Which molecular tool is used for cutting the DNA molecule in genetic engineering?

Chapter Summary

Recombinant DNA is the method of joining two or more DNA molecules to create a hybrid. The technology is made possible by two types of enzymes, restriction endonucleases and ligase. A restriction endonuclease recognizes a specific sequence of DNA and cuts within, or close to, that sequence. By chance, a restriction enzyme's recognition sequence will occur every (¼)n bases along a random DNA chain.

The amount of fragment ends (in moles) generated by cutting DNA with a restriction enzyme is given by the equation

molesofDNAends=2×(gramsofDNA)(numberofbp)×( 660g/molbp)

The amount of ends generated by a restriction enzyme digest of circular DNA is given by the equation

moles ends=2×(molesDNA)×(numberof restrictionsites)

When a linear molecule is digested with a restriction endonuclease, the amount of ends generated is calculated by the following equation.

molesends=[2×(molesDNA)×(numberofrestriction sites)]+[2×(molesDNA)]

DNA fragments generated by digestion with a restriction endonuclease can be joined together again by the enzyme ligase. The likelihood that two DNA molecules will ligate to each other is dependent on the concentration of their ends; the higher the concentration of compatible ends, the greater the likelihood that two termini will meet and be ligated. This parameter is designated by the term i and is defined as the total concentration of complementary ends in the ligation reaction. For a linear fragment of duplex DNA with cohesive ends, i is given by the formula

i=2N0M×10−3ends/mL

where N0 is Avogadro's number (6.022 × 1023) and M is the molar concentration of the DNA.

Ligation of DNA molecules can result in their circularization. The amount of circularization is dependent on the parameter j, the concentration of same-molecule ends in close enough proximity to each other that they can effectively interact. For any DNA fragment, j is a constant value dependent on the fragment's length. It can be calculated as

j=(32πlb)32 ends/mL

where l is the length of the DNA fragment, b is the minimal length of DNA that can bend around to form a circle, and π is the number pi. For bacteriophage lambda DNA, j has a value of 3.22 × 1011 ends/mL. The j value for any DNA molecule can be calculated in relation to jλ by the equation

j=jλ(MWλMW) 1.5ends/mL

where jλ is equal to 3.22 × 1011 ends/mL and “MW” represents molecular weight. Under circumstances in which j is equal to i (j = i, or j/i = 1), the end of any particular DNA molecule is just as likely to join with another molecule as it is to interact with its own opposite end. If j is greater than i (j > i), intramolecular ligation events predominate and circles are the primary product. If i is greater than j (i > j), intermolecular ligation events are favored and hybrid linear structures predominate.

Ligation of fragments to plasmid vectors may be most efficient when i is greater than j by two-to threefold—a ratio that will favor intermolecular ligation but will still allow for circularization of the recombinant molecule. In addition, the concentration of the termini of the insert (iinsert) should be approximately twice the concentration of the termini of the linearized plasmid vector (iinsert = 2ivector).

Transformation efficiency is a measure of how many bacteria were able to take in recombinant plasmids. It is expressed as transformants/μg DNA.

The likelihood of finding a particular clone within a randomly generated recombinant library can be estimated by the following equation

N=ln(1−P)ln[1−(IG)]

where I is the size of the average cloned insert, in base pairs, G is the size of the target genome, in base pairs, N is the number of independent clones, and P is the probability of isolating a specific DNA segment.

The number of clones that need to be screened to obtain the recombinant of a rare mRNA in a cDNA library at a certain probability is given by the equation

N=ln(1−P)ln[1−(nT)]

where N is the number of cDNA clones in the library, P is the probability that each mRNA type will be represented in the library at least once (P is usually set to 0.99; a 99% chance of finding the rare mRNA represented in the cDNA library), n is the number of molecules of the rarest mRNA in a cell, and T is the total number of mRNA molecules in a cell.

When constructing an expression library, the probability of obtaining a recombinant clone with the insert fragment positioned correctly within the vector is given by the equation

P=codingsequence size(inkb)genomesize(in kb)×6

Hybridization of a probe to a recombinant library should be carried out at a temperature (Ti) 15°C below the probe's Tm:

Ti=Tm−15°C

Tm is calculated using the equation

Tm=16.6log[M]+0.41[PGC]+81.5−Pm−B/L−0.65[Pf]

where M is the molar concentration of Na+, PGC is the percent of G and C bases in the oligonucleotide probe, Pm is the percent of mismatched bases, Pf is the percent formamide, B is equal to 675 (for probes up to 100 nucleotides in length), and L is the probe length in nucleotides. The Tm of probes longer than 100 bases can be calculated using the following formula

Tm=81.5°C+16.6(log[ Na+])+0.41(%GC)−0.63(%formamide )−600/L

The number of positions on a genome having a certain complexity to which an oligonucleotide probe will hybridize is given by the equation

P0=(14)L×2C

where P0 is the number of independent perfect matches, L is the length of the oligonucleotide probe, and C is the target genome's complexity.

Hybridization of an oligonucleotide probe to the DNA of a recombinant library is typically performed at 2–5°C below the oligonucleotide's Tm. The approximate length of time allowed for the hybridization reaction to achieve half-completion is given by the equation

t1/2=ln2kC

where k is a first-order rate constant and C is the molar concentration of the oligonucleotide probe (in moles of nucleotide per liter).

The rate constant, k, represents the rate of hybridization of an oligonucleotide probe to an immobilized target nucleic acid in 1-M sodium ion and is given by the equation

k=3×105L0.5L/mol/s N

where k is calculated in liters/mole of nucleotide per second, L is the length of the oligonucleotide probe in nucleotides, and N is the probe's complexity.

Complexity, as it relates to an oligonucleotide, is calculated as the number of different possible oligonucleotides in a mixture. The total number of different oligonucleotides in a mixture, its complexity, is calculated by multiplying the number of possible nucleotides at all positions.

Hybridization to recombinant clone DNA can be performed using a dsDNA probe prepared by nick translation. If you use a hybridization temperature of 68°C in aqueous solution or 42°C in 50% formamide, the following equation to estimate the amount of time to achieve half-complete hybridization can be used.

t1 /2=1X×Y5×Z10×2

where X is the amount of probe added to the hybridization reaction (in μg), Y is the probe complexity, which for most probes is proportional to the length of the probe [in kilobases (kb)], and Z is volume of the hybridization reaction (in mL).

Nearly complete hybridization is achieved after three times the t1/2 period.

Recombinant clones can be identified by a restriction digest that removes the insert (or a characterized piece of it). The generated fragments are sized by electrophoresis on a gel also carrying a size ladder. The ladder is used to generate a standard curve and regression line equation that can be used to determine the size of the fragments from the recombinant clones.

Deletions of dsDNA can be prepared by the BAL 31 nuclease. The incubation time required to produce the desired deletion can be estimated using the equation

Mt=M0−2MnVmaxt[Km+ (S)0]

where Mt is the molecular weight of the duplex DNA after t minutes of incubation, M0 is the original molecular weight of the duplex DNA, Mn is the average molecular weight of a mononucleotide (taken as 330 Da), Vmax is the maximum reaction velocity (in moles of nucleotide removed/liter/minute), t is the length of time of incubation (in minutes), Km is the Michaelis–Menten constant (in moles of dsDNA termini/liter), and S0 is the moles of dsDNA termini/L at t = 0 min.

Recombinant DNA

rDNA, the combining of genetic material in juxtapositions not found in nature, is a traditional area of research methodology overseen by the IBCs; these activities – the original target of IBC oversight – have a less direct relationship to security issues. The use of rDNA techniques in the production of therapeutic molecules (insulin, interleukins, hormones, etc.) is generally well accepted; the use of genetic modification in environmentally (e.g. bioremediation) or agriculturally (GMO) important arenas remains controversial in segments of society, as is the use of rDNA in the treatment of human disease, or gene therapy, despite a number of successes (e.g. alipogene tiparvocec, trade name Glybera, for the treatment of severe pancreatitis caused by lipoprotein lipase deficiency) [1]. However, rDNA techniques are often used in studies of virulence mechanisms, e.g. in the generation of chimeric bacteria and viruses.

Introduction

Recombinant DNA has been a transformative technology, providing tools that not only have enabled tremendous understanding of life at the most fundamental levels, but that have also led to a myriad of medical and agricultural applications. Progress in recombinant DNA research continues to revolutionize approaches to life science research and biotechnology and has been possible because scientists taking the lead in developing this technology had the foresight to recognize that the promise of recombinant DNA could only be realized if they assumed responsibility for addressing the safety and ethical concerns that it raised.

The current system of oversight of recombinant DNA research was established almost 40 years ago when the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (NIH Guidelines) were first written [1]. At the federal level, the NIH Guidelines were initially administered by the Office of Recombinant DNA Activities (ORDA) that later became the Office of Biotechnology Activities (OBA) within the Office of Science Policy.

The NIH Guidelines outline the requirements for local oversight, including the establishment of an institutional oversight committee. The first NIH Guidelines articulated the requirements for “Institutional Biohazards Committees” that were later renamed “Institutional Biosafety Committees” (IBCs) to more clearly reflect their role. IBCs must review recombinant and synthetic nucleic acid molecule research for conformity with the NIH Guidelines. In addition, they assess the research for potential risks to health and the environment. This is accomplished by reviewing physical and biological containment for the research and ensuring that researchers are adequately trained to conduct the research they are proposing safely.

The hallmarks of this oversight system from its inception were public participation and transparency. Attention to the concerns of the community and local interests is a major theme that carries forward in the system of biosafety oversight today. This key element has served to preserve public trust in the safety of the life sciences research enterprise. In retrospect, the risks of recombinant DNA technology that were feared early in its evolution did not materialize. That fact notwithstanding, the development of a scientifically based oversight system with the IBCs as the centerpiece permitted the safe development of recombinant DNA as an essential technology in research. Over the years, oversight by IBCs has proven critically important to ensuring safety throughout various research fields – medical, occupational, environmental – as well as in promoting responsible scientific practice. Due to the dynamic nature of the life sciences there remains an ongoing need to assess biosafety dimensions of the research being conducted and to manage any risks associated with work. As life sciences research continues to advance, many lines of research, particularly involving highly pathogenic organisms, continue to generate public concern. Financial support for life sciences research comes primarily from publicly derived tax dollars, and so the life sciences community must demonstrate to the public that it is being a responsible steward of those funds. IBCs today remain critically important in preserving public trust and thus facilitating continued scientific progress. The National Institutes of Health (NIH) and the institutions it funds must continue to ensure that IBCs are equipped to fulfill their responsibilities so that biosafety risks are responsibly managed and public safety and trust are preserved.

Applications

Recombinant DNA has revolutionized biological research and has had huge impact on the pharmaceutical and biotechnology industries. These techniques have provided the foundation for sequencing the human genome and hundreds of other genomes by providing genomic libraries. This information is invaluable for the molecular understanding of how cells and organisms function. Genomic sequence information allows a clear understanding of the evolutionary relationships between species and genes. Genomic information also allows an understanding of how the environment impacts cellular physiology and genetic composition. This is particularly true with microorganisms as they are found in a plethora of environments, for example, soils, oceans, streams, and animal and plant hosts. These environmental conditions can vary greatly with respect to temperature, pH, osmolarity, oxygen tension, radiation levels, or nutrient availability. Knowing the genomic sequence of bacteria found in different environments provides a molecular understanding of how genes and organisms adapt to their environment.

In the pharmaceutical industry, recombinant DNA methods are common tools used in drug discovery and development. These techniques help to find and elucidate interactions between therapeutic compounds and target macromolecules such as protein or RNA. Potential effects on cellular physiology, toxicologically, along with the mechanism of compound action and resistant profiles can be tested with molecular genetic or biochemical methodologies. Natural product therapeutics, derived from microbial secondary metobolites, are optimized by recombinant methods to improve yields or alter chemical structures.

The biotechnology industry uses recombinant DNA to isolate, improve, and produce protein therapeutics such as insulin, human growth factor, interferon, erythropoietin (Epogen), and filgrastim (Neupogen). Many other therapeutic recombinant proteins are clinically used or are under development. Recombinant DNA technology has also resulted in improved vaccines that simply contain isolated antigen and no other bacterial or viral component that often leads to adverse side effects. In agriculture, recombinant DNA has improved plant growth by increasing nitrogen fixation efficiencies, by cloning bacterial genes, and inserting them into plant cells. Other plants have been engineered to be resistant to caterpillar, pests, and viruses by inserting resistant genes into plant genomes. In humans, gene therapy may one day improve health. Here, cells are removed and transformed with altered genes and replaced back into the patient to provide missing functions. Gene therapy has the potential to replace drug therapy, which takes many years to develop safe and efficacious molecules for treating human genetic disorders. Finally, recombinant DNA methods are used to isolate, develop, and purify catalytic enzymes used in industrial processes. Enzymes have practical implications in many chemical synthesis processes by improving the rate and specificity of reactions. Such enzymes are used for pulp bleaching in paper making, oil processing, household detergent ingredients, and conversion of biomass or oils into ethanol or biofuels.

In forensic science, recombinant DNA techniques help law enforcement agencies test relationships between biological crime samples and individuals to prove guilt or innocence. These methods are also used to understand family heredity and to screen individuals for the presence or absence of genetic diseases. One technique is DNA fingerprinting by restriction fragment length polymorphisms. Here, unique repeating DNA sequences in individuals are characterized by restriction digestion analysis in which specific bands on blots are probed with labeled nucleic acids.

In biological research, recombinant DNA methods are omnipresent. They are used to study gene function by constructing mutant alleles to test phenotypes. Site-specific mutations are readily engineered into primers used in PCR amplification. This approach is particularly attractive when structural information is available or when evolutionarily conserved sequences/motifs are found within proteins. Thus site-specific changes in gene sequences directly test structure–function relationships. Alternatively, random mutagenesis of a cloned gene is used in an equally informative and unbiased approach to study gene function. DNA regulatory elements, such as promoters, can be studied in vivo by fusing them to reporter proteins, such as β-galactosidase. These reporters are used to test gene expression responses to environmental or cellular changes. Reporters can be conveniently vector encoded or integrated into the chromosome to allow accurate physiological measurements. Subcellular localization of proteins is visualized by fusing a gene product to a reporter, such as the green fluorescent protein, and observed by fluorescent microscopy. Regulatable expression vectors allow a gene product or alleles thereof to be conditionally expressed in cells. Such systems test biological responses to gene products or provide a means for protein overexpression.

When biochemical methods are used to study biological processes, the investigator may identify the protein product before the gene is known. In these cases reverse genetics is used to isolate the corresponding gene and to construct mutants to study protein function in vivo. Here the partial protein amino acid sequence is determined. With this information, a degenerative DNA sequence is deduced for every combination of codons to each amino acid. If the genome sequence is available, the corresponding gene is found with bioinformatic tools. If the genome sequence is not available, then degenerative oligos are synthesized to the peptides and a gene fragment is PCR amplified. Alternatively, the oligos (or PCR product) is labeled as a hybridization probe to identify the corresponding clone from a genomic library. Recombinant DNA and genetic techniques provide the means to insert mutant alleles into the host to study mutant phenotypes. Depending on the genetic tools available for a host a variety of mutants can be constructed such as a null, insertion, truncations, point, or promoter mutations. Recombinant DNA methods continue to improve and will inevitably serve as a foundation to approach biological and commercial problems in the foreseeable future.

Hormones and the Regulation of Transcription

Recombinant DNA techniques have been very important in identifying and characterizing the proteins involved in the mechanisms of action of hormones (receptors, G proteins, protein kinases, etc.). In many cases they have also been invaluable for studying hormone action more directly, specifically when the hormone action involves regulation of expression of specific genes. Steroid and thyroid hormones have been most intensively studied in this respect. It is now clear that these hormones bind to intracellular receptors which themselves bind to specific DNA sequences (hormone-response elements, HREs) close to the 5’ end of the target genes (usually, but not always, upstream of the gene promoter) (Cato et al., 1992; Truss and Beato, 1993). Binding of the hormone–receptor complex leads to activation (or in some cases inactivation) of gene transcription. Many hormones that bind to membrane-associated receptors also alter transcription of specific genes, but here the effect is less direct. There is no clear evidence that in these cases the hormone–receptor complexes can bind directly to DNA. However, second messengers (e.g. cyclic AMP) in association with specific binding proteins may do so. Alternatively, protein kinases or phosphatases, activated following hormone–receptor binding, may activate or inactivate transcription factors, leading in turn to altered gene expression.

The application of recombinant DNA techniques has had a major impact on our understanding of many aspects of such transcriptional control. cDNAs and genes for many steroid hormone and thyroid hormone receptors have been cloned, leading to a much improved understanding of the nature of these proteins and the ways that they bind hormones, DNA, and other proteins in the complexes involved in transcriptional regulation (Evans, 1988). Recombinant DNA methods have also provided a basis for characterizing the HREs to which hormone–receptor complexes bind, a key aspect which will be considered in some detail.

Biohazards and laboratory safety

Potential or real biohazards include recombinant DNA molecules, pathogenic (to animals, humans, and/or plants) microorganisms (viruses, bacteria, fungi, parasitic agents), and chemicals that are radioactive or potentially toxic (including carcinogenicity) to animals and humans. A variety of federal guidelines or standards exist for the handling and disposal of these agents.

Recombinant DNA and organisms containing recombinant DNA molecules. The first significant research efforts with recombinant deoxyribonucleic acid (DNA) material occurred in the early 1970s. As noted by Fredrickson [211], the first few years of recombinant DNA research were marked by controversy. The potential hazards of inserting foreign genetic material into common gut bacteria, such as Escherichia coli, were either overstated or misunderstood. Cautious progress in this field of research has given scientists and federal officials more realistic perspectives that have been used to prepare the most recently adopted NIH Guidelines [212] that have less force of law than the regulations governing human and animal research, but are nevertheless followed strictly by all institutions because violation of the NIH Guidelines threatens NIH funding for all research involving recombinant DNA work. The NIH Guidelines are administered by the Office of Biotechnology Activities (OBA) in the Office of Science Policy ([212]; within the Office of the Director of NIH), and are designed to ensure appropriate attention to biosafety practices and procedures, and ethical principles in research, involving recombinant DNA and organisms containing recombinant DNA molecules. The guidelines are very detailed, but generally instruct researchers in the following steps necessary for work with recombinant DNA:

Risk assessment – differentiated through Risk Group (RG) scaling of RG1 through RG4, beginning with agents not known to cause human disease (RG1), and moving through agents anticipated to cause greater degrees of human illness (RG2 and RG3), up through those that may cause incurable illness (RG4).

Categorization of experiments – using the RG-scale to delineate the types of experiments anticipated from the use of recombinant DNA material, outside of living organisms, through use of such materials in plants, animals, and humans.

Defining the roles of investigators, the institution, and possibly the NIH in research – based on the assessments mentioned earlier, determining the likely roles of investigators, the institution’s Biosafety Officer, Institutional Biosafety Committee (IBC; given later) and IRB, the OBA, and the federally commissioned NIH Recombinant DNA Advisory Committee (RAC) in the design and conduct of experiments.

Directing necessary containment systems – determining and using appropriate containment systems, as categorized under the Biosafety Level scaling of BL1 through BL4, with rank-order correlation to the RG categories noted earlier, and with physical containment systems and facilities commonly developed (in increasing stringency of containment) under the Biosafety Level system (BL1–BL4).

As suggested by the steps mentioned earlier, considerable thought, planning, and compliance consciousness must be invoked in all work with recombinant DNA and organisms containing recombinant DNA molecules. Fortunately, knowledgeable senior investigators, biosafety officers, and the chair or members of the institution’s IBC should be available for consultation.

Akin to the IRB and IACUC, the IBC is appointed by the university’s president, but oversight of the committee’s actions is delegated to the chief research officer of the university. The IBC [212] consists of no fewer than five members who collectively have expertise in recombinant DNA or synthetic nucleic acid molecule technology. They must also be capable of assessing the safety of recombinant DNA research, and the risks of this research to public health and the environment. At least two members of the IBC must not be affiliated with the university (other than their membership on the IBC), and should represent public health and environmental interests of the surrounding community. Additionally, at least one member each should be appointed, with relevant expertise in plant pathogens (or pests) or animal diseases, if these areas are the focus of research subject to review. Also, the institution’s Biosafety Officer must be a member of the IBC, if research is conducted at BL3 or BL4 levels, or in cases of large-scale (>10 L) operations. The IBC on many campuses will be responsible for all biohazards (given later), not just those associated with recombinant DNA molecules.

The IBC’s responsibility in recombinant DNA research is to evaluate proposals for potential hazards, and to insure that suitable precautions are adopted. The IBC chairperson is a good source of information and advice. He or she may be consulted, if work is planned with any recombinant DNA, or other potential biohazards that are defined as the responsibility of the IBC.

Microbiological hazards. Etiologic agents and oncogenic viruses are two biohazards that require special handling. Federal regulations have not been promulgated in these areas, although standards have been published by the Centers for Disease Control and Prevention (CDC) and the NIH, in a document (Biosafety in Microbiological and Biomedical Laboratories (BMBL), 2009) that can be downloaded free of charge. The BMBL contains a wealth of information under the following topic headings:

Biological Risk Assessment

Principles of Biosafety

Laboratory Biosafety Level Criteria (BL1–BL4)

Vertebrate Animal Biosafety Level Criteria for Vivarium Research Facilities

Principles of Laboratory Biosafety

Occupational Health and Immunoprophylaxis

Agent Summary Statements: Bacteria, Fungi, Parasites, Rickettsia, Viruses, Toxins, and Prions

All graduate researchers working with potential microbiological hazards should have a copy of the BMBL for appropriate reference work.

Typically, universities have safety officers with oversight and compliance responsibilities for devices emitting ionizing radiation or radioactive materials, toxic chemicals (including carcinogens), and scheduled drugs in research. Links to these individuals, the offices they serve, and the relevant policies, should be cross listed on the website of the institution’s office of research services or equivalent.

Radiation hazards. A university’s radiation safety officer will most frequently report to the institution’s chief research officer, and will coordinate efforts with a radiation safety committee (RSC) that is appointed analogously to the IRB, IACUC, and IBC. The radiation safety program is devised in conjunction with the RSC, but is maintained on a day-to-day basis by a radiation safety officer who is responsible for the:

termination of activities causing radiation hazards;

inspection of areas where sources of radiation (including radiation producing equipment) are stored or used in research;

enforcement of a program of procurement and record keeping, required of all authorized users of radioactive sources or materials;

maintenance of systems for the proper disposal of radioactive wastes;

management of educational programs on safety precautions and procedures;

assuring that new radiation sources are kept in compliance with federal and state regulations; and

service as liaison between university officials and federal and state officials, in order to assure fulfillment of radiation safety and licensure requirements.

To use radioactive sources and materials, researchers have to obtain approval through the RSC and the radiation safety officer. Usually, an advisor will be the authorized user who may supervise relevant activities by students. This magnifies the students’ responsibilities, and requires that they become well informed, as required through training available through the university’s radiation safety office or equivalent.

A copy of the university’s radiation safety manual should be available electronically through the radiation safety office. Enrollment in a radiation safety course(s) may be required before your certification for use of regulated sources or radioactive materials. Even if one’s background is in physics or chemistry, the practical insights gained through a radiation safety course will be valuable.

Lasers. The rapid increase in the use of lasers (light amplification by the stimulated emission of radiation) over the last several decades, for many medical applications, and in both basic and applied research in STEM disciplines, as diverse as chemistry, several areas of engineering, physics, and even geosciences and biology, has been the impetus for universities to develop Laser Safety Programs. In some academic settings, a separate Laser Safety Committee (LSC) is created, but in many colleges and universities, the use of lasers falls under the purview of the RSC (often leading to a renaming of the committee to RLSC). There are two major types of lasers, pulsed and continuous wave, that differ in the type of energy generated. Hazard potential for lasers is normally characterized according to “class” by the American National Standards Institute (ANSI) [214], and the International Electrotechnical Commission (IEC) [215]. Class I lasers represent the lowest level of hazard under normal operating conditions while, at the other end of the scale, Class IV lasers can not only produce retinal damage, but also can pose skin radiation and fire hazards. Or, more specifically:

Class I – lasers or laser systems that do not under normal operating conditions pose a hazard.

Class II – low power, visible light lasers or systems that because of the normal human aversion responses (blinking, eye movements, etc.) do not normally present a hazard. They may present some potential for hazard if viewed directly for extended periods of time (similar to many conventional light sources).

Class IIIA – lasers or systems that normally would not injure the eye, if viewed for only momentary periods with the unaided eye, but may present a greater hazard if viewed using collection optics. All Class IIIA lasers must have a caution label, and some must have a DANGER label.

Class IIIB – lasers or systems that will produce eye damage, if viewed directly.

Class IV – lasers or systems that produce retinal damage from direct viewing or reflected viewing. Such lasers may produce significant eye and skin radiation hazards, as well as fire hazards.

As with other types of occupational hazards, working with lasers requires all users to have state or federally approved Environmental Health and Safety (EHS) training, and regular equipment inspection by the LSO. You need to be aware exactly what type and class of laser is being used in the lab that you are joining, so that you can get appropriate training and safety guidelines.

Dangerous and toxic chemicals. Universities commonly have EHS offices that are responsible for the inspection and monitoring of laboratories where dangerous (potential causes of fires or explosions) and toxic chemicals are used. EHS offices will also assist in the training of new researchers who must use dangerous or toxic chemicals in their work. Universities’ EHS efforts may also be augmented by the work and Institutional Laboratory Safety Committee (ILSC), charged and constituted in parallel to the IRB, IACUC, and IBC.

EHS offices are responsible for the pick up and proper disposal of potentially dangerous and toxic chemicals, and it is important for graduate researchers to be well informed about waste containers and safety cabinets used for temporary containment. Researchers should also be informed about proper routines for requesting permanent disposal of dangerous and toxic materials, and should assiduously avoid disposal of chemicals in sinks or common sewer drains.

Researchers should become well informed on dangerous and toxic chemicals, especially those that may be used in their own research. We recommend that all graduate researchers obtain a copy of Prudent Practices in the Laboratory [216] that can be downloaded without cost through the National Research Council’s website. In it, you will find up-to-date descriptions of the following topics:

The Culture of Laboratory Safety

Environmental Health and Safety Management System

Emergency Planning

Evaluating and Assessing Risks in the Laboratory

Management of Chemicals

Working with Chemicals

Working with Laboratory Equipment

Management of Waste

Laboratory Facilities

Laboratory Security

Safety Laws and Standards Pertinent to Laboratories

You may complete your laboratory safety electronic file by downloading a copy of the US Department of Labor Occupational Safety and Health Administration (OSHA) Toxic and Hazardous Substances [217] regulations, also available without cost.

Scheduled drugs. Various potent drugs are useful tools in biological research. Certain types of these drugs have a high abuse potential that causes them to be categorized as scheduled drugs by the US Drug Enforcement Administration (DEA), under the Controlled Substances Act of 1970. The following are examples of drugs or drug-containing dosage forms listed under Schedules I through V, with drugs of greatest abuse potential in Schedule I [218].

Schedule I

Opiates, such as acetylmethadol

Opium derivatives, such as heroin

Hallucinogenic substances, such as lysergic acid diethylamide (LSD), 3,4-methylenedioxyamphetamine (ecstasy)

Depressants, such as methaqualone

Stimulants, such as N,N-dimethylamphetamine

Schedule II

Certain substances of vegetable origin or chemical synthesis (or salts or chemically equivalent substances), such as codeine, opium extracts

Opium poppy and poppy straw

Coca leaves and any salt, compound, derivative or preparation of coca leaves, such as cocaine, ecgonine

Schedule III

Less potent stimulants, such as chlorphentermine

Less potent depressants, such as pentobarbital

Anabolic steroids, such as norethandrolone

Schedule IV

Weak depressant drugs, such as chloral hydrate, meprobamate

Weak stimulants, such as pemoline

Schedule V

Miscellaneous drugs of abuse, such as mixtures or pharmaceutical preparations containing no more than 200 mg of codeine per 100 mL, or per 100 g

The use of scheduled substances in laboratory research, excluding humans, requires the permission of an authorized user, such as an advisor who may be registered by the DEA. Alternatively, internal authorization procedures may be possible through the university’s EHS office. Regardless of the authorization procedure, use of scheduled drugs requires strict accounting procedures, security measures, and assurance of nonuse in humans, except under conditions strictly defined and enforced through the university’s IRB. Researchers should make sure they understand all these procedures before taking on responsibilities.

The conduct of special types of research, including human subjects, animals, biohazards, and other potentially hazardous materials, is assisted by the information offered in this chapter. Furthermore, the understanding of these special areas of research can be vital in effective grant proposal development.

GENE THERAPY

Recombinant DNA technology is already used in the manufacture of therapeutic products, such as opioid peptides, which have relevance to clinical neurology (Thompson, 1984). Another rather indirect example is the availability of genetically engineered human growth hormone (GH) which has assumed great importance since it was recognized that the administration of GH from pooled postmortem pituitary tissue carried a risk of transmitting Creutzfeldt–Jakob disease to the deficient recipients (Powell-Jackson et al., 1985).

The prospect of replacing defective genes in patients is futuristic but already theoretically possible (Emery, 1984). There are numerous technical problems, but it is likely that this approach to treatment will become available in the next decade or so, albeit in a limited number of diseases. The main difficulties arise not from cloning the gene, but introducing it into the patient in such a way that it will be expressed in the appropriate tissue.

Foreign genes could be transferred to a human host by viral vectors, such as the retroviruses, but there would be no guarantee that the foreign gene would not be incorporated into other important genes and this could clearly be hazardous to the recipient. If DNA sequences are microinjected into germ cells (germ cell gene therapy), there is also random incorporation of them into the genome, and the genes may not be expressed in the target tissue unless accompanied by a tissue specific promotor. Nevertheless, Palmiter et al. (1982) produced transgenic ‘supermice’ by microinjecting the rat growth hormone gene into mouse ova soon after fertilization (see Messing, Chapter 9 of this volume).

It is technically possible to apply the same techniques to somatic cells (somatic cell gene therapy). For example, bone marrow stems cells from patients with haemoglobinopathies could be microinjected with globin genes and replaced, with the hope that the transformed cells would multiply, express the gene, and eventually replace the mutant cells. There are obvious problems in using this approach in neurological disorders. However, in rare instances, such as the Lesch–Nyhan syndrome, neurological dysfunction results from a generalized metabolic defect but is not associated with gross structural abnormalities in the brain. In these circumstances replacing the deficient enzyme might be beneficial. The hypoxanthine-guanine phosphoribosyl transferase gene, linked to retroviral or promotor sequences, has been transferred to mouse bone marrow stem cells with subsequent expression of the gene (Stein and Morrison, 1985). It remains to be seen whether this type of approach will be successful in patients with the Lesch–Nyhan syndrome, or any other neurological disease.

Comings (1980) predicted 7 years ago that recombinant DNA techniques would have a major impact on clinical medicine and basic research, referring to their application as ‘the new genetics’. The new genetics have entered the arenas of clinical neurology and neurobiology and are providing a precise means of investigating inherited neurological diseases. More generally, these techniques are already enhancing our understanding of the development and function of the normal human nervous system.

84 Discuss the role of molecular biology in the diagnosis and investigation of genetic disorders

Recombinant DNA techniques are used routinely to identify single gene mutations. The sensitivity of these methods has been dramatically improved by the use of the polymerase chain reaction (PCR), through which DNA and RNA samples can be amplified several million–fold. Molecular biology (molecular diagnostics) can be used to:

Identify known point mutations, translocations, and deletions

Infer mutations on the basis of restriction fragment length polymorphisms

Investigate the pathogenesis of genetic diseases; for example, it can be used to:

Clone genes using direct and positional cloning methods (Direct methods rely on an understanding of the abnormal gene product [e.g., abnormal globin in patients with thalassemias]. Positional cloning relies on insights concerning the location of a gene on a particular chromosome.)

Produce pure proteins through recombinant DNA technology (e.g., human clotting factors for hemophiliacs)

Analyze patterns of gene expression using DNA chip technology (By analyzing the expression of thousands of genes, researchers can gain insights into the role of the entire genome in controlling cellular and organismal phenotype.)

Websites

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed

http://www-medlib.med.utah.edu/WebPath/webpath.html#MENU

http://medgen.genetics.utah.edu/thumbnails.htm

http://www.ncbi.nlm.nih.gov/disease/index.html

Use of Transgenic and Knockout Mice to Define Gene Function

Recombinant DNA technology has resulted in the identification of many disease-related genes. To advance the understanding of the disease related to a previously unknown gene, the function of the protein encoded by that gene must be verified or identified, and the way changes in the gene's expression influence the disease phenotype must be characterized. Analysis of the role of these genes and their encoded proteins has been made possible by the development of recombinant DNA technology that allows the production of mice that are genetically altered at the cloned locus. Mice can be produced that express an exogenous gene and thereby provide an in vivo model of its function. Linearized DNA is injected into a fertilized mouse oocyte pronucleus and reimplanted in a pseudopregnant mouse. The resultant transgenic mice can then be analyzed for the phenotype induced by the injected transgene. Placing the gene under the control of a strong promoter that stimulates expression of the exogenous gene in all tissues allows the assessment of the effect of widespread overexpression of the gene. Alternatively, placing the gene under the control of a promoter that can function only in certain tissues (a tissue-specific promoter) elucidates the function of that gene in a particular tissue or cell type. A third approach is to study control elements of the gene by testing their capacity to drive expression of a “marker” gene that can be detected by chemical, immunologic, or functional means. For example, the promoter region of a gene of interest can be joined to the cDNA encoding green jellyfish protein and activity of the gene assessed in various tissues of the resultant transgenic mouse by fluorescence microscopy. Use of such a reporter gene demonstrates the normal distribution and timing of expression of the gene from which the promoter elements are derived. Transgenic mice contain exogenous genes that insert randomly into the genome of the recipient. Expression can thus depend as much on the location of the insertion as it does on the properties of the injected DNA.

In contrast, any defined genetic locus can be specifically altered by targeted recombination between the locus and a plasmid carrying an altered version of that gene (Fig. 1.9). If a plasmid contains that altered gene with enough flanking DNA identical to that of the normal gene locus, homologous recombination can occur, and the altered gene in the plasmid will replace the gene in the recipient cell. Using a mutation that inactivates the gene allows the production of a null mutation, in which the function of that gene is completely lost. To induce such a mutation, the plasmid is introduced into an embryonic stem cell, and the rare cells that undergo homologous recombination are selected. The “knockout” embryonic stem cell is then introduced into the blastocyst of a developing embryo. The resultant animals are chimeric; only a fraction of the cells in the animal contain the targeted gene. If the new gene is introduced into some of the germline cells of the chimeric mouse, then some of the offspring of that mouse will carry the mutation as a gene in all of their cells. These heterozygous mice can be further bred to produce mice homozygous for the null allele. Such knockout mice reveal the function of the targeted gene by the phenotype induced by its absence. Genetically altered mice have been essential for discerning the biologic and pathologic roles of large numbers of genes implicated in the pathogenesis of human disease.

Recombinant DNA Technology

By the end of the 1970s only a few dozen genes had been mapped to autosomes because the number of usable markers was so few. With the discovery of recombinant DNA technology, all that changed. By recombinant DNA (rDNA), we mean a combination of DNAs from different origins, that is, different organisms (such as bacterial and human).

Recombinant DNA technology depends on five “tools.” These are:

Restriction enzymes are bacterial enzymes that cut DNA, like a scissors, at specific sites, producing fragments of different sizes (Figure 6.19); some restriction enzymes recognize a four-nucleotide sequence (e.g., AGGA), others a six-nucleotide one (GAATTC). These fragments can be run out by electrophoresis (where electrically charged molecules are allowed to migrate through a fluid or gel under the influence of an electric field, thereby being separated), producing a particular pattern of fragments arrayed by size, from largest to smallest (Figure 6.20).

Molecular cloning is the process by which DNA fragments are spliced into viral or bacterial vectors, purified, and amplified (Figure 6.21).

Hybridization probes are single-stranded, purified DNA sequences of varying length (25 to several thousand nucleotides) that are labeled with a radioactive isotope or fluorescent dye. Complementarity allows them to hybridize with, and thereby identify, corresponding sequences in cloned collections of DNA fragments.

Polymerase chain reaction (PCR) uses a specific bacterial polymerase to amplify a piece of DNA up to a billion-fold or more. This is a powerful means of obtaining sufficient DNA for a variety of purposes (Figure 6.22).

DNA sequencing reveals the order of base pairs in an isolated DNA molecule or fragment. As shown in Figure 6.23, the Sanger sequencing method deploys fluorescent-labeled analogues of A, T, G, and C that interrupt the synthesis of a DNA strand complementary to the template molecule. From the sequence of terminated fragments, the sequence of the original template can be determined—a once painstaking technique that has become fully automated.