An Introduction to Molecular Biology Chapter Abstracts

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The typical biological research scientist can be reasonably described as being concerned primarily with the question "Why does it work like that?" Achieving an understanding of the mechanisms by which cells function has for years been a primary goal of biological research. Research can be described as years of very tedious investigation, punctuated by moments of brilliant insight, followed by more years of tedious investigation, all driven by a compulsion to understand the solution to a problem.

In our technologically oriented society, scientific research is often viewed as a source of new information that can help provide solutions to problems, innovations to improve the quality of life, or new and improved commercial products. The scientific knowledge that develops as a result of biological research is by itself neither good nor bad, it is merely information. The uses to which this information is applied, however, can clearly be either helpful or harmful to society in general. It is the application of knowledge rather than the knowledge itself that should be of concern to all members of society.

New technology can clearly have long-lasting effects that are not readily apparent during the initial development and application of the methods. The internal combustion engine, for example, utilized a new hydrocarbon energy resource to mechanize and industrialize world economy. The generation of hydrocarbon smog and the ultimate dependence of world economies on the availability of oil, however, were complications of the new technology that were not readily forseen. In a similar manner, the development of atomic energy was hailed as a source of cheap, clean, readily available electric power that would revolutionize the world. In the wake of the nuclear accidents that occurred at Cheronobyl and Three Mile Island and with the accumulation of intensely radioactive wastes that cannot be easily stored or detoxified, many people now question the concept of nuclear power as a safe, viable energy source. Even a technological development as trivial as the fluorocarbon propellants used in aerosol cans has been accompanied by a growing concern that the propellants may be causing significant damage to the ozone layer that protects the earth from intense ultraviolet radiation. The list of real or potential problems that have arisen as a direct result of technology is long and continues to grow.

This does not mean that all technology should simply be abandoned for fear of potentially harmful future effects. The potential harm associated with the application of new technologies must be compared with the potential benefits of the methods. It is therefore appropriate for the general public to become sufficiently scientifically educated to be able to evaluate the potential risks and benefits of new technologies. The growing concern for the quality of the environment is an excellent example of how public education and awareness have led to public assessment and re-evaluation of long-established environmental policies. The incidents at Love Canal and many other toxics disposal sites have resulted in the modification of traditional waste disposal techniques, for example. Public awareness and understanding of the problems under consideration, however, are fundamental to accurate evaluation of potential problems associated with scientific technology.

The technology that has developed in the field of molecular biology has done far more than provide tremendous information about the workings of the cell. These methods have been rapidly assimilated by the industrial sector and applied to a wide variety of problems. Hormones and proteins are currently being manufactured by microbes grown in vats, viruses have been engineered to allow the production of vaccines, DNA detection methods have been used to detect disease-causing organisms, and DNA "fingerprints" have been used in court to establish guilt or innocence of suspects. The technology is so powerful and has such widespread application that, it will not simply go away if ignored long enough. Since the methods are likely to be a long-lasting, fundamental aspect of biological research, biomedical technology, and bioindustry, it seems appropriate to obtain a basic understanding of the principles and methods that make up this extremely powerful technology.

To understand why recombinant DNA methods and molecular biology have risen to such a level of scientific significance, it is helpful to first examine the basic process by which scientific research is conducted. The human race has for centuries tried to understand the principles that direct the functioning of the world and has developed different approaches for explaining these principles. Explanations that account for natural phenomena might be thought of as falling into one of three general categories: myth, religion, and scientific explanation.

Summary

1. Scientific explanations of observations are based on the scientific method, a process in which a hypothesis is proposed, experimentally tested, and modified to be consistent with the experimental observations.

2. Scientific explanations are no more valid than the tests to which they have been subjected.

3. The hypothesis that the structure and function of nucleic acids controlled the physical characteristics of cells helped drive the development of the recombinant DNA methods that enabled the isolation and analysis of nucleic acids.

Chapter 2. DNA and RNA as Genetic Material

Why do cells function the way they do? Cells must possess several fundamental properties in order to be considered "living" - they must be capable of catabolism, or breaking down complex compounds to derive energy, of anabolism, or the synthesis of complex compounds from simple substrates, and of reproduction, or the production of progeny with traits similar to those of the parental cells. The cells must be able to sustain the variety of chemical processes necessary for the maintenance or survival of the cell itself and for the ultimate production of offspring.

By 1970, biochemical research into the chemical processes that take place in living cells had contributed greatly to the understanding of the cell theory that had been formulated based on the results of genetic experiments. This chapter provides a description of the basic hypothesis of cell structure and function and how the development of recombinant DNA technology was vital to the testing and refining of these basic notions.

Summary

1. Genetic information is stored in the chromosomes of organisms. Chromosomes are composed of nucleic acids and proteins.

2. Proteins serve primarily structural and regulatory functions in the chromosome. Genetic information is stored as the sequence of the nucleotide bases present in DNA.

3. The double-stranded, complementary structure of DNA allows accurate production of daughter copies of the genes for transmission to progeny.

4. Information stored in DNA is converted to protein products through the molecule mRNA, which is synthesized using DNA as a template.

5. Determination of the sequence of bases in DNA is important to analysis of gene structure and function.

Chapter 3. Isolation of DNA

Because of the need to pass on phenotypic characteristics to offspring, all living organisms must possess a system for storing and transmitting genetic information. With the exception of certain specialized systems that use RNA, such as some viruses, genetic information was by 1970 widely believed to be stored by means of the order of the nucleotide bases present in chromosomal DNA. Testing this hypothesis requires the purification of a DNA fragment that contains a particular gene and the determination of the order or sequence of the nucleotides in the DNA fragment.

This relatively simple goal of isolating and analyzing a DNA fragment must begin with the purification of DNA. Unfortunately, DNA is present in the cell as an association of DNA with many proteins, called a nucleoprotein complex. The DNA must be separated away from these proteins prior to characterization. RNA and polysaccharides can interfere with DNA characterization methods, so the DNA must also be purified away from these macromolecules. In addition, a single cell may contain several different types of DNA molecule. For example, a plant cell will generally contain both mitochondria involved in energy metabolism and chloroplasts involved in carbon fixation. In addition to the DNA present in the chromosomes of the nucleus, both the mitochondria and chloroplasts contain specific, specialized circular DNA molecules that carry genes required for the function of the mitochondria and chloroplasts (Figure 3.1). A simple bacterium, such as Escherichia coli, contains only a single, circular chromosomal DNA molecule that carries all the genes necessary for growth and cell division. However, bacteria can also contain extrachromosomal DNA elements, or mini-chromosomes, involved in sexual conjugation or mating, the generation of resistance to antibiotics, or specialized metabolic functions (Figure 3.2). Viruses or bacteriophage that are composed of DNA or RNA may also be present as linear or circular molecules. The analysis of DNA requires not only the purification of DNA away from contaminating proteins and other macromolecules, but often requires the separation of different types of DNA molecules.

Summary

1. DNA present in cells is associated with many proteins.

2. DNA must be purified away from other cellular macromolecules prior to analysis of the DNA.

3. DNA can exist in different forms or conformations with different purification properties.

4. DNA can be quantified by its absorbance of light at wavelength 260 nm.

5. Small samples of DNA can be rapidly prepared using miniscreen procedures.

Chapter 4. Specific Cleavage of DNA

Identification of a point of reference on a DNA molecule as it is isolated from a cell is a technical innovation that was crucial to the development of recombinant DNA methodology. Chromosomal DNA purified from cells is frequently obtained as a randomly nicked or sheared population of fragments of 30,000 to 200,000 base pairs in size. In principle, each fragment has different ends than every other fragment in the population and no two fragments are identical. If you can imagine yourself standing on one of these fragments, since all of the fragments look alike, it can be difficult to determine precisely which DNA fragment lies under your feet. Some sort of molecular reference point is necessary to determine the physical location of genes on a DNA molecule.

As an additional complication, the fragments of DNA are not even the same size and, because the fragments are generated through random breakage, a desired gene may be present on fragments of many different sizes. A typical gene of interest might be 3000 base pairs long, while the purifed DNA preparation containing the gene might be an average size of 50,000 base pairs, with each fragment different from every other fragment (Figure 4.1). Some fragments will contain the desired gene, others will contain a part of the gene, and many fragments will not contain any of the desired gene sequence. It is important to be able to define or identify reference points on the DNA molecules to generate a map so that the molecules can be aligned and compared with one another.

Summary

1. A genetic map allows the ordering of genes on a DNA molecule but the order of the genes does not necessarily correlate precisely with the physical distance between genes.

2. Restriction endonucleases cleave DNA in a site-specific manner and allow DNA fragments of random sizes to be converted to specific fragments.

3. Restriction endonucleases can be used to generate a physical restriction map of DNA that positions restriction cleavage sites on the DNA molecule.

4. Alignment of a restriction map with the genetic map of the same DNA molecule can help identify DNA fragments that contain specific genes.

Chapter 5. Making New DNA Molecules

The basic principles of genetic engineering have for centuries been applied to the breeding of plants and animals for specific, desired phenotypic traits: breed two plants or animals to facilitate recombination of genes, search through the offspring for the desired combination of phenotypic traits, then repeat the breeding process with the "improved" progeny. Disease resistance, milk production, wool characteristics, meat yield, and size of seed kernel are all genetic traits that have been specifically selected using conventional genetic breeding methods. While these conventional genetic approaches have been extremely successful in the development of agriculturally significant plant and animal strains, the methods can be very slow and remarkably inefficient. During normal sexual reproduction, the genetic traits of the parents mix in a fairly random manner and obtaining the desired combination of phenotypic characteristics may require searching through thousands of progeny. At times, the appearance of the desired trait may depend on the occurrence of a spontaneous change in DNA sequence, a mutation, that can occur at extremely low frequencies. Mutation rates of 1 in 106 copies of a gene are not unusual, and finding a mutation that occurs at this frequency might require examining millions of individuals looking for the desired phenotype. Conventional genetic methods are also limited by existing genetic barriers that make transfer of a desired trait from one species to another extremely difficult, if at all possible.

As early as the mid-1960's, long before the advent of recombinant DNA methods, science fiction writers had begun to speculate that advances in genetic technology would allow the specific manipulation of DNA to generate novel, desired genetic arrangements. The discovery of restriction enzymes that cleave DNA in a site-specific manner to generate discrete gene fragments might be considered the first crucial element in the development of the ability to make novel recombinant DNA molecules in vitro, or in a test tube. The second crucial contribution to recombinant DNA technology was the development of the ability to seal the breaks made in DNA by restriction enzymes and make new arrangements of DNA fragments.

Summary

1. DNA fragments can be joined together and specifically modified to produce new DNA molecules.

2. DNA ligase is a nick-sealing enzyme that allows the joining of DNA fragments.

3. T4 DNA ligase is capable of ligating either cohesive or blunt end fragments and is most frequently used for manipulation of DNA fragments in the test tube.

4. The use of other DNA modifying enzymes like Klenow fragment and S1 nuclease helps manipulation of the ends of DNA fragments.

5. Short, commercially available DNA fragments called oligonucleotide linkers or adaptors allow specific restriction enzyme sites to be conveniently added to the ends of DNA fragments.

Chapter 6. Vectors

Restriction enzymes give researchers the ability to take a DNA sample containing fragments of random sizes and generate specific fragments. Since these fragments can be separated from one another by gel electrophoresis, it might seem that the restriction enzymes alone are sufficient to allow the isolation of genes. In the simplest strategy, the DNA would be purified, digested with a restriction enzyme, the fragments separated by electrophoresis on a gel, and the desired fragment cut out of the gel.

This scheme will actually work with small genomes like those of plasmids and viruses. However, as the size of the genome increases, several problems complicate this approach. First, as the genome increases in size, more DNA fragments are generated by the restriction enzyme digest. A 5,000 base plasmid genome might give 2 DNA fragments, a 50,000 base bacteriophage genome might yield 25 DNA fragments, while a 5,000,000,000 base eukaryotic genome might generate thousands of individual DNA fragments. As the number of individual DNA fragments increases, the probability that two different DNA fragments will have the same length also increases. Thus, a DNA band on an agarose gel may contain more than one DNA fragment of the same size. In addition, as the number of bands increases, the individual fragments become increasingly difficult to resolve by gel electrophoresis. It is difficult to separate two fragments that are only a few base pairs different in size, causing fragments of very similar size to migrate at the same position in an agarose gel.

A second complication involves the yield of the desired fragment relative to the total amount of DNA. As the size of the genome increases, the relative proportion of the DNA that is the desired gene decreases. A 3000-base gene represents 3000/50,000, or 6%, of a 50,000base bacteriophage genome. For each 1 milligram of starting DNA, a maximum of 0.06 milligram will be the gene of interest. Assuming no problems with the gel isolation and 100% recovery of the desired DNA, this might be a workable yield of the desired DNA fragment. However, a 3000-base gene represents only 3000/5,000,000,000, or .06%, of a typical eukaryotic genome. A yield of .0006 milligram of desired DNA fragment per milligram of starting DNA is too low to allow isolation and analysis of a specific gene.

A third complication regards the ability to identify the desired DNA fragment in the background of all the other DNA fragments. In the absence of other information, it is not usually possible to simply look at a restriction enzyme digest of a DNA sample and be able tell which DNA fragment contains a desired gene. To be certain that the desired gene has been purified, it might be necessary to purify each of the individual DNA fragments in the digested DNA sample. While possible for small genomes, this is a technically and financially unacceptable approach for general use.

Summary

1. Vectors are DNA molecules that can reproduce themselves in a host cell and can accomodate the insertion of extra DNA.

2. Vectors allow the production of large amounts of a desired fragment.

3. Insertional inactivation of a marker gene allows a recombinant vector to be differentiated from a non-recombinant vector.

4. Vectors allow the molecular cloning of DNA fragments away from other fragments in a preparation.

Chapter 7. Transformation

Contrary to the perception that the recombinant DNA molecules that result from in vitro manipulation of DNA give rise to new life forms, these new DNA molecules are lifeless bits of nucleic acid. They cannot propagate or reproduce themselves any more readily than can a grain of sand. However, because nucleic acids are virtually universally recognized as genetic information by living cells, these new DNA molecules can, if introduced into and maintained in an already living cell, confer new phenotypic traits on the host cell. It would be more appropriate to say that these new molecules modify existing organisms rather than generate new life forms.

Because the new recombinant molecule cannot reproduce by itself, once a DNA fragment has been ligated to a vector in vitro, it is necessary to introduce the construct into a host where it can replicate and produce many copies of itself. The process of introducing a DNA molecule into a host cell is called transformation; when the introduced DNA is a type of viral or bacteriophage DNA, the process is often referred to as transfection. The transformation step and the subsequent biological amplification of the inserted DNA molecule was the second major innovation of recombinant DNA methodology that was critical to improving the ease of the isolation and analysis of specific DNA molecules.

Summary

1. DNA must be introduced into a host cell to allow replication of the recombinant molecules.

2. Various chemical, mechanical, and electrical treatments can be used to make host cells competent to take up DNA.

3. Vectors derived from bacteriophage lambda DNA can be placed in empty bacteriophage heads and used to transfect host cells, a more efficient process than transformation.

4. Transformation procedures exist for both prokaryotes and eukaryotes, including many types of plant and animal cells.

Chapter 8. Identifying Recombinants

Following transformation of competent cells with a ligated DNA sample, transformant colonies or plaques are obtained that must be characterized to identify the transformant that contains the DNA fragment of interest. Each of the transformants generally looks just like every other transformant and the transformant that contains a specific DNA sequence often cannot readily be identified in the population of transformants.

Summary

1. Transformants must be characterized and desired genes identified.

2. Positive selection requires that the cloned gene produce an active gene product that gives the transformant a growth advantage under certain conditions.

3. Antibodies against a protein are often used to identify recombinants. Expression vectors and gene fusions are used to produce hybrid fusion proteins for detection by antibodies.

4. Nucleic acid hybridization is used in a variety of forms to identify and characterize DNA molecules.

Chapter 9. Characterizing Genes

Molecular biology, the study of the interactions of the macromolecules present in living cells, is a rapidly evolving field of biological investigation. The methods that allow the isolation and manipulation of DNA fragments, the tools of molecular biology researchers, developed out of scientific interest in testing important hypothesis regarding the structure and function of genes. Recombinant DNA methods provide extremely powerful tools for asking very detailed questions about the biological processes functioning in living cells. The current state of understanding of gene function is constantly changing as a result of the application of these methods to very complex biological questions. Analysis of gene structure and function can appear to be technically rather complicated, but most of the analytical approaches rely on the methods described in previous chapters.

Summary

1. A primary goal of genetic research is the understanding of gene structure and gene regulatory mechanisms.

2. Nucleotide sequencing is used to determine the chemical structure of an isolated gene and the corresponding mRNA and helps reveal genetic information stored in DNA.

3. Comparison of mRNA structure with genomic DNA structure helps clarify gene structure by providing information about introns and exons.

4. Analysis of protein-DNA interaction is fundamental to the study of gene regulation. The interaction of proteins with DNA can be directly visualized by several types of in vitro assay.

5. It is important to compare in vitro observations with in vivo patterns of gene expression to be certain that results obtained with purified components are indicative of regulatory interactions in the cell.

Chapter 10. PCR Manipulation of Nucleic Acids

Although recombinant DNA technology proven extremely powerful in the isolation and analysis of the structure and function of nucleic acids, many of the methods came to be considered slow and tedious. Although automation was able to improve some of the more monotonous tasks, such as preparation of miniscreen DNA and nucleotide sequence analysis, a great deal of recombinant DNA methodology was supplanted by the discovery of a process that allowed the amplification of a specific DNA fragment outside of any biological host. The speed and utility of this process, called the Polymerase Chain Reaction, has created a second technological revolution in the analysis of nucleic acids.

Summary

1. The Polymerase Chain Reaction (PCR) is a very powerful DNA method that uses a heat-stable DNA polymerase to amplify DNA or RNA from very small amounts of starting material.

2. PCR requires some information regarding the nucleotide sequence of the target DNA to allow the design and synthesis of oligonucleotide primers that direct the amplification of the desired target gene.

3. The speed, convenience, and decreased costs associated with PCR have helped to supplant some of the more conventional methods previously used to isolate, characterize, and manipulate nucleic acids.

Chapter 11. The Application of Molecular Biology

The methods called recombinant DNA developed out of the scientific desire to address the fundamental hypothesis that the physical traits of a cell are encoded by the nucleic acids present in the chromosomes of the cell and changes in the structure and function of nucleic acid result in changes in cellular characteristics.

How has this new methodology succeeded in application to testing of fundamental scientific theories? Application of these methods to the study of developmental biology has provided significant support for the cell theory, which suggests that all organisms are composed of many individual cells, by allowing the investigation of the changes in gene expression that accompany the development of a mature organism. The ability to isolate, modify, and re-introduce specific genes has been instrumental in providing overwhelming evidence in support of the chromosomal theory of heredity, which suggests that the chromosomes within the individual cells control the physical traits of the cells. The isolation and sequence analysis of related genes from several different species and the study of the occurrence of alleles in wild populations have provided information that supports elements of the theory of evolution by natural selection, which suggests that complex organisms are derived from more primitive organisms by a process of accumulation of changes in physical traits of cells.

Development and continued refinement of the methods of molecular biology have been critical in the explosion of information regarding the role of nucleic acids in the control of the physical traits of cells. The increased understanding of the structure and function of the genome has also resulted in the appearance of molecular biology in everyday life.

It is important to understand that scientific research can be divided into two general categories: basic and applied research. Basic research might be described as the search for a basic understanding of a problem area, while applied research can be thought of as applying the results of basic research to generate a useful product.

The scientific methods and the research topics that make up the field of molecular biology are obviously of interest to the scientific community involved in basic research, but why should these topics generate widespread interest and controversy among people who are not members of the scientific community? The increasing use of molecular biology methodology in applied research has been accompanied by the appearance of biomedical and production innovations that are of increasing public notice.

Understanding of scientific theory is rarely necessary for everyday application of a scientific principle. After all, a person need not understand either electricity or the internal combustion engine to be fairly confident that an automobile with a full tank of gas and a good battery will start when the key is turned in the ignition. It is not really necessary to understand the mechanics of technology to be able to use the products of technology. Consider that the vast majority of people in the United States understand how to use fire to cook and heat, yet very few of these people can start a fire in the absence of technological devices (matches or lighters, for example).

Recombinant DNA technology developed out of the desire to obtain large amounts of a purified gene to address various important hypotheses regarding gene structure and function. The concepts that allow recombinant DNA technology are fairly simple. Restriction endonucleases are proteins that cleave DNA in a site-specific manner to generate specific, reproducible fragments of the DNA present in a chromosome. DNA fragments can be separated according to size by gel electrophoresis and stained with dyes that allow the detection of as little as 5 x 10-9 gram of DNA. Digestion of a DNA fragment with several different enzymes can be used to establish a physical map of the positions of cleavage sites relative to one another - a restriction map of the DNA fragment. Restriction maps can provide direct correlation between the physical structure of a chromosome and the order of the genes present on the chromosomal DNA molecule.

DNA fragments can be inserted into cloning vectors designed to propagate in a suitable host. The vector serves two functions: a fragment inserted into the vector has been molecularly cloned away from other DNA fragments and once cloned, can be amplified by purification of the recombinant molecule. Large amounts of a very pure DNA fragment can be obtained by cloning the fragment in a vector. Molecular cloning of DNA fragments overcomes many of the problems associated with the isolation of rare DNA molecules and the preparation of sufficient amounts of DNA for many experiments.

As discussed in the previous chapter, these methods are of tremendous value in the analysis of the biological mechanisms that are involved in regulating gene expression and have contributed to a virtual revolution in molecular biology. The new information accumulates a rate that causes a continuing re-evaluation of scientific theories regarding gene structure and function. The scientific debate that arises is part of the normal process of scientific evaluation. Experiments are designed to test theories, results are interpreted, and the results are compared with existing theories to determine whether the theories are consistent with the experimental results. Controversy is accepted among scientists as a normal part of the investigation process.

Recombinant DNA methods and their application to molecular biology problems have generated not only scientific debate, controversy, and re-evaluation of theories regarding gene structure and function, but have also generated significant public debate regarding safety, ethics, and economic potential of this type of research. These methods have been perceived as having tremendously powerful positive and negative social impacts, with both benefactors and detractors making misleading statements. Understanding how these methods can be applied to various specific problems can help illustrate why recombinant DNA methodology is no longer merely an investigative tool for molecular biologists, but is experiencing increasing application to common problems.

Summary

Molecular biology methods have tremendous value not only in the investigation of basic scientific questions, but also in application to a wide variety of problems affecting the overall human condition. Disease prevention and treatment, generation of new protein products, and manipulation of plants and animals for desired phenotypic traits are all applications that are routinely addressed by the application of molecular biology methods. Because of the wide applicability of these methods, they are rapidly becoming a pervasive - some would argue invasive - aspect of our technologically based society. The public concerns that address the application of these methods should be addressed by informed public discussion and debate. While scientists can be extremely critical of the quality, interpretation, and significance of experimental results, they have a rather remarkable tendency to be non-judgmental of the relative social merits of many applications of scientific research. It remains a public responsibility to be sufficiently well-informed to critically assess the merits of applied science research and participate in a communal decision-making process regarding the extent to which a new technology will be allowed to affect society.

Chapter 12. Introductory Experiments in Recombinant DNA

The exercises that contained in this chapter have been chosen to demonstrate the basic principles in recombinant DNA: digestion of DNA with a restriction endonuclease, gel electrophoresis of DNA samples, insertion of DNA into cells can change their growth characteristics, and DNA can be rearranged to cause changes in genetic properties. The exercises are intended to be an example of the principles that DNA equals genes and that changes in DNA cause changes in genetic properties.

Each exercise is preceded by a short discussion of the principles involved and the specific goals of the procedure. Solutions that must be prepared for each exercise are detailed in a Materials section. While some of these "recipes" can be changed considerably without affecting the outcome of the exercise, many of the "minor" details are crucial to the success of the protocol. For example, a restriction digestion buffer may contain only 6 mM NaCl relative to 50 mM TRIS-Cl buffer, an apparently trivial amount of salt in the overall scheme of things. However, many enzymes are very specific about salt concentrations required for activity, and deleting the NaCl from the buffer may inactivate or actually change the recognition properties of the enzyme. Please do not arbitrarily change recipes and expect exercises to work properly.

The recipes assume a simple knowledge of chemistry and use standard abbreviations regarding concentrations:

M = moles/liter = molar

mM = millimoles/liter = 10-3 moles/liter = millimolar

l = liter

ml = milliliter = 10-3 liter

µl = microliter = 10-6 liter

When pH of a solution is indicated, there is generally an allowance of about 0.5 pH unit. If a pH of 7.5 is indicated, a pH of 7.0 to 8.0 will generally suffice. If possible, use a pH meter in the preparation of solutions, otherwise, use pH paper and be as accurate as reasonable.

The instructions often require the addition of water, sometimes indicated specifically as distilled water (dH2O). For electrophoresis, media, and general solutions, tap water will often suffice. For reactions involving enzymes, distilled water should always be used rather than tap water. Many enzymes can be inhibited by heavy metal salts present at low levels in tap water. Distilled water purchased for use in a steam iron can be used in the event that there is no access to a water still or deionizer.

Certain solutions must be sterilized (media for culture of bacteria, for example). An electric hot plate and a standard pressure cooker can be used as a substitute for an autoclave. Heat media at 16 lb pressure for 20-30 minutes to sterilize small volumes of liquids (less than 500 ml solution per container). While a microwave oven is used for melting agarose in nearly every molecular biology research lab, a hot plate with a boiling water bath or a gas burner will accomplish the same thing. Agarose solutions have a tendency to superheat and boil violently when the agarose granules are first melting. A solution of agarose that has been previously melted, then allowed to cool and solidify is less likely to boil violently when melted the second time.

As a note of caution, realize that molecular biology uses a number of noxious chemicals: organic solvents (phenol, chloroform, ether) are quite toxic and certain compounds (ethidium bromide and UV light, for example) are known mutagens capable of causing genetic changes. The use of such compounds has been kept to a minimum in these exercises and where such compounds are used, precautions are noted in directions for the exercises. During electrophoresis, although low voltages are used, severe injury is possible. Electrophoresis boxes should have an interlock mechanism - a device that prevents the current from being applied to the buffer when the buffer is exposed. Most commercial gel boxes include this safety design feature, as do the plans that accompany these exercises. COMMON SENSE IS REQUIRED IN ALL LAB EXERCISES. Food and drinks should be prohibited from the work area and lab coats should be worn. Power units should be turned off while loading gels or handling gel boxes. All spills should be cleaned up when they occur.

Having previously indicated that these exercises should not be arbitrarily altered, I now emphasize that these protocols should not be considered inviolate. There are about 113 ways to accomplish the same thing in any molecular biology exercise. As your understanding of these methods increases, you will recognize how protocols can be altered to suit a particular circumstance. Other protocols will appear that seem easier or more reproducible in your own situation. While this is one of the aspects of molecular biology that makes the methods so powerful, this variability also tends to unnecessarily confuse novices in the field. Before modifying a protocol, be certain that you understand the changes and that these alterations will not adversely affect the outcome of an exercise.

The bacteriophage lambda, pBR322, and pUC19 plasmid DNA samples used in these exercises are available from a number of commercial suppliers. The bacterial DNA was prepared by a simple procedure (see Appendix) that uses detergent lysis and phenol/chloroform extraction to prepare high quality DNA. Commercially available calf thymus DNA can be substituted. Plasmid recombinants containing E. coli DNA in pUC19 were chosen specifically to work entirely with bacterial DNA and minimize any misperception of biohazard potential. Competent cells are commercially available from several sources, but acceptable competent cells can also be prepared with a minimum of expertise (see Appendix).

Appendix

I. Plans for simple submerged gel electrophoresis box

II. Manual pipet device

III. Bacterial DNA purification

IV. Preparation of a pUC19:E. coli chromosomal library

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