Recombinant DNA Technology - Midlands State University 402...  · Web viewThe word biotechnology has come from two words namely bio (meaning biology) and technology (meaning technological

- 1 -

BIOTECHNOLOGY

The word biotechnology has come from two words namely bio (meaning biology) and technology (meaning technological application). Thus biotechnology is defined as the industrial application of living organisms and their biological processes such as biochemistry, microbiology, genetic engineering, etc. in order to make best use of the microorganisms for the benefit of mankind.

Biotechnology is applied in many areas to produce foods and medicines, in the development of new diagnostic tools, gene therapy, DNA finger-printing for forensic purposes etc.

Applications of Biotechnology

1. Health and medicine

Fighting infectious diseases : Biotechnology is used extensively in the study of infectious diseases such as SARS (Severe Acute Respiratory Syndrome), influenza, As a result more effective pharmaceuticals have been developed. Development of vaccines and antibiotics : Using technology, microorganisms are used to develop antibiotics and vaccines to cure diseases. For example, bacteria Bacillus polymysea is used to produce polymyxin B antibiotic (used to cure urinary tract infections), fungus Penicillium notatum is used to produce penicillin (used to cure fever, pneumonia, etc.) Treating genetic disorders : Disease can occur when genes become defective due to mutations. With advance in biotechnology it will in the near future be possible to use gene therapy to replace an abnormal or faulty gene with a normal copy of the same gene. It may be used to treat ailments such as heart disease, inherited diseases such as SCID, Thallesemia. In forensic science : With the help of new techniques such as DNA fingerprinting, it has now become easy to identify criminals and have many other applications.

2. Environment

Cleaning up and managing the environment : Cleaning up the environment using living organisms is called bioremediation. Naturally occurring, as well as genetically modified microorganisms, such as bacteria and fungi, and enzymes are used to break down toxic and hazardous substances present in the environment.

3. Agriculture

Biotechnology has helped in production of crops with improved disease resistance; herbicide-tolerance and insecticide-resistance. Plants with improved nutritional value for livestock etc. have also been bred through biotechnology.

Control of pests : One application of biotechnology is in the control of insect pests. The genetic make-up of the pest is changed by causing some mutations. These pests become sterile and cannot produce next generation.

/...

- 2 -

Manufacturing and bio-processing : With the help of new biological techniques it has become possible to grow on large scale, the plants that produce compounds for use in detergents, paints, lubricants and plastics etc.

Food and drinks : With biotechnology, it has now become easy to process foods and their products. Preservation and storing of food for consumption later has become easy and cheap with the help of biotechnology. Seedless grapes and seedless citrus fruits have been developed using biotechnology.

4. Industry

Biotechnology has been used in the industry to produce new products for human consumption. Food additives have been developed which help in the preservation of food. Microorganisms are used in the mass production of items such as cheese, yoghurt, alcohol, etc.

TOOLS AND TECHNIQUES IN RECOMBINANT DNA TECHNOLOGY

What is a Recombinant DNA ?

DNA molecules constructed outside the living cells that is in vitro by joining natural or

synthetic DNA segments that can replicate in a living cell

Recombinant DNA Technology

Techniques for - Isolation - Digestion - Fractionation - Purification of the TARGET fragment - Cloning into vectors - Transformation of host cell and selection - Replication - Analysis - Expression of DNA

How do we obtain DNA and how do we manipulate DNA?

Quite straightforward to isolate DNA For instance, to isolate genomic DNA

1. Remove tissue from organism 2. Homogenise in lysis buffer containing guanidine thiocyanate (denatures

proteins)

/...

- 3 -

3. Mix with phenol/chloroform - removes proteins 4. Keep aqueous phase (contains DNA) 5. Add alcohol (ethanol or isopropanol) to precipitate DNA from solution 6. Collect DNA pellet by centrifugation 7. Dry DNA pellet and resuspend in buffer 8. Store at 4°C

Goals of Recombinant DNA Technology

a) To isolate and characterize a gene

b) To make desired alterations in one or more isolated genes

c) To return altered genes to living cells

The first step in making recombinant DNA is to isolate donor and vector DNA. General protocols for DNA isolation were available many decades before the advent of recombinant DNA technology. With the use of such methods, the bulk of DNA extracted from the donor will be nuclear genomic DNA in eukaryotes or the main genomic DNA in prokaryotes; these types are generally the ones required for analysis.

Recombinant DNA technology is a “cut and paste” technology. Specific nucleotide sequences are cut from the DNA of humans, other animals or plants and “pasted” into plasmids. DNA of the plasmid carrying nucleotide sequence of another organism is the recombinant DNA. It is then inserted into bacteria. Bacteria divide repeatedly and a clone of bacteria with the recombinant DNA is obtained.

Five requirements for recombinant DNA technology are:

(i) Cell culture

(ii) Restriction endonuclease enzyme

(iii) Plasmids

(iv) Ligases

(v) Host bacteria

(i) Cell culture : Cultured cells of an animal or plant (or even a bacterium) carrying the required gene (nucleotide sequence of DNA) in its nucleus.

(ii) The enzyme Restriction endonuclease : Restriction endonucleases cut short specific DNA sequences. There are many different restriction endonucleases found in bacteria. Each of these enzymes very specifically recognises a particular DNA sequence (usually 4 to 6 bases) and cuts it. These enzymes are the “molecular scissors”. Either they cut both the strands at the same place or at different places so that the two DNA strands hang out at the two ends. Two cuts at

/...

- 4 -

the two ends of a DNA segment releases the cut part as the restriction fragment. The ends are single stranded and called sticky ends. Thus a piece of DNA containing a particular gene can be obtained by selecting a particular restriction endonuclease. The principle is simply that, if two different DNA molecules are cut with the same restriction enzyme, both will produce fragments with the same complementary sticky ends, making it possible for DNA chimeras to form. Hence, if both vector DNA and donor DNA are cut with EcoRI, the sticky ends of the vector can bond to the sticky ends of a donor fragment when the two are mixed.

Example: the restriction enzyme EcoRI (from E. coli) recognizes a six-nucleotide-pair sequence in the DNA of any organism. This type of segment is called a DNA palindrome, which means that both strands have the same nucleotide sequence but in antiparallel orientation. Many different restriction enzymes recognize and cut specific palindromes. The enzyme EcoRI cuts within this sequence but in a pair of staggered cuts between the G and the A nucleotides. This staggered cut leaves a pair of identical single-stranded “sticky ends.” The ends are called sticky because they can hydrogen bond (stick) to a complementary sequence. Most of the type II restriction enzymes recognize 4 to 6 base pair long palindromic (have two folded rotational symmetry) sequences and cleave within or near to these sequences.

E Escherichia (genus)

co coli (species)

R RY13 (strain)

I First identified order in bacterium

Restriction enzymes recognise a specific short nucleotide sequence

This is known as a Restriction Site

/...

- 5 -

The phosphodiester bond is cleaved between specific bases, one on each DNA strand

Examples of restriction enzymes and the sequences they cleaveSource microorganism Enzyme Recognition Site Ends produced

Arthrobacter luteus Alu I AGCT Blunt

Bacillus amyloiquefaciens H Bam HI GGATCC Sticky

Escherichia coli Eco RI GAATTC Sticky

Haemophilus gallinarum Hga I GACGC(N)5 Sticky

Haemophilus infulenzae Hind III AAGCTT Sticky

Providencia stuartii 164 Pst I CTGCAG Sticky

Nocardia otitiscaviaruns Not I GCGGCCGC Sticky

(iii)Plasmids : Plasmids are extra chromosomal DNA molecules in a bacterial cell which have sequences matching those of the required gene and can be similarly cut by the same restriction enzymes. Plasmids can readily enter bacteria, yeast or other speedily reproducing cells. i)

(iv) DNA ligase : It is an enzyme which can seal one DNA fragment with another DNA segment, both having sticky ends. Ligase is the “molecular glue”.

(v) Host Bacteria : Host bacteria are the bacteria whose plasmid is used for carrying foreign DNA.

Other Enzymes used in RDT:

i) DNA ligase is used for joining DNA molecules.

ii) Alkaline phosphatase is used for dephosphorylation of the vector i.e. removal of 5’

phosphate to avoid recircularization of the cut vector.

iii) S1 nuclease is used for cutting single stranded nucleic acids.

iv) Terminal transferase is used for adding homopolymer tails.

v) Reverse transcriptase is used for cDNA synthesis.

/...

- 6 -

Sequences of steps in recombinant DNA technology:

1. Specific restriction enzyme is selected.

2. Cell culture with required gene in the cells is obtained.

3. Restriction enzyme cuts the DNA at two ends of the specific gene and a

restriction fragment is obtained

4. Same restriction enzyme cuts a matching DNA sequence from a plasmid

5. Ligase joins the restriction fragment in the place vacated by the cut DNA segment of the plasmid. The plasmid becomes a recombinant plasmid containing a foreign DNA fragment . Its DNA is the recombinant DNA. Since plasmids can carry foreign DNA, they are called clonal vectors. Bacteriophages (viruses) can also function as clonal vectors.

6. The recombinant plasmids then enter the bacteria.

7. Bacteria divide. Recombinant plasmids replicate along with bacterial DNA.

8. A large population of bacteria (more than a million) containing recombinant DNA can be obtained in less than ten hours.

9. Multiple identical copies of DNA fragments inserted into plasmids or bacteriophage (bacterial virus) then obtained and preserved in a DNA library.

10. These DNA fragments are the cloned DNA )

/...

- 7 -

/...

- 8 -

Vectors

Vector is an agent that can carry a DNA fragment into a host cell in which it is capable of replication. If it is used only for reproducing the DNA fragment, it is called a cloning vector. If it is used for expression of foreign gene, it is called an expression vector. Properties of a good vector:

(1) It should be autonomously replicating i.e. it should have ori region.

(2) It should contain at least one selectable marker e. g. gene for antibiotic resistance.

(3) It should have unique restriction enzyme site (only one site for one RE) for different REs to

insert foreign DNA.

(4) It should be preferably small in size for easy handling.

(5) It should have relaxed control of replication so that multiple copies can be obtained.

Plasmid vectors

Plasmids are autonomously replicating circular, double stranded DNA molecules found in bacteria. They have their own origin of replication (ori region), and can replicate independently of the host chromosome. The size of plasmids ranges from a few kb to 200 kb. Plasmid vectors are often used for cloning DNA segments of small size (upto 10 kilobases). Some of the commonly used plasmid vectors are described below:

pBR322

/...

- 9 -

The first plasmid vector that has been constructed artificially is pBR322. It is named after the scientists Bolivar and Rodriguiz who constructed it in 1977. It is 4362bp in size. It has an origin of replication derived from a colicin-resistance plasmid (ColE1). This origin allows a fairly high copy number, about 100 copies of the plasmid per cell. Plasmid pBR322 carries two selectable markers viz. genes for resistance to ampicillin (Apr) and tetracycline (Tcr ). Several unique RE sites are present within these genes for insertion of foreign DNA . When a foreign DNA segment is inserted in any of these genes, the antibiotic resistance by that particular gene is lost. This is called insertional inactivation. For instance, insertion of a restriction fragment in the SalI site of the Tcr gene inactivates that gene. One can still select for Apr colonies, and then screen to see which ones have lost Tcr

pUC

A series of small plasmids (about 2.7 kb) have been developed at the University of California and hence the name pUC e.g. pUC7, 8, 18 and 19 etc. . These are high copy number plasmids that carry an ampicillin resistance gene and an origin of replication, both from pBR322.

They also have a multiple cloning site (MCS) – a sequence of DNA that carries unique sites for many REs. The MCS contains a portion of lacZ gene that codes for the enzyme β-galactosidase. When such plasmids are introduced into E. coli, the colonies are blue on plates containing X-gal (substrate for β- galactosidase) and IPTG (isopropyl thiogalactoside, an inducer). When a foreign DNA is introduced in MCS, the β-galactosidase activity is lost. Thus cells containing recombinant plasmids form white (not blue) colonies.

ii) Phage vectors

Bacteriophages or phages are viruses that specifically infect bacteria. The phage particle attaches to the outer surface of bacterium and injects its DNA into the cell. The phage DNA is then replicated inside the host and its genes are expressed to make phage capsid proteins and new phage particles are assembled and released from the bacterium.

Phage vectors can accommodate more DNA (upto 25 kb) than plasmids and are often used for preparation of genomic libraries. They also have higher transformation efficiency as compared to plasmids. Two bacteriophages namely, Lambda (λ) and M13 have been commonly used for construction of vectors for cloning in E. coli.

GENE DELIVERY METHODS

Gene delivery is the process of introducing foreign DNA into host cells. There are many different methods of gene delivery developed for a various types of cells and tissues, from bacterial to mammalian. Generally, the methods can be divided into two categories, viral and non-viral.

/...

- 10 -

Virus mediated gene delivery utilizes the ability of a virus to inject its DNA inside a host cell. A gene that is intended for delivery is packaged into a replication-deficient viral particle.

Non-viral methods include physical methods such as microinjection, gene gun, hydrostatic pressure, electroporation, continuous infusion, and sonication and chemical, such as lipofection.

Ca Cl2+ Mediated Transformation

It is the process by which plasmids (or other DNA) are introduced into a host cell. The bacterial cells are made competent by incubation in the presence of divalent cations (usually Ca2+) and a brief heat shock (42°C) is given which induces the E. coli cells to take up the foreign DNA. The efficiency of transformation is calculated as the number of transformants/µg of input DNA

Microinjection refers to the process of using a glass micropipette to insert substances at a microscopic or borderline macroscopic level into a single living cell. It is a simple mechanical process in which a needle roughly 0.5 to 5 micrometers in diameter penetrates the cell membrane and/or the nuclear envelope. The desired contents are then injected into the desired sub-cellular compartment and the needle is removed. Microinjection is normally performed under a specialized optical microscope setup called a micromanipulator. The process is frequently used as a vector in genetic engineering and transgenics to insert genetic material into a single cell. Microinjection can also be used in the cloning of organisms, and in the study of cell biology and viruses. Microcapillary and microscopic devices are used to deliver DNA into a protoplast.carriers (polyplexes).

Electroporation, or electropermeabilization, is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field. Electroporation is a dynamic phenomenon that depends on the local transmembrane voltage at each point on the cell membrane. It is generally accepted that for a given pulse duration and shape, a specific transmembrane voltage threshold exists for the manifestation of the electroporation phenomenon (from 0.5 V to 1 V). This leads to the definition of an electric field magnitude threshold for electroporation (Eth). That is, only the cells within areas where E≧Eth are electroporated. If a second threshold (Eir) is reached or surpassed, electroporation will compromise the viability of the cells, i.e., irreversible electroporation.

In molecular biology, the process of electroporation is often used for the transformation of bacteria, yeast, and plant protoplasts. In addition to the lipid membranes, bacteria also have cell walls which are different from the lipid membranes and are made of peptidoglycan and its derivatives. However, the walls are naturally porous and only act as stiff shells that protect bacteria from severe environmental impacts. If bacteria and plasmids are mixed together, the plasmids can be transferred into the cell after electroporation. Several hundred volts across a distance of several millimeters are typically used in this process. Afterwards, the cells have to be handled carefully until they have had a chance to divide producing new cells that contain reproduced plasmids.

A gene gun or a biolistic particle delivery system, originally designed for plant transformation, is a device for injecting cells with genetic information. The payload is an elemental particle of a

/...

- 11 -

heavy metal coated with plasmid DNA. This technique is often simply referred to as bioballistics or biolistics.

This device is able to transform almost any type of cell, including plants, and is not limited to genetic material of the nucleus: it can also transform organelles, including plastids.

Lipofection (or liposome transfection) is a technique used to inject genetic material into a cell by means of liposomes, which are vesicles that can easily merge with the cell membrane since they are both made of a phospholipid bilayer. Lipofection generally uses a positively charged (cationic) lipid to form an aggregate with the negatively charged (anionic) genetic material. A net positive charge on this aggregrate has been assumed to increase the effectiveness of transfection through the negatively charged phospholipid bilayer. This transfection technology performs the same tasks as other biochemical procedures utilizing polymers, DEAE dextran, calcium phosphate, and electroporation. The main advantages of lipofection are its high efficiency, its ability to transfect all types of nucleic acids in a wide range of cell types, its ease of use, reproducibility, and low toxicity. In addition, this method is suitable for all transfection applications (transient, stable, co-transfection, reverse, sequential or multiple transfections…). High throughput screening assay and has also shown good efficiency in some in vivo models.

Using A. tumefaciens to genetically modify plant cells

Agrobacterium tumefaciens causes crown gall disease in a wide range of dicotyledonous plants. (Dicotyledonous plants, are also known as dicots, have broad leaves with branching veins. The infection normally occurs at the site of a wound in the plant. The disease gains its name from the large tumour-like swellings, or galls, that occur on the stem, branches or roots of the plant. The galls often occur at the crown of the plant, the point where the main roots join the stem . During an infection, the bacterium transfers part of its DNA into the plant's cells. The DNA becomes integrated into the plant's genome, causing the production of galls and changes in cell metabolism.

A. tumefaciens can be modified to allow foreign genes to be incorporated into the genome of plant cells. In order to understand the processes involved, it is important to understand how ‘natural’ infection occurs.

/...

- 12 -

Most of the genes involved in crown gall disease are not borne on the chromosome of A. tumefaciens but on a plasmid, termed the Ti (tumour-inducing) plasmid. A plasmid is a circle of DNA separate from the chromosome, capable of replicating independently in the cell and of being transferred from one bacterial cell to another.

The Ti plasmid is large, between 200 and 800 kb in size. However, a relatively small (12–24 kb) region of the Ti plasmid, called the transfer DNA (T-DNA), is integrated into a host plant chromosome during the infection process. This region contains the genes coding for both gall formation and for the synthesis of opines. Opines are modified amino acids. They are synthesised by plant cells within the crown gall and provide a source of carbon (and sometimes nitrogen) for A. tumefaciens, but cannot be used by the plant itself. Essentially, the bacteria hijack the biochemical machinery of the plant cells, using them to generate a food source that only it can utilise. The genes encoding bacterial enzymes used in opine catabolism (i.e. its breakdown) are also present in the Ti plasmid, but they are located outside the T-region.

The genes responsible for the transfer of the T-DNA into the host are also located outside the T-DNA region itself. These genes make up the virulence region and they encode proteins that facilitate the transfer of the T-DNA, and its integration into the plant cell's genome

An overview of the events in crown gall formation.

/...

- 13 -

How A. tumefaciens genetically transforms plants: . A. tumefaciens contains a tumour-inducing (Ti) plasmid, which contains both virulence (vir) genes and a transfer-DNA (T-DNA) region. The bacterium attaches to a plant cell, and the T-DNA and Vir proteins are transferred to the plant through a transport channel. Inside the plant cell, the Vir proteins promote the integration of the T-DNA into the plant genome.

An enlarged representation of the Ti plasmid. The T-DNA has left and right borders at its extremities and includes genes that produce tumours and opines. Outside the T-DNA is the virulence region. This is a cluster of genes that encode proteins that facilitate the transfer of the T-DNA into the host. The origin of DNA replication (ORI) is a sequence specific to A. tumefaciens at which DNA copying starts, allowing the plasmid to be copied within the bacterium.

Genetic engineers have capitalised on the fact that part of the DNA from the Ti plasmid of A. tumefaciens is integrated into the plant genome during the infection process. Ti plasmids can be isolated and a foreign gene spliced in at an appropriate point, making it possible to transfer the novel gene into the plant.

The principle underlying the use of the Ti plasmid as a vector for plant transformation is that any gene placed between the left and right border sequences (i.e. within the T-DNA region) will be transferred into the infected plant cell. However, the Ti plasmid is rather large and, as such, difficult to manipulate. Special procedures have been devised that allow the use of a much smaller ‘artificial’ Ti plasmids i.e. the binary vector system.

.

.

/...

- 14 -

The production of transgenic plants using A. tumefaciens. (a) The artificial Ti plasmid. The T-DNA region will be transferred to the plant cell, and contains the foreign gene and a plant selectable marker gene. The marker gene will allow the identification of plant cells that have taken up the foreign gene. (b) The artificial Ti plasmid is generated in E. coli, and then transferred to A. tumefaciens containing a helper vector. This second plasmid contains the vir region which encodes the proteins that facilitate DNA transfer from the bacterium into plant cells. (c) The T-DNA section is transferred from this modified A. tumefaciens into the plant cell's genome.

The modified A. tumefaciens containing both the artificial Ti plasmid and the helper vector is then used to infect the target plant cells. On infection, the virulence genes are activated and the DNA between the left- and right-hand borders of the artificial Ti plasmid is transferred to a plant chromosome.

SELECTION OF TRANSFORMANTS

Methods for Selection and/or Screening of Desired Recombinants

Selection refers to applying conditions that allow the desired cells or phages (containing vector or vector and insert) to replicate while preventing others from replicating.

Typical selections include:

/...

- 15 -

antibiotic sensitivity and resistance; nutrient requirements; plaque formation.

Screening allows all cells to grow, but tests the resulting clones for particular properties. Often these are indicative of the presence of an insert in the vector. Screening properties used include:

antibiotic sensitivity and resistance; nutrient requirements; plaque type; blue-white selection (β-galactosidase activity); specific (by nucleic acid hybridization, PCR with gene specific primers or antibodies)

A host cell having foreign DNA introduced in it is called a transformant. At the end of transformation experiment, we get bacterial cells that may contain non-recombinant vector, desired recombinant vector or undesired recombinant vector or may not contain any vector i.e. non-transformants. To identify the clone containing desired piece of DNA from among several others, screening is carried out.

Selection of clones carrying recombinants

The selection of recombinants is generally done on the basis of marker genes present in the vector. There are two types of marker genes, selectable marker and a reporter gene or scorable marker.

Selectable markers

A selectable marker gene codes for a function which enables only those cells which possess it to survive under suitable conditions. For example, genes conferring resistance to antibiotics like ampicillin, tetracycline and kanamycin are good selectable markers. When a population of bacterial cells is plated on an ampicillin containing medium, only those cells that have ampicillin resistance genes survive and form colonies.

Reporter genes

A reporter gene produces a protein product whose activity can be assayed and permits either an easy selection or quick identification of cells in which it is present. Therefore it is also called as scorable marker. Among the more commonly used reporter genes are gus (codes for βglucuronidase which produces blue colour in the presence of suitable substrate), lux ( luciferase, produces phosphorescence, gfp (green fluorescence protein, fluoresces on irradiation with U.V.). Some examples making use of selectable and or scorable markers to identify recombinant clones are listed below:

i) Insertional inactivation of antibiotic resistance gene

/...

- 16 -

This can be explained with the help of pBR322 which has two selectable markers i.e. Apr and Tcr. Both these genes have unique cloning sites in them. Insertion of foreign DNA in any of the sites causes inactivation of that gene. The recombinants thus become susceptible to one of the antibiotics while non-recombinants are resistant to both.

ii) Insertional inactivation of lacZ gene

Some vectors contain a gene or sometimes only part of a gene, which complements a function missing in their host cells, e.g. lacZ gene ( encodes fragment of β-galactosidase) in the pUC vectors, M13 and some λ phage vectors which complements defective lacZ gene (encodes part of β- galactosidase) in E. coli host strains. Insertion of foreign gene in the vector causes inactivation of lacZ gene. The recombinants are identified by formation of white colonies /plaques while nonrecombinants form blue colonies /plaques

SCREENING

There are several methods for screening DNA libraries. Some of the commonly used methods are described below:

1. Methods based on nucleic acid hybridization

2. Immunochemical methods

3. Screening DNA libraries using PCR

Methods based on nucleic acid hybridization

a) Colony/ plaque hybridization

This method was given by Grunstein and Hogness (1975). It is used for screening both genomic as well as cDNA libraries and is the most common method of library screening. The procedure has following steps:

1. The recombinant bacterial colonies or phage plaques to be screened are transferred from the culture plate on to a nitrocellulose filter paper by replica plating (Fig. 20).

2. The filter with colony replicas is treated with NaOH to lyse the cells/ phages and to denature DNA.

3. The filter is then baked at 800C for two hours in vacuum oven to fix the DNA.

4. The filter is allowed to hybridize with a labeled probe.

5. The filter is washed to remove the unbound excess probe, dried and then subjected to

autoradiography if the probe is radioactively labeled.

SOME IMPORTANT TECHNIQUES USED IN CHARACTERIZATION OF GENES/GENE PRODUCTS

/...

- 17 -

A number of techniques are used for analyzing the genes/gene products. Some of these techniques are blotting and hybridization, DNA finger printing and Micro-array analysis.

These are briefly discussed below.

Blotting and hybridization techniques

These techniques are used for detecting a specific gene sequence or its expression, number of mcopies of a gene, relatedness among organisms etc. There are three types of blotting techniques: Southern, Northern and Western blotting.

Southern blotting

Southern blotting is a technique for transferring DNA molecules from agarose gel to a solid support such as nitrocellulose paper or nylon membrane. Blotting is required to carry out hybridization with a probe so that specific DNA fragment in a complex mixture of DNA fragments can be detected. The technique was invented in 1975 by Edward Southern and is named after its inventor. Following are the steps of this technique:

1. The DNA to be analyzed is digested with a restriction enzyme and then separated by agarose gel electrophoresis.

2. The DNA fragments in the gel are denatured with alkali. Denaturation of DNA fragments prior to blotting is essential so that the probe can hybridize.

3. Denatured DNA is then transferred to a nitrocellulose filter or nylon membrane by blotting. A buffer saturated Whatman No.1 filter paper is placed on top of a support such as glass plate and two edges of the filter are dipped in buffer solution. Alternatively sponge dipped in buffer can be used as support. The gel is laid on top of the filter paper placed on the support

4. A sheet of nitrocellulose membrane cut to the size of the gel is placed on top of the gel. A stack of dry rough filter papers of the size of the gel are then placed on top of the nitrocellulose filter. A weight of about 0.5 kg is then placed on top of this. The DNA molecules move upward by capillary action of buffer and on coming in contact with the nitrocellulose filter they get bound to it.

5. The membrane is then heated at 80 ºC for about 2h in a vacuum oven or exposed briefly (3-5 minutes) to UV radiations for firm binding of DNA to the nitrocellulose filter.

6. This nitrocellulose filter can be used for hybridization with a labeled probe. This is known as Southern hybridization. The location of the DNA fragment that hybridizes with the probe can be

/...

- 18 -

detected by autoradiography (when probe is radioactively labeled).

ELECTROPHORESIS

Electrophoresis is the standard method for analyzing, identifying, and purifying fragments of DNA or RNA that differ in size, charge, or conformation. in operation. When charged molecules are placed in an electric field, they migrate toward the positive (anode, red) or negative (cathode, black) pole according to their charge. In contrast to proteins, which can have either a net positive or net negative charge, nucleic acids have a consistent negative change due to their phosphate backbone, and they migrate toward the anode. Proteins and nucleic acids are separated by electrophoresis within a matrix or “gel.” Most commonly, the gel is cast in the shape of a thin slab, with wells for loading the sample. The gel is immersed within an electrophoresis buffer that provides ions to carry a current and some type of buffer to maintain the pH at a relatively constant value.

The gels used for electrophoresis are composed either of agarose or polyacrylamide. Agarose gels are used in a horizontal gel apparatus, while polyacrylamide gels are used in a vertical gel apparatus. These two differ in resolving power. Agarose gels are used for the analysis and of fragments between 100 and 50,000 bp in size with moderate resolution, and polyacrylamide gels are used for the analysis and preparation of small molecules with single nucleotide resolution. This high resolution is required for applications such as for DNA sequencing.

In electrophoretic gel, nucleic acids migrate through the pores; thus fragments separate by size with the smallest pieces moving the fastest and farthest through the gel. Because DNA by itself is not visible in the gel, the DNA is stained with a fluorescent dye such as ethidium bromide (EtBr). EtBr intercalates between the bases causing DNA to fluoresce orange when the dye is illuminated by ultraviolet light. Fragments of linear DNA migrate through agarose gels with amobility that is inversely proportional to the log10 of their molecular weight.

Polymerase Chain Reaction (PCR)/...

- 19 -

Polymerase Chain Reaction (PCR) PCR is a robust, speedy and flexible method, conceived by and put in practice by Kary Mullis in 1985. It is used to generate microgram quantities of DNA (up to billions of copies) of the desired DNA (or RNA), from a single copy in few hours.

Components:

• Sterile deionised water

• 10X PCR buffer

• dNTP mix

• Primer

• Taq DNA polymerase

• MgCl2

• Template DNA

A PCR cycle comprises of basically three repeated reaction including denaturation of thetemplate DNA followed by primer annealing to target sequence & extension of the primersby DNA polymerase.

DNA denaturationThe optimum temperature for DNA denaturation is determined by the % GC content of eachparticular template DNA. The more is the proportion of GC, higher temperatures arerequired to denature the parental duplex strands. At lower temperature, only A-T regionsstart melting. Longer DNA templates require longer denaturation time to separate themcompletely. Routinely 950C is the temperature used for denaturation, since it is the maximumtemperature at which Taq Polymerase can exhibit its activity

Annealing

The temperature which is required to anneal primers to the template DNA is critical. Itshould not be too high to avoid poor annealing and even not too low which facilitatenonspecific annealing, resulting in accumulation of unwanted sequences(55-650C). As such there is no proper formula to calculate annealing temperature. Conditions are optimized by performing several hit & trial PCRs at temperature ranging from 2-100C below the melting temperature, calculated from the oligonucleotide primer sequence. The most commonly used formula to determine the Tm from primer sequence is: 4(G+C) + 2(A+T), where G/C/A/T denotes, number of times these bases are present.

ExtensionExtension reaction is usually carried out at temperature favorable to the themostablepolymerase used in the Reaction. For Taq polymerase 72-780C is the optimum temperature.

/...

- 20 -

In 1st two cycles, polymerase extends beyond the sequence complementary to the binding site of the primer but after the 3rd cycle, only sequence that is defined & limited by the binding of primers are accumulated in a geometrical progression. As a thumb rule, for every 1000 base pair, 1 minute is used.

No. of cyclesNumber of cycles that are to be used to amplify a particular region from the target DNAdepends on the initial concentration of template present and the efficiency of primerextension and amplification.apprx 30-40 cycles. The reaction products are separated by gel electrophoresis. Depending on the quantity produced and the size of the amplified fragment, the reaction products can be visualised directly by staining with ethidium bromide or a silver-staining protocol.

/...

- 21 -

The exponential amplification of the gene in PCR.

Verification of the PCR product on gel.

The ladder is a mixture of fragments with known size to compare with the PCR fragments. Notice that the distance between the different fragments of the ladder is logarithmic. Lane 1 : PCR fragment is approximately 1850 bases long. Lane 2 and 4 : the fragments are approximately 800 bases long. Lane 3 : no product is formed, so the PCR failed. Lane 5 : multiple bands are formed because one of the primers fits on different places.

/...

- 22 -

APPLICATIONS OF BIOTECNOLOGY TO FOOD SCIENCE

What are genetically-modified foods?

The term GM foods or GMOs (genetically-modified organisms) is most commonly used to refer to plants, animals or products created for human or animal consumption using the latest molecular biology techniques. These organisms have been modified in the laboratory to enhance desired traits such as increased resistance to herbicides or improved nutritional content.

QUALITATIVE APPLICATIONS

GOLDEN RICE (VITAMINS)

Vitamin A, more properly known as retinol, is an important chemical intermediate in a number of biochemical processes in mammals. It is involved in vision, and is found in the rod cells of the retina of the eye. These cells are particularly important in seeing at low light levels, and night blindness is a symptom of vitamin A deficiency (VAD). Vitamin A is also involved in the proper functioning of the immune system. Children suffering from VAD are prone to serious infections, and often die from relatively minor illnesses, like diarrhoea or measles. The World Health Organisation in 2003 estimated that between 100 and 140 million children worldwide were vitamin A deficient, of whom between 250 000 and 500 000 become blind each year. Of these, half died within 12 months of losing their sight.

Many plants and bacteria can produce vitamin A from simpler molecules, but mammals cannot. Humans can either ingest vitamin A directly, or produce it by the chemical cleavage of one of a group of molecules called carotenoids. Carotenoid molecules contain 40 carbon atoms, and mammals can chemically cleave a number of them to produce either one or two molecules of the 20-carbon retinol. A number of related carotenoid molecules are found in the human diet. The ones that can be converted into vitamin A are referred to as the provitamin A carotenoids. The commonest of these is β-carotene. VAD is a disease of poverty, found where people are unable to afford an appropriate diet. It is prevalent in countries where rice is a staple, particularly in South Asia. The rice plant itself does contain carotenes: they are found in both the leaves and the husks. Rice that has not been milled, brown rice, can therefore be an important source of both dietary fibre and carotenes.

The science

The series of reactions that produces β-carotene in plants begins with the compound isopentenyl diphosphate (abbreviated as IPP). A common intermediate in many of the biochemical pathways from IPP, geranylgeranyl diphosphate (GGPP), is present in rice endosperm, but conversion to β-carotene was expected to require a four-stage process, involving four separate enzymes.

/...

- 23 -

1) GGPP would be converted to phytoene in the normal way, catalysed by phytoene synthase produced by a gene from a daffodil. (2) Phytoene would be converted directly to lycopene, catalysed by bacterial phytoene desaturase. (3) Lycopene would be converted to β-carotene, catalysed by lycopene β-cyclase, again produced by a daffodil gene.

The team undertook two experiments, in each case using A. tumefaciens and a binary vector system. The technique involved the infection of immature rice embryos, rather than fragments of mature plants.

Experiment 1: The team produced A. tumefaciens with an artificial Ti plasmid containing the series of sequences necessary to introduce active phytoene synthase and the bacterial phytoene desaturase. They attempted to infect around 800 immature rice embryos, of which 50 were found to have taken up the sequences. These embryos would be expected to produce only the first two of the enzymes required, those needed to convert GGPP to lycopene.

/...

- 24 -

Experiment 2: The team produced two types of modified A. tumefaciens. Type A contained all the sequences necessary for active phytoene synthase and the bacterial phytoene desaturase enzyme, as previously. Type B contained the series of sequences necessary to introduce the final enzyme in the biosynthesis, lycopene β-cyclase. 500 immature rice embryos were infected with both types of A. tumefaciens at once. Sixty embryos could be shown to have been infected by type A, but only 12 to have been infected by both types of A. tumefaciens.

The team was able to grow the 50 rice embryos from Experiment 1 and the 12 doubly infected embryos from Experiment 2 into mature rice plants. They allowed the plants to self-fertilise, and go on to produce a crop of rice

IMPROVING HEALTH CHARACTERISTICS

FLAVONOID BIOSYNTHESIS IN TOMATO(ANTIOXIDANTS)

Modern biotechnology is proving to be a powerful tool to improve the nutritional quality of crop foods when combined with traditional plant breeding and genetic resource management. A biotechnological approach was used for increasing health-promoting compounds in tomato. Flavonoids comprise a large and diverse group of polyphenolic compounds that are ubiquitous in plants. Flavonoids are involved in many aspects of plant growth and development, such as pathogen resistance, pigment production and UV light protection. In addition, due to their antioxidant properties they are thought to be beneficial to human health . Several epidemiological studies showed that increased consumption of flavonoids could help to protect against chronic diseases, such as cardiovascular disease. Many major food crops however, contain only small amounts of flavonoids in their edible parts or produce flavonoids that do not have optimal antioxidant characteristics.

Most of the enzymes involved in the biosynthesis of the different flavonoids have been well characterized, and their encoding and regulatory genes have already been isolated. The knowledge and availability of these genes gives us the tools to genetically up-regulate the overall flavonoid biosynthesis or to engineer the pathway towards new flavonoid species in crop plants.

Tomatoes are the excellent sources of carotenoids lycopenes. They also contain small amounts of flavonoids in their peel [5-10mg /kg fresh weight, mainly naringenin chalcon and the flavonol rutin(quercetin glycoside)]. The flavonoids form a large family of low molecular weight polyphenolic compounds, which occur naturally in plant tissues and include the flavonols, flavones, flavanones, catechins, anthocyanins, isoflavonoids, dihydroflavonols, and stilbenes.The tomato, which is an important food crop worldwide, contains small amounts of

/...

- 25 -

flavonols in the peel of its fruit. Flavonols are a group of flavonoids that are very good antioxidants and are thought to protect against cardiovascular diseases. Tomato is thus an excellent candidate for transgenic enhancement of flavonoid content.

Carotenoids are lipophylic while flavonoids are hydrophilic and thus presence of both in larger quantities increases the nutritional benefits of tomatoes with regard to protection against cardiovascular diseases and cancer. Both biochemical and gene expression data of tomato have suggested that one of the rate-limiting steps in flavonol biosynthesis in the peel could lie at the level of chalcone isomerise (CHI), a key enzyme in flavonoid biosynthesis. Flavonol biosynthesis was up-regulated in tomato fruit peel by overexpressing the CHI gene from Petunia in transgenic tomatoes, resulting in a more than seventyfold increase of the flavonol quercetin glycoside . Such tomato lines may offer opportunities for tomato-based products with an

expanded range of health benefiting properties. It involves transformation of tomato with the

petunia (Petunia hybrida) CHI gene encoding chalcone isomerase under the control of CaMV35S promoter.

Phenylalanine 4 Caumaroyl malcnyl CoA

chalcone synthase

Narigenin Chalcone

chalcone isomerase(CHI)

Naringenin

Dihdroflavonol reductase flavanone-3-hydroxylase

Anthocyanins Dihydrokaempferol Dihydroquercetin

flavonol synthase flavonol synthase

kaempferol Quecetin

FLAVR SAVR TOMATO

Most tomatoes that have to be shipped to market are harvested before they are ripe. Otherwise, ethylene synthesized by the tomato causes them to ripen and spoil before they

/...

- 26 -

reach the customer. In tomatoes, it takes about 45-55 days for the fruit to reach full maturity. After which, it starts to undergo the ripening process. The production of ethylene within the fruit in turn signals the activity of different enzymes resulting in physiological changes such as the change of color from green to red, the softening of the fruit, and the development of its distinct taste and aroma.

Normally, farmers pick their produce while they are still green. The ripening process is then induced by spraying the fruits or vegetables with ethylene gas when they reach their destination. For long hauls, fruits and vegetables are refrigerated to prevent damage and delay their ripening.

However, there are drawbacks to these postharvest practices. Fruits that have been harvested prematurely may result in poor taste and quality despite appearing as fully ripened ones. Fruits transported for long periods under refrigeration also have the tendency to lose their quality.

Transgenic tomatoes have been constructed that carry in their genome an artificial gene (DNA) that is transcribed into an antisense RNA complementary to the mRNA for an enzyme involved in ethylene production. These tomatoes make only 10% of the normal amount of the enzyme.

Antisense RNA

Messenger RNA (mRNA) is single-stranded. Its sequence of nucleotides is called "sense" because it results in a gene product (protein). Normally, its unpaired nucleotides are "read" by transfer RNA anticodons as the ribosome proceeds to translate the message.

However, RNA can form duplexes just as DNA does. All that is needed is a second strand of RNA whose sequence of bases is complementary to the first strand; e.g.

5´ C A U G 3´ mRNA3´ G U A C 5´ Antisense RNA

The second strand is called the antisense strand because its sequence of nucleotides is the complement of message sense. When mRNA forms a duplex with a complementary antisense RNA sequence, translation is blocked.

/...

- 27 -

This may occur because

the ribosome cannot gain access to the nucleotides in the mRNA or

duplex RNA is quickly degraded by ribonucleases in the cell .

With recombinant DNA methods, synthetic genes (DNA) encoding antisense RNA molecules can be introduced into the organism. The translation process is thus blocked and no functional enzymes are produced.

Regulation of Ethylene Production

The amount of ethylene produced can be controlled primarily by “switching off” or decreasing the production of ethylene in the fruit and there are several ways to do this. They include:

Suppression of ACC synthase gene expression. ACC (1-aminocyclopropane-1-carboxylic acid) synthase is the enzyme responsible for the conversion of S-adenosylmethionine (SAM) to ACC; the second to the last step in ethylene biosynthesis. Enzyme expression is hindered when an antisense (“mirror-image”) or truncated copy of the synthase gene is inserted into the plant’s genome.

Insertion of the ACC deaminase gene. The gene coding for the enzyme is obtained from Pseudomonas chlororaphis, a common nonpathogenic soil bacterium. It converts ACC to a different compound thereby reducing the amount of ACC available for ethylene production.

Insertion of the SAM hydrolase gene. This approach is similar to ACC deaminase wherein ethylene production is hindered when the amount of its precursor metabolite is reduced; in this case SAM is converted to homoserine. The gene coding for the enzyme is obtained from E. coli T3 bacteriophage.

Suppression of ACC oxidase gene expression. ACC oxidase is the enzyme which catalyzes the oxidation of ACC to ethylene, the last step in the ethylene biosynthetic pathway. Through anti-sense technology, down regulation of the ACC oxidase gene results in the suppression of ethylene production, thereby delaying fruit ripening.

Suppression of Polygalacturonase Activity

Polygalacturonase (PG) is the enzyme responsible for the breakdown of pectin, the substance that maintains the integrity of plant cell walls. Pectin breakdown occurs at the start of the ripening process resulting in the softening of the fruit. To produce a fruit with delayed ripening trait using this method, scientists insert an anti-sense or a truncated copy of the PG gene into the plant’s genome resulting in a dramatic reduction of the amount of PG enzyme produced

/...

- 28 -

thereby delaying pectin degradation. The tomato was made more resistant to rotting by adding an antisense gene which interferes with the production of the enzyme polygalacturonase. Flavr Savr tomatoes, on the other hand, could be allowed to ripen on the vine, without compromising their shelf-life. The intended effect of slowing down the softening of Flavr Savr tomatoes would allow the vine-ripe fruits to be harvested like green tomatoes without greater damage to the tomato itself

BIOTECHNOLOGICAL PRODUCTION OF ENZYMES

RECOMBINANT CHYMOSIN

Cheeses represent a traditional way of preserving a perishable foodstuff, milk. Cheese is made by addition of milk to a starter culture of lactic acid bacteria whic acidify the milk to about pH 5.5. In addition, milk clotting enzymes called rennet are added, resulting in a coagulated protein gel trapping other proteins and fat. Traditionally the rennet used is a preparation of several enzymes isolated from calf stomach. Modern cheeses making, however, increasingly relies upon microbial sources of the most important enzyme, the protease chymosin. This is now produced by recombinant micro-organisms.

Milk Proteins

Milk consists of water, fat, protein, phosphate, lactose, citric acid and inorganics such as calcium phosphate. The protein component of milk can be divided into two groups, the casein fraction and the whey proteins.The casein proteins are the ones that will form the curd during cheese making. Casein proteins tend not to have a particularly compact globular structure and they tend to be rather susceptible to proteolysis. As they are all phosphorylated, they bind the calcium content of the milk and exisit in the form of casein micelles.

Chymosin Reaction

The chymosin content of the rennin causes a specific and rapid cleaveage of the kappa-casein component of the casein micelles. This protein stabilises the micelles and after cleavage, the casein proteins precipitate under the influence of the calcium ions. Chymosin specifically recognises the sequence from His 98 to Lys 111 and cleaves the peptide bond between Phe 105 and Met 106 in the kappa-casein chain:

/...

- 29 -

In addition to its role in milk clotting, chymosin is also involved in the proteolytic changes occurring during ripening.

Chymosin Structure

Bovine chymosin is an acid protease produced in the fourth stomach of the cow in the form of a precursor. After a series of proteolysis events, the mature chymosin has a molecular weight of 35600 Da and exisits in one of two allelic forms, A and B, differing in the nature of the amino acid at position 244 (Asp or Gly). The A form has a slightly higher specific activity for kappa-casein but is marginally less stable than the B form.

Microbial alternatives to bovine chymosin are very desirable due to their lower price and more stable availability as the supply of calf-rennet is in decline. In addition, vegetarians find the use of bovine rennet unacceptible. Suitable candidates are the proteases from Mucor miehei, Mucor pusillis and Endothia parasitica. All of these are acid proteases of similar structure to the bovine enzyme and all have the neccessary narrow specificity for kappa-casein.

Recombinant Chymosin Production

/...

- 30 -

Genetic engineering has allowed cloning the bovine gene into a suitable production strain and producing the enzyme by fermentation. There are several possibilities for a production host:

Eschrichia coli.

E. coli is the favourite organism of molecular biologists and is most frequently used in gene cloning experiments. The problem with E. coli, however, is that recombinant proteins are frequently synthesised as intracellular inclusion bodies, increasing process costs cosiderably. Another issue with E. coli is that it is not generally recognised as safe for human consumption.

Bacillus sp.

Bacillus species are non-pathogenic and are used industrially to produce several enzymes used in food processing such as amylases. B. licheniformis could produce the chymosin, but the signal sequences did not allow the secretion of the enzyme into the medium.

Lactococcus lactis. This host was chosen as it is already used in starter cultures, but production levels were found to be very low.

Saccharomyces cerevisiae. Difficulties were experienced in achieving high secretion rates in yeast.

Kluveromyces lactis.

K. lactis is used for the production of dairy grade beta-galactosidase and its fermentation properties are well understood. It was found that the chymosin could be produced in this host and good levels of secretion into the medium were achieved.

The chymosin gene was inserted into the K. lactis chromosome and the yeast is grown by fed-batch fermentation. After fermentation, the yeast is killed by addition of benzoic acid and the chymosin is isolated by filtration. When grown in a bioreactor, the yeast release chymosin into the culture medium. Afterwards, the enzyme is extracted and purified yielding a product that is 80 to 90 percent pure. Natural rennin contains only 4 to 8 percent active enzyme.

QUANTITATIVE APPLICATIONS

HIGHER BIOMASS THROUGH RESISTANCE TO DISEASES AND PESTS

GENETICALLY MODIFIED MAIZE

Genetically modified maize (corn) is an artificial mutant with agronomically desirable traits. Traits that have been engineered into corn include resistance to herbicides and resistance to insect pests, the latter being achieved by incorporation of a gene that codes for the Bacillus thuringiensis (Bt) toxin. Hybrids with both herbicide and pest resistance have also been produced. In 2009, transgenic maize was grown commercially in 11 countries, including the United States (where 85% of the maize crop was genetically modified), Argentina (83% GM),

/...

- 31 -

Brazil (36% GM), South Africa (57% GM), Canada (84% GM), the Philippines (19% GM) and Spain (20% GM)

Bacillus thuringiensis (or Bt) is a Gram-positive, soil-dwelling bacterium, commonly used as a biological pesticide; alternatively, the Cry toxin may be extracted and used as a pesticide. During sporulation, many Bt strains produce crystal proteins (proteinaceous inclusions), called δ-endotoxins, that have insecticidal action. This has led to their use as insecticides, and more recently to genetically modified crops using Bt genes. The European corn borer, Ostrinia nubilalis, destroys corn crops by burrowing into the stem, causing the plant to fall over. Bt corn is a variant of maize, genetically altered to express the bacterial Bt toxin, which is poisonous to insect pests

Cry toxins have specific activities against insect species of the orders Lepidoptera (moths and butterflies), Diptera (flies and mosquitoes), Coleoptera (beetles), Hymenoptera (wasps, bees, ants and sawflies) and nematodes. Thus, B. thuringiensis serves as an important reservoir of Cry toxins for production of biological insecticides and insect-resistant genetically modified crops. When insects ingest toxin crystals, the alkaline pH of their digestive tract denatures the insoluble crystals, making them soluble and thus amenable to being cut with proteases found in the insect gut, which liberate the cry toxin from the crystal. The Cry toxin is then inserted into the insect gut cell membrane, forming a pore. The pore results in cell lysis and eventual death of the insect. Expressing the toxin was achieved by inserting a gene from the microorganism Bacillus thuringiensis into the corn genome. This gene codes for a toxin that causes the formation of pores in the Lepidoptera larval digestive tract. These pores allow naturally occurring enteric bacteria, such as E. coli and Enterobacter, to enter the hemocoel, where they multiply and cause sepsis

/...

- 32 -

GM PRODUCTS: BENEFITS AND CONTROVERSIES

Crops

Enhanced taste and quality

Reduced maturation time

Increased nutrients, yields, and stress tolerance

Improved resistance to disease, pests, and herbicides

New products and growing techniques

Safety

Potential human health impact: allergens, transfer of

antibiotic resistance markers, unknown effects.

Potential environmental impact: unintended transfer

of transgenes through cross-pollination, unknown

effects on other organisms (e.g., soil microbes),

and loss of flora and fauna biodiversity

/...

- 33 -

Animals

Increased resistance, productivity, hardiness, and

feed efficiency

Better yields of meat, eggs, and milk

Access and Intellectual Property

Domination of world food production by a few companies

Biopiracy—foreign exploitation of natural resources

Environment

"Friendly" bioherbicides and bioinsecticides

Conservation of soil, water, and energy

Better natural waste management

Ethics

Violation of natural organisms' intrinsic values

Tampering with nature by mixing genes among species

Labeling

Not mandatory in some countries

Society

Increased food security for growing populations

Society

New advances may be skewed to interests of rich countries

/...

- 34 -

BIOSAFETY

CARTAGENA PROTOCOL ON BIOSAFETY

Article 1

OBJECTIVE

In accordance with the precautionary approach contained in Principle 15 of the Rio Declaration on Environment and Development, the objective of this Protocol is to contribute to ensuring an adequate level of protection in the field of the safe transfer, handling and use of living modified organisms resulting from modern biotechnology that may have adverse effects on the conservation and sustainable use of biological diversity, taking also into account risks to human health, and specifically focusing on transboundary movements.

/...

- 35 -

Annex II I

RISK ASSESSMENT

Objective

1. The objective of risk assessment, under this Protocol, is to identify and evaluate the potential adverse effects of living modified organisms on the conservation and sustainable use of biological diversity in the likely potential receiving environment, taking also into account risks to human health.

Use of risk assessment

2. Risk assessment is, inter alia, used by competent authorities to make informed decisions regarding living modified organisms.

General principles

3. Risk assessment should be carried out in a scientifically sound and transparent manner, and can take into account expert advice of, and guidelines developed by, relevant international organizations.4. Lack of scientific knowledge or scientific consensus should not necessarily be interpreted as indicating a particular level of risk, an absence of risk, or an acceptable risk.5. Risks associated with living modified organisms or products thereof, namely, processed materials that are of living modified organism origin, containing detectable novel combinations of replicable genetic material obtained through the use of modern biotechnology, should be considered in the context of the risks posed by the non-modified recipients or parental organisms in the likely potential receiving environment.6. Risk assessment should be carried out on a case-by-case basis. The required information may vary in nature and level of detail from case to case, depending on the living modified organism concerned, its intended use and the likely potential receiving environment.

Methodology

7. The process of risk assessment may on the one hand give rise to a need for further information about specific subjects, which may be identified and requested during the assessment process, while on the other hand information on other subjects may not be relevant in some instances.8. To fulfil its objective, risk assessment entails, as appropriate, the following steps:

(a) An identification of any novel genotypic and phenotypic characteristics associated with the living modified organism that may have adverse effects on biological diversity in the likely potential receiving environment, taking also into account risks to human health;

(b) An evaluation of the likelihood of these adverse effects being realized, taking into account the level and kind of exposure of the likely potential receiving environment to the living modified organism;

/...

- 36 -

(c) An evaluation of the consequences should these adverse effects be realized; (d) An estimation of the overall risk posed by the living modified organism based

on the evaluation of the likelihood and consequences of the identified adverse effects being realized;

(e) A recommendation as to whether or not the risks are acceptable or manageable, including, where necessary, identification of strategies to manage these risks; and

(f) Where there is uncertainty regarding the level of risk, it may be addressed by requesting further information on the specific issues of concern or by implementing appropriate risk management strategies and/or monitoring the living modified organism in the receiving environment.

Points to consider

Identification of harmful properties (hazard) of the GMM

The risk assessment process requires the identification of any potentially harmful properties of the GMM as a result of the genetic modification or any alteration of the recipient organisms' existing properties. Potentially harmful properties associated with the GMM must be determined. This should be done by consideration of the recipient organism, the donor organism, the characteristics and location of the inserted genetic material and any vector. It is important to appreciate that the genetic modification of a micro-organism can affect its ability to cause harm to human health and the environment. Genetic modifications can result in a decreased, unchanged or increased ability to cause harm.

Aspects that should be considered where relevant are:

The recipient organism

nature of pathogenicity and virulence, infectivity, allergenicity, toxicity and vectors of disease transmission,

nature of indigenous vectors and adventitious agents, where they could mobilise the inserted genetic material, and the frequency of mobilisation,

nature and stability of disabling mutations, if any, any prior genetic modifications, host range (if relevant), any significant physiological traits which may be altered in the final GMM and if

relevant their stability, natural habitat and geographic distribution, significant involvement in environmental processes (such as nitrogen fixation or pH

regulation); interaction with, and effects on, other organisms in the environment (including likely

competitive, pathogenic or symbiotic properties) ability to form survival structures (such as spores or sclerotia).

The donor organism (for fusion experiments or 'shotgun' experiments where the insert is not well characterised)

/...

- 37 -

nature of pathogenicity and virulence, infectivity, toxicity and vectors of disease transmission,

nature of indigenous vectors: Sequence, frequency of mobilisation and specificity, presence of genes which confer resistance to anti-microbials including antibiotics host range, other relevant physiological traits.

The insert

specific identity and function of the insert (genes), level of expression of inserted genetic material, source of the genetic material, identity of the donor organism(s)and characteristics

where appropriate, history of prior genetic modifications if appropriate, location of inserted genetic material (possibility of insertional activation/deactivation

of host genes).

The vector

nature and source of the vector, structure and amount of any vector and/or donor nucleic acid remaining in the final

construction of the modified micro-organism, if present in the final GMM frequency of mobilisation of inserted vector and/or

capability for transfer of genetic material.

The resulting GMM

Human health considerations

expected toxic or allergenic effects of the GMM and/or its metabolic products, comparison of the modified micro-organism to the recipient or (where appropriate)

parental organism regarding pathogenicity, expected capacity for colonisation, if the micro-organism is pathogenic to humans who are immunocompetent: diseases caused and mechanism of transmission including invasiveness and

virulence, infective dose, possible alteration of route of infection or tissue specificity, possibility of survival outside of human host, biological stability, antibiotic-resistance patterns, allergenicity, toxigenicity, availability of appropriate therapies and prophylactic measures.

/...

- 38 -

Environmental considerations

ecosystems to which the micro-organism could be unintentionally released from the contained use,

expected survivability, multiplication and extent of dissemination of the modified micro-organism in the identified ecosystems,

anticipated result of interaction between the modified micro-organism and the organisms or micro-organisms

which might be exposed in case of unintentional release into the environment, known or predicted effects on plants and animals such as pathogenicity, toxicity,

allergenicity, vector for a pathogen,altered antibiotic-resistance patterns, altered tropism or host specificity, colonisation,

known or predicted involvement in biogeochemical processes.

/...