What Do Scientists Hope to Accomplish Using Recombinant Dna?

Recombinant DNA

Recombinant Deoxyribonucleic acid

Frank H. Stephenson , in Calculations for Molecular Biological science and Biotechnology (Third Edition), 2016

Chapter Summary

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

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

$\text{moles}\text{of}\text{DNA}\text{ends}=\frac{\mathrm{ii}\times \left(\text{grams}\text{of}\text{Deoxyribonucleic acid}\right)}{\left(\text{number}\text{of}\text{bp}\right)\times \left(\frac{660\text{g/mol}}{\text{bp}}\right)}$

The amount of ends generated past a restriction enzyme digest of circular Deoxyribonucleic acid is given past the equation

$\text{moles}\text{ends}=\mathrm{two}\times \left(\text{moles}\text{DNA}\right)\times \left(\text{number}\text{of}\text{brake}\text{sites}\right)$

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

$\text{moles}\text{ends}=\left[2\times \left(\text{moles}\text{Dna}\right)\times \left(\text{number}\text{of}\text{restriction}\text{sites}\right)\right]+\left[\mathrm{two}\times \left(\text{moles}\text{DNA}\right)\right]$

Deoxyribonucleic acid fragments generated by digestion with a restriction endonuclease can exist joined together once more by the enzyme ligase. The likelihood that 2 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 2 termini will see and be ligated. This parameter is designated by the term i and is divers as the total concentration of complementary ends in the ligation reaction. For a linear fragment of duplex Deoxyribonucleic acid with cohesive ends, i is given by the formula

$i=2{\mathrm{North}}_{0}M\times {10}^{-\mathrm{three}}\text{ends/mL}$

where North ₀ is Avogadro's number (6.022   ×   10²³) and M is the molar concentration of the Dna.

Ligation of DNA molecules tin 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 tin finer interact. For any Dna fragment, j is a constant value dependent on the fragment'southward length. It can be calculated as

$j={\left(\frac{\mathrm{iii}}{2\pi \mathrm{fifty}b}\right)}^{\frac{3}{2}}\text{ends/mL}$

where l is the length of the Dna fragment, b is the minimal length of Dna that can bend effectually to class a circle, and π is the number pi. For bacteriophage lambda Deoxyribonucleic acid, j has a value of iii.22   ×   10¹¹  ends/mL. The j value for any Deoxyribonucleic acid molecule can be calculated in relation to j _λ past the equation

$j=j_{\text{λ}}{\left(\frac{{\text{MW}}_{\text{λ}}}{\text{MW}}\right)}^{1.5}\text{ends/mL}$

where j _λ is equal to 3.22   ×   ten¹¹  ends/mL and "MW" represents molecular weight. Under circumstances in which j is equal to i (j  = i, or j/i  =   1), the terminate of whatever particular DNA molecule is just as likely to join with another molecule equally information technology is to interact with its ain 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 volition still let for circularization of the recombinant molecule. In add-on, the concentration of the termini of the insert (i _insert) should exist approximately twice the concentration of the termini of the linearized plasmid vector (i _insert  =   twoi _vector).

Transformation efficiency is a measure of how many leaner were able to have in recombinant plasmids. Information technology is expressed every bit transformants/μg Dna.

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

$N=\frac{\text{ln}(1-P)}{\text{ln}\left[1-\left(\frac{I}{\mathrm{Thou}}\right)\right]}$

where I is the size of the average cloned insert, in base pairs, G is the size of the target genome, in base of operations pairs, N is the number of independent clones, and P is the probability of isolating a specific Deoxyribonucleic acid 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 past the equation

$\mathrm{North}=\frac{\text{ln}\left(\mathrm{one}-P\right)}{\text{ln}\left[1-\left(\frac{\mathrm{north}}{T}\right)\right]}$

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 normally set to 0.99; a 99% take chances 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 jail cell.

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

$P=\frac{\text{coding}\text{sequence}\text{size}\left(\text{in}\text{kb}\right)}{\text{genome}\text{size}\left(\text{in}\text{kb}\right)\times \mathrm{vi}}$

Hybridization of a probe to a recombinant library should be carried out at a temperature (T _i ) 15°C below the probe's T _thousand :

$T_i=T_k-15°\text{C}$

T _m is calculated using the equation

$T_{\mathrm{chiliad}}=16.6\log \left[M\right]+0.41\left[P_{\text{GC}}\right]+81.5-P_m-B/L-0.65\left[P_f\right]$

where Chiliad is the molar concentration of Na⁺, P _GC is the percent of G and C bases in the oligonucleotide probe, P _grand is the percent of mismatched bases, P _f is the per centum formamide, B is equal to 675 (for probes up to 100 nucleotides in length), and 50 is the probe length in nucleotides. The T _m of probes longer than 100 bases tin be calculated using the following formula

$T_m=81.5°\text{C}+\mathrm{16.half-dozen}\left(\log \left[{\text{Na}}^{+}\right]\right)+0.41(\%\text{GC})-0.63(\%\text{formamide})-600/L$

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

$P_0={\left(\frac{1}{4}\right)}^L\times \mathrm{two}C$

where P ₀ is the number of contained 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–v°C beneath the oligonucleotide's T _grand . The approximate length of time allowed for the hybridization reaction to attain one-half-completion is given by the equation

$t_{1/\mathrm{ii}}=\frac{\ln 2}{gC}$

where m is a first-guild charge per unit constant and C is the molar concentration of the oligonucleotide probe (in moles of nucleotide per liter).

The charge per unit abiding, k, represents the rate of hybridization of an oligonucleotide probe to an immobilized target nucleic acid in 1-Thou sodium ion and is given by the equation

$m=\frac{\mathrm{iii}\times {\mathrm{ten}}^5{L}^{0.5}\text{L}/\text{mol}/\text{due south}}{\mathrm{Northward}}$

where k is calculated in liters/mole of nucleotide per second, 50 is the length of the oligonucleotide probe in nucleotides, and N is the probe's complication.

Complication, as it relates to an oligonucleotide, is calculated equally 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 Deoxyribonucleic acid 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 fifty% formamide, the following equation to approximate the corporeality of fourth dimension to accomplish half-complete hybridization can exist used.

$t_{1/2}=\frac{1}{\mathrm{Ten}}\times \frac{Y}{\mathrm{v}}\times \frac{Z}{\mathrm{ten}}\times \mathrm{ii}$

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 book of the hybridization reaction (in mL).

Nearly complete hybridization is achieved after 3 times the t _ane/2 menstruum.

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 bend and regression line equation that tin 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

${\mathrm{Yard}}_t=M_0-\frac{2M_n{V}_{\max }t}{\left[{\mathrm{Thousand}}_m+{\left(\mathrm{South}\right)}_0\right]}$

where G _t is the molecular weight of the duplex DNA after t minutes of incubation, One thousand ₀ is the original molecular weight of the duplex DNA, M _northward is the average molecular weight of a mononucleotide (taken as 330   Da), V _max is the maximum reaction velocity (in moles of nucleotide removed/liter/minute), t is the length of time of incubation (in minutes), One thousand _m is the Michaelis–Menten constant (in moles of dsDNA termini/liter), and S ₀ is the moles of dsDNA termini/L at t  =   0   min.

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Biosecurity challenges for the IBC

Ryan N. Burnette , Nancy D. Connell , in Ensuring National Biosecurity, 2016

Recombinant Dna

rDNA, the combining of genetic material in juxtapositions not found in nature, is a traditional expanse of research methodology overseen by the IBCs; these activities – the original target of IBC oversight – have a less direct relationship to security issues. The apply of rDNA techniques in the production of therapeutic molecules (insulin, interleukins, hormones, etc.) is generally well accepted; the employ of genetic modification in environmentally (due east.g. bioremediation) or agriculturally (GMO) important arenas remains controversial in segments of society, as is the use of rDNA in the treatment of homo disease, or gene therapy, despite a number of successes (eastward.g. alipogene tiparvocec, trade name Glybera, for the treatment of severe pancreatitis acquired past lipoprotein lipase deficiency) [1]. However, rDNA techniques are oftentimes used in studies of virulence mechanisms, e.1000. in the generation of chimeric bacteria and viruses.

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IBCs – A cornerstone of public trust in research

Kathryn L. Harris , ... Carrie D. Wolinetz , in Ensuring National Biosecurity, 2016

Introduction

Recombinant Dna has been a transformative technology, providing tools that non but have enabled tremendous understanding of life at the most fundamental levels, but that accept also led to a myriad of medical and agronomical 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 hope of recombinant Dna could only be realized if they assumed responsibleness for addressing the safety and ethical concerns that it raised.

The electric current organisation of oversight of recombinant Dna research was established nearly 40 years ago when the NIH Guidelines for Enquiry Involving Recombinant or Synthetic Nucleic Acrid 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 after became the Office of Biotechnology Activities (OBA) inside 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 afterwards renamed "Institutional Biosafety Committees" (IBCs) to more clearly reflect their role. IBCs must review recombinant and synthetic nucleic acid molecule enquiry for conformity with the NIH Guidelines. In addition, they assess the inquiry for potential risks to health and the environment. This is accomplished by reviewing physical and biological containment for the enquiry and ensuring that researchers are adequately trained to conduct the inquiry they are proposing safely.

The hallmarks of this oversight system from its inception were public participation and transparency. Attending to the concerns of the community and local interests is a major theme that carries forrad in the system of biosafety oversight today. This key chemical element has served to preserve public trust in the safe of the life sciences research enterprise. In retrospect, the risks of recombinant Dna technology that were feared early in its development did not materialize. That fact notwithstanding, the evolution of a scientifically based oversight organisation with the IBCs as the centerpiece permitted the safe development of recombinant Deoxyribonucleic acid as an essential technology in research. Over the years, oversight by IBCs has proven critically important to ensuring condom throughout various research fields – medical, occupational, environmental – equally well equally in promoting responsible scientific exercise. Due to the dynamic nature of the life sciences there remains an ongoing need to assess biosafety dimensions of the research beingness conducted and to manage whatsoever risks associated with work. As life sciences enquiry continues to accelerate, many lines of research, particularly involving highly pathogenic organisms, continue to generate public concern. Financial support for life sciences enquiry comes primarily from publicly derived taxation dollars, and so the life sciences customs 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 information technology funds must continue to ensure that IBCs are equipped to fulfill their responsibilities and then that biosafety risks are responsibly managed and public rubber and trust are preserved.

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Recombinant Deoxyribonucleic acid, Basic Procedures

D. Wall , in Encyclopedia of Microbiology (Tertiary Edition), 2009

Applications

Recombinant DNA has revolutionized biological research and has had huge impact on the pharmaceutical and biotechnology industries. These techniques accept 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 part. Genomic sequence data allows a clear understanding of the evolutionary relationships betwixt species and genes. Genomic data as well allows an understanding of how the environment impacts cellular physiology and genetic limerick. This is particularly true with microorganisms as they are found in a plethora of environments, for case, soils, oceans, streams, and animal and found hosts. These environmental conditions can vary profoundly 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 Deoxyribonucleic acid 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 every bit protein or RNA. Potential effects on cellular physiology, toxicologically, along with the mechanism of chemical compound activeness 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 manufacture uses recombinant Deoxyribonucleic acid to isolate, improve, and produce protein therapeutics such equally 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 too 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 exist resistant to caterpillar, pests, and viruses past inserting resistant genes into establish genomes. In humans, gene therapy may i mean solar day improve wellness. 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 chemic synthesis processes past 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 assistance police force enforcement agencies test relationships between biological crime samples and individuals to prove guilt or innocence. These methods are likewise used to empathize 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 inquiry, recombinant DNA methods are omnipresent. They are used to report cistron 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 data is available or when evolutionarily conserved sequences/motifs are found within proteins. Thus site-specific changes in gene sequences directly test construction–function relationships. Alternatively, random mutagenesis of a cloned cistron is used in an equally informative and unbiased approach to report cistron function. DNA regulatory elements, such every bit promoters, can be studied in vivo by fusing them to reporter proteins, such every bit β-galactosidase. These reporters are used to test cistron 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 cistron product to a reporter, such equally the green fluorescent protein, and observed by fluorescent microscopy. Regulatable expression vectors allow a factor 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 poly peptide product before the gene is known. In these cases reverse genetics is used to isolate the corresponding cistron and to construct mutants to report poly peptide function in vivo. Here the fractional poly peptide amino acrid 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 respective factor is establish with bioinformatic tools. If the genome sequence is not bachelor, then degenerative oligos are synthesized to the peptides and a factor fragment is PCR amplified. Alternatively, the oligos (or PCR product) is labeled as a hybridization probe to place the corresponding clone from a genomic library. Recombinant Dna and genetic techniques provide the ways to insert mutant alleles into the host to study mutant phenotypes. Depending on the genetic tools bachelor for a host a variety of mutants tin can exist constructed such as a null, insertion, truncations, point, or promoter mutations. Recombinant Dna methods continue to improve and volition inevitably serve equally a foundation to arroyo biological and commercial issues in the foreseeable future.

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Molecular and Cellular Endocrinology

Michael Wallis , in Principles of Medical Biological science, 1997

Hormones and the Regulation of Transcription

Recombinant Deoxyribonucleic acid techniques have been very important in identifying and characterizing the proteins involved in the mechanisms of action of hormones (receptors, One thousand proteins, protein kinases, etc.). In many cases they accept also been invaluable for studying hormone activity more directly, specifically when the hormone activeness involves regulation of expression of specific genes. Steroid and thyroid hormones accept been most intensively studied in this respect. Information technology is now articulate that these hormones demark to intracellular receptors which themselves demark to specific DNA sequences (hormone-response elements, HREs) close to the 5' terminate of the target genes (usually, but non always, upstream of the cistron promoter) ( Cato et al., 1992; Truss and Beato, 1993). Binding of the hormone–receptor circuitous leads to activation (or in some cases inactivation) of factor transcription. Many hormones that bind to membrane-associated receptors likewise alter transcription of specific genes, but here the consequence is less straight. In that location is no articulate bear witness that in these cases the hormone–receptor complexes tin bind directly to DNA. However, second messengers (east.m. cyclic AMP) in clan 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 take been cloned, leading to a much improved understanding of the nature of these proteins and the ways that they bind hormones, Deoxyribonucleic acid, and other proteins in the complexes involved in transcriptional regulation (Evans, 1988). Recombinant Deoxyribonucleic acid methods have also provided a ground for characterizing the HREs to which hormone–receptor complexes bind, a central aspect which will be considered in some detail.

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Research Involving Human Subjects and Animals and Biohazards and Laboratory Safety

Robert V. Smith , ... Edward F. Lener , in Graduate Research (Fourth Edition), 2016

Biohazards and laboratory prophylactic

Potential or existent biohazards include recombinant Deoxyribonucleic acid molecules, pathogenic (to animals, humans, and/or plants) microorganisms (viruses, leaner, 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 beginning significant research efforts with recombinant deoxyribonucleic acid (Deoxyribonucleic acid) material occurred in the early 1970s. Equally noted by Fredrickson [211], the first few years of recombinant DNA research were marked by controversy. The potential hazards of inserting strange genetic material into common gut bacteria, such equally 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 forcefulness of law than the regulations governing human and animal enquiry, 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 Role of Biotechnology Activities (OBA) in the Office of Science Policy ([212]; inside the Function of the Director of NIH), and are designed to ensure appropriate attention to biosafety practices and procedures, and upstanding principles in research, involving recombinant DNA and organisms containing recombinant DNA molecules. The guidelines are very detailed, but more often than not instruct researchers in the following steps necessary for work with recombinant DNA:

•

Risk assessment – differentiated through Chance Group (RG) scaling of RG1 through RG4, start with agents not known to crusade human being illness (RG1), and moving through agents anticipated to crusade greater degrees of human illness (RG2 and RG3), upwards through those that may crusade incurable affliction (RG4).

•

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

•

Defining the roles of investigators, the establishment, and peradventure 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 carry of experiments.

•

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

Equally suggested by the steps mentioned earlier, considerable idea, planning, and compliance consciousness must be invoked in all piece of 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 bachelor for consultation.

Akin to the IRB and IACUC, the IBC is appointed past the university'southward president, but oversight of the committee's actions is delegated to the primary research officer of the university. The IBC [212] consists of no fewer than five members who collectively have expertise in recombinant Dna or constructed nucleic acid molecule technology. They must besides be capable of assessing the safety of recombinant DNA inquiry, and the risks of this enquiry to public wellness and the environs. At least ii members of the IBC must not exist 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 creature diseases, if these areas are the focus of research bailiwick to review. As well, the institution'southward Biosafety Officer must be a member of the IBC, if enquiry is conducted at BL3 or BL4 levels, or in cases of big-scale (>10 Fifty) 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 inquiry is to evaluate proposals for potential hazards, and to insure that suitable precautions are adopted. The IBC chairperson is a proficient source of information and communication. He or she may exist consulted, if piece of work is planned with any recombinant Deoxyribonucleic acid, or other potential biohazards that are divers as the responsibility of the IBC.

Microbiological hazards. Etiologic agents and oncogenic viruses are 2 biohazards that crave special treatment. Federal regulations have not been promulgated in these areas, although standards have been published past the Centers for Affliction Control and Prevention (CDC) and the NIH, in a document (Biosafety in Microbiological and Biomedical Laboratories (BMBL), 2009) that can be downloaded costless of charge. The BMBL contains a wealth of data under the post-obit topic headings:

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Biological Hazard Assessment

•

Principles of Biosafety

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Laboratory Biosafety Level Criteria (BL1–BL4)

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Vertebrate Creature 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 advisable reference work.

Typically, universities have rubber officers with oversight and compliance responsibilities for devices emitting ionizing radiation or radioactive materials, toxic chemicals (including carcinogens), and scheduled drugs in enquiry. 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.

Radiations hazards. A university'due south radiation rubber officer will near frequently study to the institution's chief inquiry officeholder, and will coordinate efforts with a radiation safety committee (RSC) that is appointed analogously to the IRB, IACUC, and IBC. The radiation safe programme is devised in conjunction with the RSC, but is maintained on a day-to-twenty-four hour period footing by a radiation rubber officer who is responsible for the:

1.

termination of activities causing radiations hazards;

2.

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

three.

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

iv.

maintenance of systems for the proper disposal of radioactive wastes;

5.

management of educational programs on prophylactic precautions and procedures;

six.

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

vii.

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

To employ radioactive sources and materials, researchers have to obtain blessing through the RSC and the radiations 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 academy's radiation rubber part or equivalent.

A re-create of the academy's radiation rubber transmission should be available electronically through the radiation safety office. Enrollment in a radiation safety class(due south) may be required earlier your certification for apply of regulated sources or radioactive materials. Even if one'due south background is in physics or chemistry, the applied insights gained through a radiation prophylactic course will be valuable.

Lasers. The rapid increment in the use of lasers (light amplification by the stimulated emission of radiations) 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, simply in many colleges and universities, the utilise of lasers falls nether the purview of the RSC (often leading to a renaming of the committee to RLSC). In that location are two major types of lasers, pulsed and continuous wave, that differ in the blazon of free energy generated. Chance potential for lasers is ordinarily characterized according to "class" by the American National Standards Institute (ANSI) [214], and the International Electrotechnical Committee (IEC) [215]. Course I lasers stand for the lowest level of hazard under normal operating conditions while, at the other end of the scale, Class 4 lasers can not only produce retinal damage, but also tin pose skin radiation and burn hazards. Or, more than specifically:

Class I – lasers or light amplification by stimulated emission of radiation systems that exercise non under normal operating weather condition pose a adventure.

Grade II – low power, visible low-cal lasers or systems that because of the normal homo aversion responses (blinking, heart movements, etc.) do non normally present a adventure. They may nowadays some potential for take chances if viewed directly for extended periods of time (similar to many conventional light sources).

Class IIIA – lasers or systems that commonly would not hurt the eye, if viewed for simply momentary periods with the unaided centre, just may present a greater hazard if viewed using drove optics. All Form IIIA lasers must have a caution label, and some must have a DANGER label.

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

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

As with other types of occupational hazards, working with lasers requires all users to take state or federally approved Ecology Health and Safety (EHS) training, and regular equipment inspection by the LSO. Yous need to be aware exactly what type and class of laser is being used in the lab that you are joining, and then that you tin go advisable training and safety guidelines.

Unsafe 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 volition 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 past the work and Institutional Laboratory Safety Commission (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 information technology is important for graduate researchers to be well informed about waste containers and safe cabinets used for temporary containment. Researchers should likewise be informed almost 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 exist downloaded without toll through the National Research Council's website. In it, you volition find upward-to-date descriptions of the following topics:

•

The Culture of Laboratory Rubber

•

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 matter

•

Laboratory Facilities

•

Laboratory Security

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Rubber Laws and Standards Pertinent to Laboratories

You may consummate your laboratory safety electronic file by downloading a re-create of the Usa Department of Labor Occupational Safety and Health Administration (OSHA) Toxic and Chancy Substances [217] regulations, also available without cost.

Scheduled drugs. Various strong 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 U.s. Drug Enforcement Administration (DEA), under the Controlled Substances Act of 1970. The post-obit 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 every bit acetylmethadol

Opium derivatives, such as heroin

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

Depressants, such as methaqualone

Stimulants, such equally Due north,N-dimethylamphetamine

Schedule 2

Sure 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 whatsoever table salt, compound, derivative or preparation of coca leaves, such as cocaine, ecgonine

Schedule III

Less potent stimulants, such equally chlorphentermine

Less stiff depressants, such every bit pentobarbital

Anabolic steroids, such every bit norethandrolone

Schedule IV

Weak depressant drugs, such as chloral hydrate, meprobamate

Weak stimulants, such equally pemoline

Schedule V

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

The use of scheduled substances in laboratory inquiry, 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'south EHS office. Regardless of the dominance procedure, employ of scheduled drugs requires strict accounting procedures, security measures, and balls of nonuse in humans, except under weather 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 being subjects, animals, biohazards, and other potentially hazardous materials, is assisted past the information offered in this chapter. Furthermore, the understanding of these special areas of enquiry tin be vital in constructive grant proposal development.

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Molecular genetics and neurological illness: bones principles and methods

A.E. Harding , Roger N. Rosenberg , in The Molecular Biology of Neurological Disease, 1988

GENE THERAPY

Recombinant Dna technology is already used in the manufacture of therapeutic products, such as opioid peptides, which take 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 information technology was recognized that the administration of GH from pooled postmortem pituitary tissue carried a gamble of transmitting Creutzfeldt–Jakob disease to the scarce recipients (Powell-Jackson et al., 1985).

The prospect of replacing defective genes in patients is futuristic merely already theoretically possible (Emery, 1984). There are numerous technical issues, merely it is likely that this approach to treatment will go available in the next decade or and so, albeit in a express number of diseases. The main difficulties arise not from cloning the factor, merely 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 man host past viral vectors, such every bit the retroviruses, just there would be no guarantee that the foreign gene would non be incorporated into other important genes and this could clearly exist chancy to the recipient. If Deoxyribonucleic acid sequences are microinjected into germ cells (germ cell gene therapy), in that location is likewise random incorporation of them into the genome, and the genes may not be expressed in the target tissue unless accompanied past 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 prison cell gene therapy). For example, bone marrow stems cells from patients with haemoglobinopathies could exist microinjected with globin genes and replaced, with the hope that the transformed cells would multiply, express the gene, and eventually supersede the mutant cells. There are obvious problems in using this approach in neurological disorders. However, in rare instances, such every bit the Lesch–Nyhan syndrome, neurological dysfunction results from a generalized metabolic defect just is not associated with gross structural abnormalities in the brain. In these circumstances replacing the deficient enzyme might be benign. 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). Information technology 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 vii years ago that recombinant Dna techniques would take a major affect on clinical medicine and basic research, referring to their awarding as 'the new genetics'. The new genetics take entered the arenas of clinical neurology and neurobiology and are providing a precise ways of investigating inherited neurological diseases. More than generally, these techniques are already enhancing our agreement of the development and function of the normal human nervous system.

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Developmental and Genetic Diseases

Bruce A. Fenderson PhD , in Pathology Secrets (Tertiary Edition), 2009

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

Recombinant Dna techniques are used routinely to identify single cistron mutations. The sensitivity of these methods has been dramatically improved by the use of the polymerase concatenation reaction (PCR), through which DNA and RNA samples tin can be amplified several 1000000–fold. Molecular biological science (molecular diagnostics) tin be used to:

▪

Place known signal mutations, translocations, and deletions

▪

Infer mutations on the basis of restriction fragment length polymorphisms

▪

Investigate the pathogenesis of genetic diseases; for instance, information technology can be used to:

○

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

○

Produce pure proteins through recombinant DNA engineering (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 proceeds insights into the office of the unabridged genome in controlling cellular and organismal phenotype.)

Websites

ane

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

2

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

three

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

iv

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

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Anatomy and Physiology of the Gene

Andrew J. Wagner , ... Edward J. BenzJr., in Hematology (Seventh Edition), 2018

Use of Transgenic and Knockout Mice to Define Gene Function

Recombinant Deoxyribonucleic acid technology has resulted in the identification of many disease-related genes. To accelerate the understanding of the affliction related to a previously unknown gene, the function of the protein encoded by that factor must be verified or identified, and the way changes in the gene'due south expression influence the affliction 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 so be analyzed for the phenotype induced by the injected transgene. Placing the cistron nether the control of a stiff promoter that stimulates expression of the exogenous gene in all tissues allows the assessment of the result of widespread overexpression of the gene. Alternatively, placing the cistron under the command of a promoter that can function only in sure tissues (a tissue-specific promoter) elucidates the function of that gene in a particular tissue or prison cell type. A third approach is to study control elements of the gene by testing their capacity to bulldoze expression of a "marker" cistron that can be detected by chemical, immunologic, or functional ways. For case, the promoter region of a gene of interest tin be joined to the cDNA encoding green jellyfish protein and activeness of the factor assessed in various tissues of the resultant transgenic mouse by fluorescence microscopy. Employ of such a reporter cistron demonstrates the normal distribution and timing of expression of the gene from which the promoter elements are derived. Transgenic mice incorporate exogenous genes that insert randomly into the genome of the recipient. Expression can thus depend equally much on the location of the insertion as information technology does on the properties of the injected DNA.

In dissimilarity, any defined genetic locus tin be specifically altered by targeted recombination between the locus and a plasmid carrying an altered version of that gene (Fig. i.ix). If a plasmid contains that altered gene with enough flanking DNA identical to that of the normal gene locus, homologous recombination tin occur, and the altered cistron in the plasmid will supersede the gene in the recipient cell. Using a mutation that inactivates the gene allows the production of a null mutation, in which the office of that factor is completely lost. To induce such a mutation, the plasmid is introduced into an embryonic stem prison 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 brute incorporate the targeted gene. If the new gene is introduced into some of the germline cells of the chimeric mouse, and then some of the offspring of that mouse will carry the mutation as a factor in all of their cells. These heterozygous mice tin be further bred to produce mice homozygous for the null allele. Such knockout mice reveal the part of the targeted factor past 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.

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Construction of Genes, Chromosomes, and Genomes

Leon E. Rosenberg , Diane Drobnis Rosenberg , in Man Genes and Genomes, 2012

Recombinant DNA Technology

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

Recombinant DNA technology depends on five "tools." These are:

1.

Restriction enzymes are bacterial enzymes that cut DNA, like a scissors, at specific sites, producing fragments of dissimilar sizes (Figure vi.19); some restriction enzymes recognize a 4-nucleotide sequence (e.one thousand., AGGA), others a six-nucleotide one (GAATTC). These fragments tin 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 half dozen.20).

FIGURE half dozen.19. Restriction enzymes cut DNA at specific sites, producing fragments. Some enzymes (e.g., Rsaone) in (A) produce fragments with blunt ends, others (e.g. Eco R1) in (B) produce fragments with a v′ overhang. Such enzymes evolved to protect bacteria from viruses that infect them.

FIGURE 6.20. The Southern blot, used to identify DNA fragments. (A) DNA is cleaved and its fragments separated by electrophoresis; (B) fragments are blotted onto a nitrocellulose filter; (C) filter is exposed to radioactive probe; (D) filter is developed by exposure to photographic motion-picture show. Bands assort with the largest fragments near the acme of the film, the smallest ones near the lesser.

2.

Molecular cloning is the process by which DNA fragments are spliced into viral or bacterial vectors, purified, and amplified (Figure vi.21).

FIGURE half-dozen.21. Creating recombinant Dna molecules with plasmid vectors. A DNA fragment, prepared past digestion with Eco R1, is joined to an Eco R1 cleaved plasmid; the recombinant plasmid then transforms a bacterial cell, where the plasmid is replicated as the bacteria proliferate; the recombinant plasmids are isolated and the cloned donor fragments of Deoxyribonucleic acid pooled.

iii.

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, respective sequences in cloned collections of Dna fragments.

4.

Polymerase concatenation reaction (PCR) uses a specific bacterial polymerase to dilate a piece of Deoxyribonucleic acid up to a billion-fold or more. This is a powerful ways of obtaining sufficient DNA for a diverseness of purposes (Figure 6.22).

FIGURE 6.22. Amplification of a Deoxyribonucleic acid sequence using the polymerase chain reaction (PCR). The isolated target sequence is mixed with a thermo-stable DNA polymerase (ordinarily Taq 1). Amplification is accomplished past using specific primers and modifying the reaction temperature. Billions of copies of the target sequence are formed.

5.

DNA sequencing reveals the guild of base pairs in an isolated DNA molecule or fragment. Every bit shown in Effigy 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 automatic.

Effigy 6.23. Sanger sequencing of Deoxyribonucleic acid. (A) The DNA fragment to be sequenced is mixed with an fluorescently-labeled primer complementary to a portion of that sequence, and then DNA polymerase and deoxynucleotides are added and the mixture is divided into four parts. (B) Into each part, a single chain-terminating, fluorescently-labeled dideoxy nucleotide is added. (C) Subsequently the dideoxy analogue has acquired termination of the growing chain, the 4 aliquots are separated by electrophoresis co-ordinate to size. The sequence is and so read starting with the smallest fragment according to its dideoxy analogue.

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