CHAPTER ELEVEN:
INTRODUCTORY ANIMAL BIOTECHNOLOGY
Kabir Mohammed Adamu Department of Biology, IBB University, Lapai |
& | Hauwa Hussaini Ndayako Department of Biology, IBB University, Lapai
|
11.1 Introduction
Biotechnology is defined as ‘the use of organisms or their components in industrial or commercial processes, which can be aided by the techniques of genetic manipulation in developing e.g. novel plants for agriculture or industry’. Biotechnology can also be defined as using scientific process to get new organisms or new products from organisms. This may involves changing organism in some way to get a desired trait. It is used to promote the production of desired products by organisms.
Biotechnology is said to be multidisciplinary. This means that it involves many disciplines, or branches of learning. All areas of the life sciences, such as microbiology, biology, biochemistry, botany, zoology, agricultural sciences, medicines, food science, and genetics are branches of biotechnology. Computer science and related computer applications are used in biotechnology. Similarly, engineering and the physical sciences such as physics, chemistry are often involved. Biotechnology is carried out in two major levels;
a. Organismic biotechnology: this is working with complete, intact organism or their cells. The organisms are not genetically changed with artificial means. A major goal of organismic biotechnology is to improve organisms and the condition in which they grow. One typical example of organismic biotechnology is cloning. Cloning is the process of producing new organisms from cell or tissue of existing organisms. It does not alter genetic makeup. It uses existing genetic characteristics to achieve desired traits in new organism.
b. Molecular biotechnology/ Recombinant Deoxyribonucleic Acid (DNA) technology: this is changing the genetic make-up of organisms. It is achieved by altering the structure and parts of cells, particularly the genetic material. Genetic engineering, molecular mapping and similar procedures are used in molecular biotechnology. Genetic engineering is changing the genetic information in a cell. Organisms produced by genetic engineering are called transgenic organisms. Transgenic means that the genetic material in an organism has been altered.
Biotechnology helps meet human needs. Three of these needs are food, clothing and shelter. The products of plants and animals are used in manufacturing food, clothing and materials for homes. Biotechnology is also used to make products that are more desirable, such as the conversion of milk into cheese.
11.2 History of Biotechnology
The nature and uses of biotechnology have changed over the years. Older uses are still important in some areas, these include making cheese, baking bread etc. these older methods have served as the foundation on which modern biotechnology has been built. The amount of science needed to use the new methods in biotechnology has greatly increased in recent years with the advent of computers, communication and other technologies where biotechnology now includes DNA structure, gene expression and recombinant DNA methods. The stages of biotechnology development are;
a. Ancient Biotechnology: this includes important development in agriculture and food production that is dated to human civilization.
b. Classical Biotechnology: this makes wide spread use of the methods from ancient biotechnology, especially fermentation. The methods were adapted to industrial production. Many of the methods that emerged through classical biotechnology are widely used today.
c. Modern Biotechnology: this is the manipulation of the genetic material within organisms. It is based on genetics and the use of microscopy, biochemical methods and related sciences and technologies. This is often known as genetic engineering.
11.3 The Roles of Animals in Biotechnology
Animals have important roles in biotechnology. As most of the biotechnology work begins in a laboratory. Researchers attempt to answer questions about animals through laboratory experiment. Without the use of animals, humans might be in danger.
11.3.1 Animal Models
Scientist have developed models for animal research. One uses live animals, in the other model do not. These helps alleviate problems that might arise about the well-being of animals. Overall, four models apply for the use of animals.
a. Model 1: living Animals: these animals are living and usually have no threat to their well-being. They are known as the laboratory or scientific animals. As seen in agriculture /biological research, where experimental group of animals are used.
b. Model 2: living animal tissues/ systems: animal tissue can be cultured in laboratory. This saves the use of the whole animal, feeding, housing, cleaning up expenses.
c. Model 3: non-living systems: this involves using non-living mechanical models that reflect animal activity. These often relates to skeletal movement and locomotion. Artificial replacement parts, such as hip joints, can be studied using non-living systems.
d. Model 4: computer and mathematical approach: computer simulations with virtual reality and other uses help in biotechnology. Computer modelling may be done with a proposed biotechnology practice before it is tested with animals.
11.3.2 Laboratory Animals
A laboratory animal is an animal used for laboratory or research purposes. Note that laboratory could be cages, pastures, or other facilities where the animals can be controlled and observed. The significance of research with animals includes:
a. Scientist having base of information about animals.
b. People know about the nutritional needs of animals.
c. People know about proper care for animals.
d. Having animals to be more productive.
11.3.3 Animals Species
Most animals are subjected of study at one time or another. The commonest species used in research laboratories are: mice, rats, hamsters, guinea pigs, rabbit, cats, dogs and non-primates. A primate is an animal with thumb and forefingers opposition whilst a non-human primate is an animal similar to humans, but is not human e.g. Monkeys and chimpanzees. In agriculture, when information is needed from research, cattle, hogs, sheep, horse, fish and others are used. Where their results were carefully studied to determine future recommendations.
11.3.4 Transgenic Animals and Products.
Several uses for animals of animals to produce drugs have been investigated. A few have been developed to the level of satisfactory use. Pigs have been used to manufacture human haemoglobin Pigs are used as sources of organ in human in xenograft. Xenograft is the practice of grafting an organ or a tissue from one species into another. Transgenic mice have been used to produce human antibodies; the antibodies are needed to fight some kinds of diseases. Transgenic mice have been used to model disease that occurs in human e.g. modelling of sickle cell anemia, atherosclerosis and prostate cancer. White cattle have been used to produce lactose in tolerance milk. They are also used to control disease such as mastitis in dairy cows. Mastitis is a disease of mammary glands that interferes with milk production.
11.3.4.1 Methods of Creating Transgenic Animal
The only proven method is by the microinjection of DNA into the embryos which are in one-cell or two-cell stages. There are three general steps well decorated for the transgenic development sheep, goats, cattle’s and pigs.
a. Step 1: collect embryos: timing is important, super ovulation is promoted in donor females. With proper stimulation, far more embryos can be obtained than would be natural result of the reproduction process.
b. Step 2: injection embryos: microscopy is used to locate embryonic cells. The pronuclei within the cells are identified. A pronucleus is the haploid (single) nucleus of the sperm or ovum that have been united in fertilization to form zygote. A zygote is the one-cell embryo formed by the union of sperm and ovum. The appropriate DNA is determined and through the injection process, picked and used using microscope.
c. Step 3: zygote culture: following microinjection, the zygotes are placed in the oviduct of a recipient female. This implies that the female must be a proper stage in the oestrous cycle. This transfer may either involve surgical or non-surgical method. The embryo develops much as a normal embryo. Some may be carried to full –term, others may be removed in various stages of development.
All of the steps in this were proven to involve hazards in the survival of the zygotes. Moving about, inspecting, injecting and other procedures injure and kill zygotes. The success rate for the birth of live transgenic animals is often low. It ranges from less than 1% in cattle to more than 1% in goats. Mice have a nearly 3% success rate.
11.4 Techniques of Biotechnology
To understand different biochemical events of prokaryotic and eukaryotic cells at molecular level and be able to characterize, isolate and manipulate molecular components of cells and organisms, wide arrays of bio-physico-chemical techniques are used thus;
a. Polymerase Chain Reaction (PCR): This is a versatile technique for copying DNA, and allows a single DNA sequence to be copied repeatedly, or altered in predetermined ways; essentially when it is used for repeated replication of a defined segment of DNA.
b. Gel Electrophoresis: This is a common method in which molecules (DNA, Ribonucleic Acid (RNA) and proteins) are separated based on the rates of their migration in an electric field. A gel, usually formed from agarose or polyacrylamide, is placed between two buffer compartments containing electrodes. The sample is then pipetted into preformed slots in the gel, and the electric field is turned on; the gel acts like a sieve, selectively retarding the movement of larger molecules. Smaller molecules therefore move through the gel more rapidly, allowing a mixture of nucleic acids to be separated on the basis of size.
c. Expression of Cloned Genes: this is the situation where molecular cloning enables the determination of the nucleotide sequences of genes that provides new approaches to obtaining large amounts of proteins for structural and functional characterization.
d. Nucleic Acid Hybridization: Nucleic acid hybridization is a method used for the detection and analyzes of sequences of homologous DNA. This enables the mapping of genes, to chromosomes, the analysis of gene expression, and the localization of proteins to sub-cellular organelles. In this way, it is possible to study genetic differences between organisms or individuals. Hybridization can be achieved by two methods:
a) Southern blotting. Southern blotting is a method for probing for the presence of a specific DNA sequence within a DNA sample and it enables a researcher to determine not only whether a particular sequence is present within a sample of DNA, knowing the sequences present and the size of the restriction fragments that contain these sequences.
b) Northern blotting is when Messenger RNA subjected to hybridization analysis; in an analogous process. It is used for the study of the expression patterns of a specific type of RNA molecules which is essentially a combination of denaturing RNA gel electrophoresis and a blot. In this process RNA is separated based on size and is transferred to a membrane that is then probed with a labeled complement of a sequence of interest. It is used to determine whether a particular gene is made into mRNA, how much of that mRNA is present, and whether the abundance of that specific mRNA changes at different stages of development or in response to certain regulatory signals that control gene expression.
e. Restriction Fragment Length Polymorphism Analysis: this is when the DNA fragments that result from cutting a particular piece of DNA with a specific restriction enzyme give a characteristic pattern of bands upon gel electrophoresis. Fact band corresponds to a DNA restriction fragment of a certain length. Such differences are called restriction fragment length polymorphisms (RFLPs) serving as genetic marker for a particular location in the genome. RFLPs analysis is important in the diagnosis of genetic disorders and in forensic applications.
11.5 Tools of Deoxyribonucleic Acid Science
The DNA code of any organism can be analyzed. This includes plants, animals, bacteria and other organisms. Most of these procedures begin with;
a. DNA isolation: is the process of extracting and separating DNA from all other cell materials. Different procedures can be used to isolate ‘clean DNA’. The procedure varies with the cells/tissues being used.
· Step 1: Break open the cell wall/membrane. This is done with liquid nitrogen and/or grinding. The step varies with the source of DNA. A mortar and pestle may be used to crack and grind seed cells, such as wheat germ. A food blender containing sucrose may be used with animal tissues, such as a thymus gland. The solution thereof is strained through cheesecloth to remove clumps and gristle (low speed centrifuging may help settle the nuclei to the bottom of a test tube and leave the cellular material in suspension).
· Step 2: Digest the cellular components: this is often done by heating with a detergent. With wheat germ, a small amount of detergent solution is mixed with the ground powder. The wheat germ solution is heated to 60oC for 10 minutes. With the thymus solution, a small amount is put into a small test tube. EDTA solution is added to weaken membranes and inactivate DNA digesting enzymes.
· Step 3: Separate the polar compounds. This involves using a detergent solution such as sodium dodecyl sulfate (SDS). The detergent dissolves the lipids (fat) in the nuclear membranes. Salts such as NaCl, may be added to the solution. Gently stirring the solution with a slow circular motion promotes the process. Procedures may vary slightly depending on the source of the DNA. Some involves using chloroform/ethanol.
b. Polymerase chain reaction (PCR): is a procedure that uses controlled temperature and the enzyme, taq polymerase to replicate pieces of DNA. This technique allows scientists to make many copies from a few target DNA molecules. Essentially, by multiplying the molecules exponentially. DNA can be made visible and thus analyzed. Taq polymerase is the DNA replication enzyme found in bacteria that live in hydrophilic vents in the ocean (Thermus aquaticus). In its natural environment, the bacteria work at very high temperatures. Thus, temperature is used to control PCR reaction. PCR is a three-step process performed in a piece of equipment called thermocycler. The machine alters the temperature at each step of the process and the process is repeated many times. The steps are:
i. Separation: heat to 95oC to separate the DNA strands
ii. Annealing: cool to 35 – 58oC for the primers to bond to complimentary DNA regions.
iii. Extension: warm to 72oC for Taq polymerase to build a new DNA strand primed region.
NB: each cycle represents an exponential step in DNA production: 1st cycle = 2n = 2 copies; where n is the number of cycles.
OR
The purpose of sequencing is to determine the order of the nucleotides of a gene. For sequencing, we don't start from gDNA (like in PCR) but mostly from PCR fragments or cloned genes. There are three major steps in a sequencing reaction (like in PCR), which are repeated for 30 or 40 cycles.
a. Denaturation at 94°C: During the denaturation, the double strand melts open to single stranded DNA, all enzymatic reactions stop (for example: the extension from a previous cycle).
b. Annealing at 50°C: In sequencing reactions, only one primer is used, so there is only one strand copied (in PCR: two primers are used, so two strands are copied). The primer is jiggling around, caused by the Brownian motion. Ionic bonds are constantly formed and broken between the single stranded primer and the single stranded template. The more stable bonds last a little bit longer (primers that fit exactly) and on that little piece of double stranded DNA (template and primer), the polymerase can attach and starts copying the template. Once there are a few bases built in, the ionic bond is so strong between the template and the primer, that it does not break anymore.
c. Extension at 60°C: This is the ideal working temperature for the polymerase (normally it is 72 °C, but because it has to incorporate dideoxynucleotides triphosphates (ddNTP's) which are chemically modified with a fluorescent label, the temperature is lowered so it has time to incorporate the 'strange' molecules. The primers, where there are a few bases built in, already have a stronger ionic attraction to the template than the forces breaking these attractions. Primers that are on positions with no exact match, come loose again and don't give an extension of the fragment. The bases (complementary to the template) are coupled to the primer on the 3'side (adding dNTP's or ddNTP's from 5' to 3', reading from the template from 3' to 5' side, bases are added complementary to the template). When a ddNTP is incorporated, the extension reaction stops because a ddNTP contains a H-atom on the 3rd carbon atom (dNTP's contain a OH-atom on that position). Since the ddNTP's are fluorescently labeled, it is possible to detect the color of the last base of this fragment on an automated sequencer.
c. Gel electrophoresis: is a process of using an electrical field in agar to separate DNA and RNA molecules based on molecular size. It provides a sequence of the DNA fragments. DNA sequencing is the process of determining the order of the nucleotides in DNA fragments. The chain termination sequencing is most commonly used. The chain has three steps DNA synthesis, gel electrophoresis and DNA detection.
OR
This is done on an acrylamide gel, which is capable of separating a molecule of 30 bases from one of 31 bases, but also a molecule of 750 bases from one of 751 bases. All this is done with gel electrophoresis. DNA has a negative charge and migrates to the positive side. Smaller fragments migrate faster, so the DNA molecules are separated on their size.
d. Sequence alignment: To compare two or more sequences, it is necessary to align the conserved and un-conserved residues across all the sequences (identification of locations of insertions and deletions that have occurred since the divergence of a common ancestor). These residues form a pattern from which the relationship between sequences can be determined with phylogenetic programs. When the sequences are aligned, it is possible to identify locations of insertions or deletions since their divergence from their common ancestor. There are three possibilities:
a) The bases match: this means that there is no change since their divergence.
b) The bases mismatch: this means that there is a substitution since their divergence.
c) There is a base in one sequence, no base in the other: there is an insertion or a deletion since their divergence.
Figure: The comparison of sequences.
A good alignment is important for the next step: the construction of phylogenetic trees. The alignment will affect the distances between 2 different species and this will influence the inferred phylogeny. There are several programs available on the net for aligning sequences. These are all based on different mathematical models to compare two or more sequences with the most optimal score for matching bases with a minimum number of gaps inserted (because you can insert a huge number of gaps, so every base will match another).
e. DNA profiling techniques: is identifying an organism based on regions of DNA that vary greatly from one organism to another. DNA profiling is also known as DNA typing/fingerprints.
11.6 DNA fingerprinting
DNA fingerprinting, also called DNA typing, DNA profiling, genetic fingerprinting, genotyping, or identity testing, in genetics. It is method of isolating and identifying variable elements within the base-pair sequence of DNA (deoxyribonucleic acid). The technique was developed in 1984 by British geneticist Alec Jeffreys, after he noticed that certain sequences of highly variable DNA (known as minisatellites), which do not contribute to the functions of gene, are repeated within genes. Jeffreys recognized that each individual has a unique pattern of minisatellites (the only exceptions being multiple individuals from a single zygote, such as identical twins). DNA fingerprinting is a law-enforcement technology in which a small sample of hair, semen, blood, other tissue can be subjected to DNA analysis and the owner identified with high certainty. DNA fingerprints depend on the genetic differences between individuals, we have called such genetic differences DNA markers; they are also called DNA polymorphisms.
The term polymorphism literally means “multiple forms”, the term DNA polymorphism refers to a wide range of variations in nucleotide base composition, length of nucleotide repeats, or single nucleotide variants. DNA polymorphisms are important as genetic markers to identify and distinguish alleles at a gene locus and to determine their parental origin, there are different types of genetic polymorphism:
a. Single Nucleotide Polymorphism (SNP): these allelic variants differ in a single nucleotide at a specific position. At least one in a thousand DNA bases differs among individuals. The detection of SNPs does not require gel electrophoresis, this facilitates large-scale detection. A SNP can be visualized in a Southern blot as a Restriction Fragment Length Polymorphism (RFLP) if the difference in the two alleles corresponds to a difference in the recognition site of a restriction enzyme. Single Nucleotide Polymorphism (SNP) in genes and non-coding parts of the genome is considered as a worthwhile tool for the biodiversity assessment.
b. Simple Sequence Length Polymorphism (SSLP): these allelic variants differ in the number of tandem repeated short nucleotide sequences in noncoding DNA. Genetic variation in the cervix mitochondrial genome has been utilized largely in population genetic analysis or phylogenetic studies. Mitochondrial DNA (mtDNA) sequence variation is highly appropriate for phylogenetic analysis amongst closely related species, as compared to nuclear DNA markers. This is because mtDNA shows more rapid evolution (especially the hypervariable D-loop region), maternal inheritance and the absence of recombination, although there is some evidence that recombination may occur in the mtDNA of animals.
11.6.1 Procedure of Creating a DNA Fingerprint
First obtaining a sample of cells, such as skin, hair, or blood cells, which contain DNA. The DNA is extracted from the cells and purified. There are several methods for DNA extraction such as: conventional method with phenol chloroform and commercial DNA extraction kit or tissue kit. The application depends on the type of samples and techniques for each laboratory. It should adjust DNA to the optimal concentration before used.
In Jeffreys’s original approach, which was based on restriction fragment length polymorphism (RFLP) technology, the DNA was then cut at specific points along the strand with proteins known as restriction enzymes. The enzymes produced fragments of varying lengths that were sorted by placing them on a gel and then subjecting the gel to an electric current (electrophoresis): the shorter the fragment, the more quickly it moved toward the positive pole (anode). The sorted double-stranded DNA fragments were then subjected to a blotting technique in which they were split into single strands and transferred to a nylon sheet. The fragments underwent autoradiography in which they were exposed to DNA probes - pieces of synthetic DNA that were made radioactive and that bound to the minisatellites. A piece of X-ray film was then exposed to the fragments, and a dark mark was produced at any point where a radioactive probe had become attached. The resultant pattern of marks could then be analyzed.
Note;
ü Genomic DNA is amplified by using single or multiplex polymerase chain reaction (PCR) technique.
ü DNA fragments of PCR products produced for each marker are separated on acrylamide gel electrophoresis or automate DNA sequencing.
ü This technique is species-specific, requiring the development of suitable genetic makers for each species.
ü More number of markers will enhance power of the analysis.
ü The set of 6-17 microsatellite markers were used in various species of animals such as horse, cow, dog, cat, bird and elephant while 12-13 markers for the DNA fingerprint were used in human for DNA fingerprint.
INTRODUCTORY ANIMAL BIOTECHNOLOGY
11.6.2 Techniques of DNA Profiling
Over past 3 decades, the fundamental DNA technology developments-restriction enzymes coupled with Southern-blot hybridization, sequencing and PCR have contributed to a burst of applications in multiple research areas, including genetic variation and diversity in chickens.
Restricted fragment length polymorphism (RFLP): originally, RFLP referred to analysis of band patterns derived from DNA cleavage using restriction endonuclease enzymes based on SNP. RFLP and related techniques are usually modifications of the Southern blot method when the whole genomic DNA or its fraction is cut with restriction enzymes, transferred to a membrane and hybridized with radiolabeled or fluorescent probes. The latter can be cloned fragments of endogenous avian viruses, particular nuclear genes, MHC genes, EST, or mitochondrial DNA (mtDNA) genes. Individual or pooled RFLP patterns can easily be compared with identify variation within and among populations studied. The technique is time consuming but might still be useful in species for which no or little sequence information is available.
PCR-Based techniques: amplification of noncoding or coding regions of a genome using PCR has revolutionized molecular genetics research and provided an impressive variety of new markers to tackle diversity problems:
a. Random amplified polymorphic DNA (RAPD): the random amplified polymorphic DNA technique employs single short primers of random sequence, usually 10-mers, which produce multiband patterns similar to DNA fingerprints. No sequencing information is needed before genotyping. Use of RAPD markers to study vertebrate genetic diversity was thought to be promising and they were heavily exploited in the 1990s. However, because of poor PCR reproducibility and dominance mode of inheritance, they are no longer markers of choice.
b. Amplified fragment length polymorphism (AFLP): AFLP molecular markers have been an important tool to enrich existing genetic maps in plants, bacteria and less widely in animal genomes. As developed by Keygene (Keygene N.V., Wageningen, The Netherlands), the amplified fragment length polymorphism technique involves the restriction of genomic DNA, followed by ligation of adaptors complimentary to the restriction sites and selective PCR amplification of a subset of the adapted restriction fragments. Although this type of markers is popular, especially among plant researchers, there are just a few examples of its application to examine genetic variation in vertebrates. Like RAPD markers, AFLP markers are characterized by a dominant nature, which is a main disadvantage of this technique. Microsatellites, these types of single-locus markers are also known as short (or simple) tandem repeats, simple sequence repeats, or simple sequence-length polymorphisms and belong to a variable number of tandem repeat loci, the most extensively used class of highly polymorphic molecular markers. Unlike all the above techniques, prior sequence information of flanking regions is necessary to develop these markers. Major advantages of microsatellites are that they are detectable by PCR representing unique sequences in the genome that can be mapped and easily be exploited for many genetic applications. Also, they show extensive allelic differences in length, mainly based on variation in the number of repeats and partly on polymorphism of flanking regions.
11.6.3 DNA Fingerprinting Applications
a. Crime scene (forensic analysis in murder, rape, and other violent crimes).
b. Human relatedness (Paternity)
c. Animal relatedness
d. Anthropology studies
e. Disease-causing organisms
f. Food identification
g. Detecting infectious species of bacteria
h. Human remains
i. Monitoring transplants
j. Analysis of old or ancient DNA.
k. Mapping the human genome
l. Tumor biology
m. Transplantation medicine
n. Medical microbiology
o. Application of DNA fingerprinting in animal breeding.
p. Estimate genetic variation
q. Population identification by mixed DNA from more one sample.
r. Identification of genes affecting traits of INTEREST
s. Evaluation of genetic diversity
t. Measure progress of selection.
u. Prediction of Hybrid Vigour (Heterosis)
v. Inbreeding calculation
w. Marker-Assisted Selection.
x. Transgenesis.
11.7 Biotechnology Laboratory Hygiene/Safety
11.7.1 Operational Guidelines
a. Food and drink are not to be stored or prepared in laboratories or chemical storerooms. All food and drink should be consumed in specially designated areas such as canteen or pantry.
b. Use appropriate personal protective equipment (laboratory coats, disposable gloves, and safety glasses).
c. Protective clothing must be removed when leaving the working areas.
d. Gloves must not be worn outside the laboratory.
e. Wash your hands regularly when working with chemical reagents, especially before meals or snack.
f. Smoking in laboratories is prohibited.
g. Do not store personal items such as street clothing, backpacks, etc. on work benches.
h. The working areas should be cleared up and kept clean. The benches must be disinfected before and after use according to the hygiene concept.
i. Only the equipment and materials actually needed must remain on the workbenches. Stocks are stored only in the designated areas or cupboards.
j. If you have long hair, ensure that it is properly tied back.
k. DO NOT mouth pipette. Always use a pipette filler or other pipetting device.
l. Wearing of contact lenses in the lab is strongly discouraged. If it is unavoidable, notify your supervisor and co-workers so that this information is known in the event of a chemical splash in the eyes.
11.7.2 Chemicals Guidelines
a. A number of chemicals used in any molecular biology/biotechnology laboratory are hazardous.
b. You are strongly urged to make use of the information on the MSDS (Material safety data sheet) prior to using a new chemical and certainly in the case of any accidental exposure or spill.
c. The instructor/lab manager must be notified immediately in the case of an accident involving any potentially hazardous reagents.
d. The following chemicals are particularly noteworthy; however, they are not harmful if used properly:
a) PHENOL - can cause severe burns
b) ACRYLAMIDE - potential neurotoxin
c) ETHIDIUM BROMIDE - carcinogen
e. Always wear gloves when using potentially hazardous chemicals and never mouth-pipette them.
f. If you accidentally splash any of these chemicals on your skin, immediately rinse the area thoroughly with water and inform the instructor.
g. Discard the waste in appropriate containers.
h. Keep all noxious and volatile compounds in the fume hood.
i. Work with foul-smelling or toxic substances and highly flammable gases must only be carried out in the fume hood. The additional safety measures required in the particular case must be observed.
11.7.3 Disposal of Buffers and Chemicals
a. Any uncontaminated, solidified agar or agarose should be discarded in the trash, not in the sink, and the bottles rinsed well.
b. Any media that becomes contaminated should be promptly autoclaved before discarding it.
c. Petri dishes and other biological waste should be discarded in Biohazard containers which will be autoclaved prior to disposal.
d. Organic reagents, e.g. phenol, should be used in a fume hood and all organic waste should be disposed of in a labelled container, not in the trash or the sink.
e. Ethidium bromide is a mutagenic substance that should be treated before disposal and should be handled only with gloves.
f. Ethidium bromide should be disposed of in a labelled container.
11.7.4 Ultraviolet light
a. Exposure to ultraviolet light can cause acute eye irritation. Since the retina cannot detect UV light, you can have serious eye damage and not realize it until 30 min to 24 hours after exposure.
b. Therefore, always wear appropriate eye protection when using UV lamps.
c. Use UV goggles and common sense when working with the UV lightbox.
11.7.5 Electricity
a. The voltages used for electrophoresis are sufficient to cause electrocution.
b. Cover the buffer reservoirs during electrophoresis.
c. Always turn off the power supply and unplug the leads before removing a gel.
11.7.6 Glassware and Plastic Ware
a. Glass and plastic ware used for molecular biology must be scrupulously clean. Dirty test tubes, bacterial contamination and traces of detergent can inhibit reactions or degrade nucleic acid.
b. Glassware should be rinsed with distilled water and autoclaved or baked at 150 degrees C for 1 hour.
c. For experiments with RNA, glassware and solutions are treated with diethyl-pyrocarbonate to inhibit RNases which can be resistant to autoclaving.
d. Plastic ware such as pipettes and culture tubes are often supplied sterile. Tubes made of polypropylene are turbid and are resistant to many chemicals, like phenol and chloroform.
e. Polycarbonate or polystyrene tubes are clear and not resistant to many chemicals.
f. Make sure that the tubes you are using are resistant to the chemicals used in your experiment.
g. Micro pipette tips and microcentrifuge tubes should be autoclaved before use.
11.7.7 Equipment Use
a. It is to everyone's advantage to keep the equipment in good working condition.
b. As a rule of thumb, don't use anything unless you have been instructed in the proper use.
c. This is true not only for equipment in the lab but also departmental equipment.
d. Report any malfunction immediately.
e. Rinse out all centrifuge rotors after use and in particular if anything spills.
f. Please do not waste supplies - use only what you need. If the supply is running low, please notify either the instructor/lab manager before the supply is completely exhausted.
g. Occasionally, it is necessary to borrow a reagent or a piece of equipment from another lab. Except in an emergency, notify the instructor.
11.7.8 Micropipettes
a. Most of the experiments you will conduct in this laboratory will depend on your ability to accurately measure volumes of solutions using micropipettes.
b. The accuracy of your pipetting can only be as accurate as your pipette and several steps should be taken to ensure that your pipettes are accurate and are maintained in good working order.
c. They should then be checked for accuracy following the instructions given by the instructor. If they need to be recalibrated, do so.
d. Ensure the use of appropriate pipette tip during pipetting.
e. Do not drop the pipette on the floor. If you suspect that something is wrong with the pipette, first check the calibration to see if your suspicions were correct, then notify the instructor.
11.7.9 Curative Measures
a. Fire extinguisher
b. First-Aid Box
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National Academy of Sciences (NAS) (2002). Animal Biotechnology: Science Based Concerns, National Academy of Sciences, Washington DC.
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