUNESKO about genome and rights of human.
12
THEMES OF PRACTICAL TREININGS “Biosafety (use of biotechnologies)”
SUBSTANTIAL MODULE I.
Practical training №1.
Structure of DNA and RNA, replication, transcription, and translation. Construction of genome and chromosome libraries (4 h)
Practical training №2.
Classical immune analysis and its use for the determination of quality and origin foods and feeds (4 h)
Practical training №3.
Monoclonal antibodies and their use in immune analysis (4 h)
Practical training №4.
Modern immune chemical analysis: varieties and its use at the providing of biosafety (4 h)
SUBSTANTIAL MODULE II.
Practical training №1.
Documents in the field of biosafety which regulate the use of genetically modified organisms in different aspects (4 h)
Practical training №2.
Familiarization with the fulfillment of ELISA-method (4 h)
Practical training №3.
Fulfillment of instrumental analysis for the revealing of individual substances in samples of water and some foods at the registration of biospecific interactions by the
optical biosensor based on the surface plasmon resonance (4 h)
Practical training №4.
DNA electrophoresis (4 h)
13
НУБіП України Ф-7.5-2.1.8-05
National University of Life and Environmental Science of Ukraine
THE CALENDAR THEMATIC PLAN For preparation of experts in direction 0514 "Biotechnology"
from a speciality 6.051401 – “Biotechnology” from course “ Biosafety (use of biotechnologies)”
1st semester
2011/2012 academic year
APPROVED: Dean of Faculty of Biotechnology J. V. Kolomiets /_________________________ Professor M.F. Starodub /_________________________ Number of weeks 16 Lections 16 Practice works 32 Independent work 16 Term paper 75 In all 146
Week Topic of lectures Hours Topic of practice works Hours. Topic of independent work under the supervision of lecturer Hours
1 2 3 4 5 6
1-2
Biosafety, its main points and tasks. General characteristics of separate directions of scientific-technical progress and possible variants of its effect on the genome of living organisms
2
Structure of DNA and RNA, replication, transcription, and translation. Construction of genome and chromosome libraries
4 Cartagena protocol and Orchuskaja convencion 2
3-4 Heredity and variability – basic abilities of living organisms. 2
Classical immune analysis and its use for the determination of quality and origin foods and feeds
4 Codex of Alimentariusa 2
5-6 Horizontal and vertical gene transfer 2 Monoclonal antibodies and
their use in immune analysis 4 Bilbao and Inujama Declarations 2
7-8 Practical achievements of modern biotechnology and genetic engineering.
2 Modern immune chemical analysis: varieties and its use at the providing of biosafety
4 General declaration of JUNESKO about genome and rights of human 2
14
9-10 Modern methods of molecular genetics. Characteristics of mutations.
2
Documents in the field of biosafety which regulate the use of genetically modified organisms in different aspects
4 Ukrainian low about fulfillment of works in the field of genetic engineering and GMO application
2
11-12
Biotechnologies of manipulation with genes. Genetically modified organisms: their main points, directions of use.
2 Familiarization with the fulfillment of ELISA-method 4
Ways of obtaining of polyclonal antibodies and their application at the analysis of food and feed qualities
2
13-14 Problems of possible ecological consequences from use of genetically modified organisms
2
Fulfillment of instrumental analysis for the revealing of individual substances in samples of water and some foods at the registration of biospecific interactions by the optical biosensor based on the surface plasmon resonance
4
New type of instrumental analytical devices – biosensors: their varieties and directions of application. Bioterrorism: peculiarities, varieties, dangerous and ways to avoid consequences
2
15-16 Main rules and agreements in the field of biosafety. Ecologic-genetical models.
2 DNA electrophoresis 4
Water resources: their use and control. Surface active substances, their potential dangerous for living organisms
2
Lecturer of the course _____________________ prof. Starodub N.F Chief of department _____________________prof. Patyka M.V.
15
НУБіП України Ф-7.5-2.1.8-03
Protocol co-ordination of working educational program of discipline “Biosafety (use of biotechnologies)”
with other disciplines 6.051401 – “Biotechnology”
Discipline and its sections which are before studying of
this discipline
Family and name, academic degree of lecturer which
implements previous discipline Signature
Next discipline and its divisions in frame of which materials of this discipline
are used
Family and name, academic degree of lecturer which
implements next discipline Signature
General and microbiology Fedelesh-Gladynets M.Y. PhD Immune genetics Starodub M.F. PhD
Cell biology Balanda O.V. PhD Methodology and
organization of scientific experiment
Starodub M.F. PhD
16
НУБіП України Ф-7.5-2.1.8-04
Structural scheme of discipline “Biosafety (use of biotechnologies)”
Number of
module Charter of discipline Topic of lectures Topic of practice works Form of
control
1 1
Biosafety, its main points and tasks. General characteristics of separate directions of scientific-technical progress and possible variants of its effect on the genome of living organisms
Cartagena protocol and Orchuskaja convencion
Tesr
1 1
Heredity and variability – basic abilities of living organisms. Molecular basis of heredity and variety.
Codex of Alimentariusa Tesr
1 1 Horizontal and vertical genes transfer.
Bilbao and Inujama Declarations Tesr
1 1 Practical achievements of modern biotechnology and genetic engineering.
General declaration of JUNESKO about genome and rights of human
Tesr
1 2
Modern methods of molecular genetics. Characteristics of mutations.
Ukrainian low about fulfillment of works in the field of genetic engineering and GMO application
Tesr
1 2
Biotechnologies of manipulation with genes. Genetically modified organisms: their main points, directions of use.
Ways of obtaining of polyclonal antibodies and their application at the analysis of food and feed qualities
Tesr
2 2
Problems of possible ecological consequences from use of genetically modified organisms.
New type of instrumental analytical devices – biosensors: their varieties and directions of application. Bioterrorism: peculiarities, varieties, dangerous and ways to avoid consequences
Tesr
2 2
Main rules and agreements in the field of biosafety.
Water resources: their use and control. Surface active substances, their potential dangerous for living organisms
Tesr
17
Modular structure of discipline Form
of control Т.1. Т.2. Т.3.
The substantial module 1
Т.4.
Т.1. Т.2. Т.3.
MO
DU
LE
The substantial module 1I
Т.4.
Test
Final control of knowledges Final test Test
18
5. THE METHODS AND SCALE OF STUDENTS KNOWLEDGE ESTIMATION
THE METHODS OF ESTIMATION: examinations while the semester; (progect; report;) total written test.
Balls distribution:
Examinations while the semester
The substantial module 1 The substantial module 1I Common balls quantity Common balls quantity
Т. 1 Т. 2 Т. 3 Т. 4 Т. 1 Т. 2 Т. 3 Т. 4
Indi
vidu
al
scie
ntifi
c pr
ojec
t
Tota
l con
trol
Sum
5 bolls 5 5 5 5 5 10 5 25 30 100
19
Criteria of appreciation of students knowledge’s on intermediate and final phases of studying
Student knowledge’s are appreciated according to system of modular-rate control. Whole programmed material of the “Biosafety” course devided on two blocks – module: Module A – “Biotechnologies for manipulation with genes”: Module B – “Lows and ecological-genetic aspects of biosafety”.
Calculated rates of discipline are equal 100 points. Educational rates – 70 points.
Taking into account volume and structure of programmed material of discipline it was divided it for two appropriate modules. Calculated rate mark of each module was taken on the level 35 points. Minimal rate mark for each module is 17,5 points.
For each module it is planed test which includes 30 questions. Each question contents 4 answers one of which is right.
Test is in writing and individual for each student. Rial rate of student with educational work will be estimated according to
obtained points at the module fulfillment. To be present at the test the student should have no less as 50% of planed rate from the educational work. According to “Rules about module-rate system of education of students and appreciation of their knowledge’s” it will be written “credit” in the student's record-book. Rate mark will be included according to system of ЕСТS (A, B, C, D, E, FX, F) in special list.
The results of studying of discipline by students content a weighted average rate. Student rate of discipline equal to sum of educational work rate and rate of test. Lecturer prof. Starodub M.F.
20
6. INDEPENDENT WORK UNDER THE SUPERVISION OF LECTURER
Task 1 (2 hours)
Cartagena protocol and Orchuskaja convencion. Codex of Alimentariusa.
Task 2 (2 hours)
General declaration of JUNESKO about genome and rights of human.
Task 3 (2 hours)
Ukrainian low about fulfillment of works in the field of genetic engineering and GMO
application.
Task 4 (2 hours)
Ways of obtaining of polyclonal antibodies and their application at the analysis of food
and feed qualities.
Task 5 (2 hours)
New type of instrumental analytical devices – biosensors: their varieties and directions
of application.
Task 6 (2 hours)
Water resources: their use and control. Polymeric packing – its advantages and lags in
the comparison with the traditional materials.
Task 7 (2 hours)
Surface active substances, their potential dangerous for living organisms.
Task 8 (2 hours)
Bioterrorism: peculiarities, varieties, dangerous and ways to avoid consequences.
Ultraviolet radiation: effect on the genetic system of living organisms. Modern mobile
connection – possible physiological and genetic consequences.
21
THE RECOMMENDED LITERATURE
Main
1. Ghimulev I.F. General and molecular genetics: School-book. – Novosibirsk,
traits only, but maize and cotton carried both herbicide tolerance and insect protection traits (the latter
largely the Bacillus thuringiensus Bt insecticidal protein). In the period 2002 to 2006, there were
significant increases in the area planted to Bt protected cotton and maize, and herbicide tolerant maize
also increased in sown area.
Lecture 6.
Modern methods of molecular genetics.
Experimental breeding. Genetically diverse lines of organisms can be crossed in such a way to
produce different combinations of alleles in one line. For example, parental lines are crossed,
producing an F1 generation, which is then allowed to undergo random mating to produce offspring that
have purebreeding genotypes (i.e., AA, bb, cc, or DD). This type of experimental breeding is the origin
of new plant and animal lines, which are an important part of making laboratory stocks for basic
research. When applied to commerce, transgenic commercial lines produced experimentally are called
genetically modified organisms (GMOs). Many of the plants and animals used by humans today (e.g.,
cows, pigs, chickens, sheep, wheat, corn (maize), potatoes, and rice) have been bred in this way.
Cytogenetic techniques. Cytogenetics focuses on the microscopic examination of genetic
components of the cell, including chromosomes, genes, and gene products. Older cytogenetic
techniques involve placing cells in paraffin wax, slicing thin sections, and preparing them for
microscopic study. The newer and faster squash technique involves squashing entire cells and studying
their contents. Dyes that selectively stain various parts of the cell are used; the genes, for example,
may be located by selectively staining the DNA of which they are composed. Radioactive and
fluorescent tags are valuable in determining the location of various genes and gene products in the cell.
Tissue-culture techniques may be used to grow cells before squashing; white blood cells can be grown
from samples of human blood and studied with the squash technique. One major application of
cytogenetics in humans is in diagnosing abnormal chromosomal complements such as Down syndrome
(caused by an extra copy of chromosome 21) and Klinefelter syndrome (occuring in males with an
extra X chromosome). Some diagnosis is prenatal, performed on cell samples from amniotic fluid or
the placenta.
Biochemical techniques. Biochemistry is carried out at the cellular or subcellular level,
generally on cell extracts. Biochemical methods are applied to the main chemical compounds of
genetics—notably DNA, RNA, and protein. Biochemical techniques are used to determine the
activities of genes within cells and to analyze substrates and products of gene-controlled reactions. In
one approach, cells are ground up and the substituent chemicals are fractionated for further analysis.
29
Special techniques (e.g., chromatography and electrophoresis) are used to separate the components of
proteins so that inherited differences in their structures can be revealed. For example, more than 100
different kinds of human hemoglobin molecules have been identified. Radioactively tagged
compounds are valuable in studying the biochemistry of whole cells. For example, thymine is a
compound found only in DNA; if radioactive thymine is placed in a tissue-culture medium in which
cells are growing, genes use it to duplicate themselves. When cells containing radioactive thymine are
analyzed, the results show that, during duplication, the DNA molecule splits in half, and each half
synthesizes its missing components.
Molecular techniques. Although overlapping with biochemical techniques, molecular genetics
techniques are deeply involved with the direct study of DNA. This field has been revolutionized by the
invention of recombinant DNA technology. The DNA of any gene of interest from a donor organism
(such as a human) can be cut out of a chromosome and inserted into a vector to make recombinant
DNA, which can then be amplified and manipulated, studied, or used to modify the genomes of other
organisms by transgenesis. A fundamental step in recombinant DNA technology is amplification. This
is carried out by inserting the recombinant DNA molecule into a bacterial cell, which replicates and
produces many copies of the bacterial genome and the recombinant DNA molecule (constituting a
DNA clone). A collection of large numbers of clones of recombinant donor DNA molecules is called a
genomic library. Such libraries are the starting point for sequencing entire genomes such as the human
genome. Today genomes can be scanned for small molecular variants called single nucleotide
polymorphisms, or SNPs (“snips”), which act as chromosomal tags to associated specific regions of
DNA that have a property of interest and may be involved in a human disease or disorder.
Lecture 7.
Characteristics of mutations.
In biology, mutations are changes to the nucleotide sequence of the genetic material of an
organism. Mutations can be caused by copying errors in the genetic material during cell division, by
exposure to ultraviolet or ionizing radiation, chemical mutagens, or viruses, or can be induced by the
organism itself, by cellular processes such as hypermutation. In multicellular organisms with dedicated
reproductive cells, mutations can be subdivided into germ line mutations, which can be passed on to
descendants through the reproductive cells, and somatic mutations, which involve cells outside the
dedicated reproductive group and which are not usually transmitted to descendants. If the organism
can reproduce asexually through mechanisms such as cuttings or budding the distinction can become
blurred. For example, plants can sometimes transmit somatic mutations to their descendants asexually
or sexually where flower buds develop in somatically mutated parts of plants. A new mutation that was
30
not inherited from either parent is called a de novo mutation. The source of the mutation is unrelated to
the consequence, although the consequences are related to which cells were mutated.
Mutations create variation within the gene pool. Less favorable (or deleterious) mutations can
be reduced in frequency in the gene pool by natural selection, while more favorable (beneficial or
advantageous) mutations may accumulate and result in adaptive evolutionary changes. For example, a
butterfly may produce offspring with new mutations. The majority of these mutations will have no
effect; but one might change the color of one of the butterfly's offspring, making it harder (or easier)
for predators to see. If this color change is advantageous, the chance of this butterfly surviving and
producing its own offspring are a little better, and over time the number of butterflies with this
mutation may form a larger percentage of the population.
Neutral mutations are defined as mutations whose effects do not influence the fitness of an
individual. These can accumulate over time due to genetic drift. It is believed that the overwhelming
majority of mutations have no significant effect on an organism's fitness. Also, DNA repair
mechanisms are able to mend most changes before they become permanent mutations, and many
organisms have mechanisms for eliminating otherwise permanently mutated somatic cells.
Mutation is generally accepted by the scientific community as the mechanism upon which
natural selection acts, providing the advantageous new traits that survive and multiply in offspring or
disadvantageous traits that die out with weaker organisms.
Lecture 8.
Biotechnologies of manipulation with genes.
Genetic engineering, recombinant DNA technology, genetic modification/manipulation (GM)
and gene splicing are terms that apply to the direct manipulation of an organism's genes. Genetic
engineering is different from traditional breeding, where the organism's genes are manipulated
indirectly. Genetic engineering uses the techniques of molecular cloning and transformation to alter the
structure and characteristics of genes directly. Genetic engineering techniques have found some
successes in numerous applications.
There are a number of ways through which genetic engineering is accomplished. Essentially,
the process has five main steps:
1. Isolation of the genes of interest
2. Insertion of the genes into a transfer vector
3. Transfer of the vector to the organism to be modified
4. Transformation of the cells of the organism
5. Selection of the genetically modified organism (GMO) from those that have not been
successfully modified
31
Isolation is achieved by identifying the gene of interest that the scientist wishes to insert into
the organism, usually using existing knowledge of the various functions of genes. DNA information
can be obtained from cDNA or gDNA libraries, and amplified using PCR techniques. If necessary, i.e.
for insertion of eukaryotic genomic DNA into prokaryotes, further modification may be carried out
such as removal of introns or ligating prokaryotic promoters.
Insertion of a gene into a vector such as a plasmid can be done once the gene of interest is
isolated. Other vectors can also be used, such as viral vectors, bacterial conjugation, liposomes, or
even direct insertion using a gene gun. Restriction enzymes and ligases are of great use in this crucial
step if it is being inserted into prokaryotic or viral vectors. Daniel Nathans and Hamilton Smith
received the 1978 Nobel Prize in Physiology or Medicine for their isolation of restriction
endonucleases.
Once the vector is obtained, it can be used to transform the target organism. Depending on the
vector used, it can be complex or simple. For example, using raw DNA with gene guns is a fairly
straightforward process but with low success rates, where the DNA is coated with molecules such as
gold and fired directly into a cell. Other more complex methods, such as bacterial transformation or
using viruses as vectors have higher success rates.
After transformation, the GMO can be selected from those that have failed to take up the vector
in various ways. One method is screening with DNA probes that can stick to the gene of interest that
was supposed to have been transplanted. Another is to package genes conferring resistance to certain
chemicals such as antibiotics or herbicides into the vector. This chemical is then applied ensuring that
only those cells that have taken up the vector will survive.
9. Genetically modified organisms: their main points, directions of use.
A genetically modified organism (GMO) or genetically engineered organism (GEO) is an
organism whose genetic material has been altered using genetic engineering techniques. These
techniques, generally known as recombinant DNA technology, use DNA molecules from different
sources, which are combined into one molecule to create a new set of genes. This DNA is then
transferred into an organism, giving it modified or novel genes. Transgenic organisms, a subset of
GMOs, are organisms which have inserted DNA that originated in a different species. Some GMOs
contain no DNA from other species and are therefore not transgenic but cisgenic.
GMOs have widespread applications. They are used in biological and medical research,
production of pharmaceutical drugs, experimental medicine (e.g. gene therapy), and agriculture (e.g.
golden rice). The term "genetically modified organism" does not always imply, but can include,
targeted insertions of genes from one species into another. For example, a gene from a jellyfish,
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encoding a fluorescent protein called GFP, can be physically linked and thus co-expressed with
mammalian genes to identify the location of the protein encoded by the GFP-tagged gene in the
mammalian cell. Such methods are useful tools for biologists in many areas of research, including
those who study the mechanisms of human and other diseases or fundamental biological processes in
eukaryotic or prokaryotic cells.
To date the broadest application of GMO technology is patent-protected food crops which are
resistant to commercial herbicides or are able to produce pesticidal proteins from within the plant, or
stacked trait seeds, which do both. The largest share of the GMO crops planted globally are owned by
Monsanto according to the company. In 2007, Monsanto’s trait technologies were planted on 246
million acres (1,000,000 km2) throughout the world, a growth of 13 percent from 2006.
In the corn market, Monsanto’s triple-stack corn – which combines Roundup Ready 2 weed
control technology with YieldGard Corn Borer and YieldGard Rootworm insect control – is the market
leader in the United States. U.S. corn farmers planted more than 17 million acres (69,000 km2) of
triple-stack corn in 2007, and it is estimated the product could be planted on 45 million to 50 million
acres (200,000 km2) by 2010. In the cotton market, Bollgard II with Roundup Ready Flex was planted
on nearly 3 million acres (12,000 km2) of U.S. cotton in 2007. Rapid growth in the total area planted is
measurable by Monsanto's growing share. On January 3, 2008, Monsanto Company (MON.N) said its
quarterly profit nearly tripled, helped by strength in its corn seed and herbicide businesses, and raised
its 2008 forecast. According to the International Service for the Acquisition of Agri-Biotech
Applications (ISAAA), of the approximately 8.5 million farmers who grew biotech crops in 2005,
some 90% were resource-poor farmers in developing countries. These include some 6.4 million
farmers in the cotton-growing areas of China, an estimated 1 million small farmers in India,
subsistence farmers in the Makhathini flats in KwaZulu Natal province in South Africa, more than
50,000 in the Philippines and in seven other developing countries where biotech crops were planted in
2005. ISAAA estimated that by 2008, 13.3 million farmers were growing GM crops, including 12.3
million in developing counties,. These comprised 7.1 million in China (Bt cotton), 5.0 million in India
(Bt cotton), and 200,000 in the Philippines.
"The Global Diffusion of Plant Biotechnology: International Adoption and Research in 2004",
a study by Dr. Ford Runge of the University of Minnesota, estimates the global commercial value of
biotech crops grown in the 2003–2004 crop year at US$44 billion. In the United States the United
States Department of Agriculture (USDA) reports on the total area of GMO varieties planted.
According to National Agricultural Statistics Service, the States published in these tables represent 81-
86 percent of all corn planted area, 88-90 percent of all soybean planted area, and 81-93 percent of all
upland cotton planted area (depending on the year). See more on the extent of adoption at:
http://www.ers.usda.gov/Data/BiotechCrops/. USDA does not collect data for global area. Estimates
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are produced by the International Service for the Acquisition of Agri-biotech Applications (ISAAA)
and can be found in the report, Global Status of Commercialized Transgenic Crops: 2007. Transgenic
animals are also becoming useful commercially. On 6 February 2009 the U.S. Food and Drug
Administration approved the first human biological drug produced from such an animal, a goat. The
drug, ATryn, is an anticoagulant which reduces the probability of blood clots during surgery or
childbirth. It is extracted from the goat's milk.
Lecture 10.
Problems of possible ecological consequences from use of genetically modified organisms.
Genetically-modified foods have the potential to solve many of the world's hunger and
malnutrition problems, and to help protect and preserve the environment by increasing yield and
reducing reliance upon chemical pesticides and herbicides. Yet there are many challenges ahead for
governments, especially in the areas of safety testing, regulation, international policy and food
labeling. Many people feel that genetic engineering is the inevitable wave of the future and that we
cannot afford to ignore a technology that has such enormous potential benefits. However, we must
proceed with caution to avoid causing unintended harm to human health and the environment as a
result of our enthusiasm for this powerful technology.
WHO will take an active role in relation to GM foods, primarily for two reasons:
(1) on the grounds that public health could benefit enormously from the potential of
biotechnology, for example, from an increase in the nutrient content of foods, decreased allergenicity
and more efficient food production; and (2) based on the need to examine the potential negative effects
on human health of the consumption of food produced through genetic modification, also at the global
level. It is clear that modern technologies must be thoroughly evaluated if they are to constitute a true
improvement in the way food is produced. Such evaluations must be holistic and all-inclusive, and
cannot stop at the previously separated, non-coherent systems of evaluation focusing solely on human
health or environmental effects in isolation.
Work is therefore under way in WHO to present a broader view of the evaluation of GM foods
in order to enable the consideration of other important factors. This more holistic evaluation of GM
organisms and GM products will consider not only safety but also food security, social and ethical
aspects, access and capacity building. International work in this new direction presupposes the
involvement of other key international organizations in this area. As a first step, the WHO Executive
Board will discuss the content of a WHO report covering this subject in January 2003. The report is
being developed in collaboration with other key organizations, notably FAO and the United Nations
Environment Programme (UNEP). It is hoped that this report could form the basis for a future
initiative towards a more systematic, coordinated, multi-organizational and international evaluation of
certain GM foods.
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Ecological risks. The potential impact on nearby ecosystems is one of the greatest concerns
associated with transgenic plants. Transgenes have the potential for significant ecological impact if the
plants can increase in frequency and persist in natural populations. These concerns are similar to those
surrounding conventionally bred plant breeds. Several risk factors should be considered:
• Is the transgenic plant capable of growing outside a cultivated area?
• Can the transgenic plant pass its genes to a local wild species, and are the offspring also
fertile?
• Does the introduction of the transgene confer a selective advantage to the plant or to
hybrids in the wild?
Many domesticated plants can mate and hybridise with wild relatives when they are grown in
proximity, and whatever genes the cultivated plant had can then be passed to the hybrid. This applies
equally to transgenic plants and conventionally bred plants, as in either case there are advantageous
genes that may have negative consequences to an ecosystem upon release. This is normally not a
significant concern, despite fears over 'mutant superweeds' overgrowing local wildlife: although hybrid
plants are far from uncommon, in most cases these hybrids are not fertile due to polyploidy, and will
not multiply or persist long after the original domestic plant is removed from the environment.
However, this does not negate the possibility of a negative impact.
In some cases, the pollen from a domestic plant may travel many miles on the wind before
fertilising another plant. This can make it difficult to assess the potential harm of crossbreeding; many
of the relevant hybrids are far away from the test site. Among the solutions under study for this
concern are systems designed to prevent transfer of transgenes, such as Terminator Technology, and
the genetic transformation of the chloroplast only, so that only the seed of the transgenic plant would
bear the transgene. With regard to the former, there is some controversy that the technologies may be
inequitable and might force dependence upon producers for valid seed in the case of poor farmers,
whereas the latter has no such concern but has technical constraints that still need to be overcome.
Solutions are being developed by EU funded research programmes such as Co-Extra and
Transcontainer.
There are at least three possible avenues of hybridization leading to escape of a transgene:
• Hybridization with non-transgenic crop plants of the same species and variety.
• Hybridization with wild plants of the same species.
• Hybridization with wild plants of closely related species, usually of the same genus.
However, there are a number of factors which must be present for hybrids to be created.
• The transgenic plants must be close enough to the wild species for the pollen to reach
the wild plants.
• The wild and transgenic plants must flower at the same time.
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• The wild and transgenic plants must be genetically compatible.
In order to persist, these hybrid offspring:
• Must be viable, and fertile.
• Must carry the transgene.
Studies suggest that a possible escape route for transgenic plants will be through hybridization
with wild plants of related species.
1. It is known that some crop plants have been found to hybridize with wild counterparts.
2. It is understood, as a basic part of population genetics, that the spread of a transgene in
a wild population will be directly related to the fitness effects of the gene in addition to the rate of
influx of the gene to the population. Advantageous genes will spread rapidly, neutral genes will spread
with genetic drift, and disadvantageous genes will only spread if there is a constant influx.
3. The ecological effects of transgenes are not known, but it is generally accepted that
only genes which improve fitness in relation to abiotic factors would give hybrid plants sufficient
advantages to become weedy or invasive. Abiotic factors are parts of the ecosystem which are not
alive, such as climate, salt and mineral content, and temperature. Genes improving fitness in relation to
biotic factors could disturb the (sometimes fragile) balance of an ecosystem. For instance, a wild plant
receiving a pest resistance gene from a transgenic plant might become resistant to one of its natural
pests, say, a beetle. This could allow the plant to increase in frequency, while at the same time animals
higher up in the food chain, which are at least partly dependent on that beetle as food source, might
decrease in abundance. However, the exact consequences of a transgene with a selective advantage in
the natural environment are almost impossible to predict reliably.
It is also important to refer to the demanding actions that government of developing countries
had been building up among the last decades.
Agricultural impact of transgenic plant. Outcrossing of transgenic plants not only poses
potential environmental risks, it may also trouble farmers and food producers. Many countries have
different legislations for transgenic and conventional plants as well as the derived food and feed, and
consumers demand the freedom of choice to buy GM-derived or conventional products. Therefore,
farmers and producers must separate both production chains. This requires coexistence measures on
the field level as well as traceability measures throughout the whole food and feed processing chain.
Research projects such as Co-Extra, SIGMEA and Transcontainer investigate how farmers can avoid
outcrossing and mixing of transgenic and non-transgenic crops, and how processors can ensure and
verify the separation of both production chains.
Lecture 11.
Main rules and agreements in the field of biosafety.
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The Cartagena Protocol on Biosafety regulates genetic engineering. It is a remarkable
achievement in international law, given the determination of GMO producer/exporter countries and the
biotechnology industry to block global regulation. The following article outlines the progress so far
and examines the challenges ahead. THE Cartagena Protocol on Biosafety (CPB) entered into force on
11 September 2003.
The EU Directive on the deliberate release of GMOs into the environment (Directive
2001/18/EC) has been applicable since 17 October 2002. It applies to the deliberate release into the
environment of GMOs and the placing on the market of GMOs as such or in products. The Directive
strengthens previous legislation, requiring more detailed pre-market scientific
evaluation and risk assessment of GMOs, and specifically refers to the Precautionary Principle.
Mandatory post-market monitoring and general surveillance will allow potential longer-term effects to
be followed.
The Regulation on GM food and feed (Regulation (EC) No 1829/2003) applies to the
evaluation, authorisation and labelling of GM food and feed. It has been in force since 7 November
2003 and will be applicable as of April 2004. This legislation extends the scope of previous regulation
to now include feed produced from GMOs and all products derived from GMOs, irrespective of
whether the DNA or protein is detectable. It also improves on the approvals process and can require
post-market monitoring. The threshold that triggers labelling has been lowered from 1.0% to 0.9%,
provided the presence of the authorised GMO in the final product is ‘technically unavoidable’.
The Regulation on traceability and labelling of GMOs and the traceability of food and feed
produced from GMOs (Regulation (EC) No 1830/2003) provides a traceability framework for GMOs,
GM food and GM feed. It entered into force on 7 November 2003. Traceability is defined as the ability
to trace GMOs and products produced from GMOs at all stages throughout the production and
distribution chains. Apart from providing the possibility of withdrawing products when problems arise,
it is also important for meaningful labelling of GMOs and in addressing liability issues.
The Regulation on trans-boundary movements of GMOs (Regulation (EC) No 1946/2003)
implements the EU’s obligations under the Protocol. In accordance with the Protocol’s AIA procedure,
no first export of GMOs intended for deliberate release into the environment can be carried out without
the prior written express consent of the importing country. The regulation recognises the flexibilities in
the Protocol that preserve the right of importing countries to take (stricter) decisions according to their
domestic regulatory frameworks, for GMOs intended for deliberate release and for food, feed and
processing. It also recognises the right of countries to set standards for contained use and to regulate
transit of GMOs. In the absence of domestic legislation, the provisions of the CPB apply and the prior
written express consent provision holds.
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In the United States the Coordinated Framework for Regulation of Biotechnology governs the
regulation of transgenic organisms, including plants. The three agencies involved are:
USDA Animal and Plant Health Inspection Service - who state that:
The Biotechnology Regulatory Services (BRS) program of the U.S. Department of
Agriculture’s (USDA) Animal and Plant Health Inspection Service (APHIS) is responsible for
regulating the introduction (importation, interstate movement, and field release) of genetically
engineered (GE) organisms that may pose a plant pest risk. BRS exercises this authority through
APHIS regulations in Title 7, Code of Federal Regulations, Part 340 under the Plant Protection Act of
2000. APHIS protects agriculture and the environment by ensuring that biotechnology is developed
and used in a safe manner. Through a strong regulatory framework, BRS ensures the safe and confined
introduction of new GE plants with significant safeguards to prevent the accidental release of any GE
material. APHIS has regulated the biotechnology industry since 1987 and has authorized more than
10,000 field tests of GE organisms. In order to emphasize the importance of the program, APHIS
established BRS in August 2002 by combining units within the agency that dealt with the regulation of
biotechnology. Biotechnology, Federal Regulation, and the U.S. Department of Agriculture, February
2006, USDA-APHIS Fact Sheet.
EPA - evaluates potential environmental impacts, especially for genes which encode for
pesticide production.
DHHS, Food and Drug Administration (FDA) - evaluates human health risk if the plant
is intended for human consumption.
Ukraine has adopted its Law ”On the State System of Biosafety in Creating, Testing,
Transporting and Using Genetically-Modified Organisms”, which regulates relations between
executive authorities, manufacturers, vendors (suppliers), developers, researchers, scholars and
consumers of genetically-modified organisms and products manufactured by technologies envisaging
their development, creation, testing, study, transportation, import, export, marketing, discharge to the
environment and use of genetically modified organisms in the Ukraine, and ensuring biological and
genetic safety. The Law shall not apply to humans, tissues and individual cells being part of a human
body. Since November 1st, 2007 Ukraine also enforced the Government’s Decree #985 from August
1st, 2007 ”On Matters Related to the Circulation of Food Products Containing Genetically Modified
Organisms and/or Microorganisms”, which enacts compulsory labeling of such products and bans
”import, manufacturing, and sales of children’s food products containing genetically modified
organisms and/or microorganisms”. According to the text of the resolution such measures are taken
”…in order to bring Ukrainian laws into compliance with the standards of the European Union”.
Law of Ukraine #1103-V “On State Safety System while Creating, Testing, Transporting and
Implementing Genetically Modified Organisms” of May 31, 2007, is an important step forward in
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regulating application of GMO to minimize biological risk and to guarantee customer’s right to make a
choice. At the same time the law is missing clear definitions of key terms and ideas (for example,
“biological safety”). There is no regulation for classifying risks. But the main disadvantage is a lack of
a competent controlling body to ensure safety measures for creating, testing, registering, transporting,
using and utilizing GMO. Greens are convinced that efficiency of law implementation depends on
developing by-law normative-legal base to regulate handling of GMO.
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NATIONAL UNIVERSITY of LIFE and ENVIROMENTAL SCIENCES of UKRAINE Faculty of biotechnology Speciality 6.05140101 “Biotechnology” Full-time department specialists in direction 0929 "Biotechnology" Educational-qualification level "Bachelor's of Science Degree " Semester - 5 Department of molecular biology, microbiology and biosafety Discipline “Biosafety (use of biotechnologies)” Lecturer – Starodub N.F. “Approved” Head of department__________Patyka M.V. “ ”__________________2011
№1 Reaction of immune diffusion is: 1 Quantitative 2 Semi-quantitative 3 Qualitative 4 None
№2 What kind of the listed approach of immune enzymatic analysis cannot be fulfilled: 1 On the plates 2 DOT 3 Blot 4 In solution
№3 What kind of label does not use in modern immune chemical analysis____________
№4 What kind of classical immune analysis is as quantitative?____________________
№5 What kind of Immune enzymatic analysis does not exist: 1 Hetero-phase 2 Solid-phase 3 Homogeny 4 Heterocyclic
№6 Data of discovering of modern immune chemical analysis is____________________
№7 Immune chemical analysis by “Dot” way is fulfilled by:_______________________
№8 “Blot” immune chemical analysis is fulfilled by:______________________________
№12 Haptens are able: 1 Induce immune response 2 To interact with antibodies 3 To polymerization 4 To spontaneous interaction with carbohydrates
№13 Classical immune analysis is based on: _____________________________________
№14 What kind of listed shortened title reflectes modern immune chemical analysis:
1 RID 2 ELISA 3 FIA 4 LIA
№15 Cells in which transformation was made are chosen accordingly to: 1 Ability to live in the presence of antibiotics or herbicides 2 Outer view 3 Ability to form specific colonies 4 Growth intensity
№16 Data of appearance of genetic engineering is:________________________________
№18 First transgenic animals were:_____________________________________________
№19 Obtaining genomic libraries does not include: 1 Preparation total DNA 2 Fragmentation by restrictases 3 Conjugation to the vectors 4 Introduce to the recipient
№20 For the discovering sex of genetic relationship it is used:_______________________
№21 Restrictases are able to split: 1 Outside DNA only 2 Own DNA only 3 Both (outside and own) DNA
№22 How much main classes restrictas are today:_________________________________
№23 What kind of abbreviators are used for antigens:______________________________
№24 Transfections is insertion of foreign DNA by:________________________________
№25 Northern blot analysis is used for check:____________________________________
№26 When firstly were transferred genes to anymals:______________________________
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№27 First successful attempt of genetic therapy in clinical practice was in:
1 1982 2 1985 3 1995 4 1990
№28 For the analysis of family relationships are used the next molecular markers:_____
№29 Sites of restriction may be protected by:____________________________________
№30 Restrictases are marked accordingly to: 1 Name of organisms from what they were obtained 2 Anyhow 3 Direction of their action