It is a discipline closely related to the fields of biochemistry, genetics and cell biology that focuses on the interactions between the various systems of a cell, the interrelationship of DNA, RNA and protein synthesis, and how they regulate each other. Although there are many types of molecules in every living being, molecular biology studies generally focus on genes and proteins.
The reason is that proteins perform a huge diversity of functions in living cells, and genes contain the information needed to make more proteins. Molecular biologists are responsible for conducting experiments that focus on investigating the structure, function, processing, regulation and evolution of biological molecules and their interactions.
Gathering this information not only provides basic knowledge about how biology works, but also provides the knowledge needed to manipulate genetic biology, which is fundamental to drug design and genetic engineering.
Genetic engineering techniques make it possible to study living beings, making it possible to manipulate genes to achieve greater agricultural productivity, develop new materials, improve the food industry, characterize materials and perform forensic engineering analyses.
For example, to analyze corrosion and stains on stainless steel , as well as to develop new energies and open up an enormous field of research in genetics and medicine. Advances in molecular biology technology and genetic engineering, as well as microbial genetic manipulations have promoted the application of microorganisms in research and the birth of some of the subdisciplines that are part of molecular biology such as the following:.
Molecular biology technology has not only broadened the horizon, but also increased the depth of research in microbial ecology applied to the materials industry , development of new energy sources , characterization of antibiofilm properties for prevention on surfaces , modification of composites , forensic engineering , improvement of the food and energy industry.
The growing amount of genomic data from microbes offers new opportunities to understand the genetic and molecular basis of the degradation genes of various bacteria that affect products, foods and materials. Similarly, genetic engineering is an essential technique for building microbes with enhanced biodegradability that can be used to control polluted environments or to ferment waste in ways that produce natural gas.
On the other hand, the key technological areas of biological transformation will be bioengineering, information and machine engineering, which will lead the way for biological transformation. In this context, neurorobotics can make a significant contribution to improving the current state of the art by linking human nerve cells with technical components.
As discussed earlier in the historical sections, molecular biologists have relied heavily on model organisms see the entry on models in science. But making inferences from a single exemplary model to general biological patterns has been cause for worry. What grounds do biologists have for believing that what is true of a mere model is true of many different organisms?
One answer, provided by Marcel Weber , is that the generality of biological knowledge obtained from studying exemplary models can be established on evolutionary grounds. According to Weber, if a mechanism is found in a set of phylogenetically distant organisms, this provides evidence that it is also likely to be found in all organisms that share a common ancestor with the organisms being compared.
Unlike the aim of exemplary models, the representative aim of a surrogate model is not necessarily to be broad. For example, biomedical researchers frequently expose surrogate models to harmful chemicals with the aim of modeling human disease. However, if a chemical proves to be carcinogenic in rats, for example, there is no guarantee that it will also cause cancer in humans. Although this problem is not unique to surrogate models, it often arises when biomedical researchers use them to replicate human disease at the molecular level.
Consequently, philosophers who write about the problem of extrapolation in the context of molecular biology often focus on such models see, for example, Ankeny ; Baetu ; Bechtel and Abrahamsen ; Bolker ; Burian b; Darden ; LaFollette and Shanks ; Love ; Piotrowska ; Schaffner ; Steel ; Weber ; Wimsatt Within the context of surrogate models, any successful solution to the problem of extrapolation must explain how inferences can be justified given causally relevant differences between models and their targets Lafollette and Shanks Cook and Campbell This method avoids the circle because it eliminates the need to know if two mechanisms are similar.
All that matters is that two outcomes are produced to a statistically significant degree, given the same intervention. For this reason, statistically significant outcomes in clinical trials are at the top of the evidence hierarchy in biomedical research Sackett et al. One problem with relying merely on statistics to solve the problem of extrapolation, however, is that it cannot show that an observed correlation between model and target is the result of intervention and not a confounder. This approach avoids the circle because the suitability of a model can be established given only partial information about the target.
For example, Steel argues that only the stages downstream from the point where the mechanisms in the model and target are likely to differ need to be compared, since the point where differences are likely will serve as a bottleneck through which the eventual outcome must be produced. One worry, raised by Jeremy Howick et al. For example, there may be an upstream difference that affects the outcome but does not pass through the downstream stages of the mechanism.
This problem is taken up again below in Section 3. The resulting big picture account of the experimental model is an aggregate of findings that do not describe a mechanism that actually exists in any cell or organism.
Instead, as a number of authors have also pointed out Huber and Keuck ; Lemoine ; Nelson , the mechanism of interest is often stipulated first and then verified piecemeal in many different experimental organisms.
These genetically engineered rodents are supposed to make extrapolation more reliable by simulating a variety of human diseases, e. As Monika Piotrowska points out, however, this raises a new problem. The question is no longer how an inference from model to target can be justified given existing differences between the two, but rather, in what way should these mice be modified in order to justify extrapolation to humans? Piotrowska has proposed three conditions that should be met in the process of modification to ensure that extrapolation is justified.
The first two requirements demand that we keep track of parts and their boundaries during transfer, which presupposes a mechanistic view of human disease, but the third requirement—that the constraints that might prevent the trait from being expressed be eliminated—highlights the limits of using a mechanistic approach when making inferences from humanized mice to humans.
As Piotrowska explains,. As our ability to manipulate biological models advances, philosophers will need to revisit the problem of extrapolation and seek out new solutions. The history of molecular biology is in part the history of experimental techniques designed to probe the macromolecular mechanisms found in living things.
Philosophers in turn have looked to molecular biology as a case study for understanding how experimentation works in science—how it contributes to scientific discovery, distinguishes correlation from causal and constitutive relevance, and decides between competing hypotheses Barwich and Baschir In all three cases, the concept of a mechanism is central to understanding the function of experimentation in molecular biology also see the entry on experimentation in biology.
Take discovery. Darden has countered with a focus on the strategies that scientists employ to construct, evaluate, and revise mechanical explanations of phenomena; on her view, discovery is a piecemeal, incremental, and iterative process of mechanism elucidation.
In the s and s, for example, scientists from both molecular biology and biochemistry employed their own experimental strategies to elucidate the mechanisms of protein synthesis that linked DNA to the production of proteins. Molecular biologists moved forward from DNA using experimental techniques such as x-ray crystallography and model building to understand how the structure of DNA dictated what molecules it could interact with; biochemists simultaneously moved backward from the protein products using in vitro experimental systems to understand the chemical reactions and chemical bonding necessary to build a protein.
Tudor Baetu builds on the contemporary philosophy of mechanism literature as well to provide an account of how different experiments in molecular biology move from finding correlations, to establishing causal relevance, to establishing constitutive relevance Baetu b. Much recent philosophical attention has been given to the transition from correlation to causal relevance.
On a manipulationist account of causal relevance, some factor X is determined to be causally relevant to some outcome Y when interventions on X can be shown to produce the change in Y. But these one-variable experiments, Baetu cautions, do not necessarily provide information about the causal mechanism that links X to Y.
Is X causally relevant to Y by way of mechanism A , mechanism B , or some other unknown mechanism? In a two-variable experiment, two interventions are simultaneously made on the initial factor and some component postulated in the mechanical link, thereby establishing both causal and constitutive relevance.
An experiment is taken to be a crucial experiment if it is devised so as to result in the confirmation of one hypothesis by way of refuting other competing hypotheses. But the very idea of a crucial experiment, Pierre Duhem pointed out, assumes that the set of known competing hypotheses contains all possible explanations of a given phenomenon such that the refutation of all but one of the hypotheses deductively ensures the confirmation of the hypothesis left standing.
Duhem actually raised two problems for crucial experiments—the problem mentioned above, as well as the problem of auxiliary assumptions, which any hypothesis brings with it; for reasons of space, we will only discuss the former here.
Marcel Weber has utilized a famous experiment from molecular biology to offer a different vision of how crucial experiments work. After Watson and Crick discovered the double helical structure of DNA, molecular biologists turned their attention to how that macromolecule could be replicated see Section 1. The focus was in part on the fact that the DNA was twisted together in a helix, and so the challenge was figuring out what process could unwind and replicate that complexly wound molecule.
Three competing hypotheses emerged, each with their own prediction about the extent to which newly replicated DNA double helices contained old DNA strands versus newly synthesized material: semi-conservative replication, conservative replication, and dispersive replication. They grew E. By then taking regular samples of the replicating E. Moreover, any hypothesis of DNA replication had to satisfy mechanistic constraints imposed by what was already known about the physiological mechanism—that DNA was a double helix, and that the sequence of nucleotides in the DNA needed to be preserved in subsequent generations.
For a critique, see Baetu An overview of the history of molecular biology revealed the original convergence of geneticists, physicists, and structural chemists on a common problem: the nature of inheritance. Conceptual and methodological frameworks from each of these disciplinary strands united in the ultimate determination of the double helical structure of DNA conceived of as an informational molecule along with the mechanisms of gene replication, mutation, and expression.
With this recent history in mind, philosophers of molecular biology have examined the key concepts of the field: mechanism, information, and gene.
Moreover, molecular biology has provided cases for addressing more general issues in the philosophy of science, such as reduction, explanation, extrapolation, and experimentation. History of Molecular Biology 1. Concepts in Molecular Biology 2. Molecular Biology and General Philosophy of Science 3. History of Molecular Biology Despite its prominence in the contemporary life sciences, molecular biology is a relatively young discipline, originating in the s and s, and becoming institutionalized in the s and s.
He concluded a essay: The geneticist himself is helpless to analyse these properties further. Weaver wrote, And gradually there is coming into being a new branch of science—molecular biology—which is beginning to uncover many secrets concerning the ultimate units of the living cell…. According to Lily Kay, Up until around molecular biologists…described genetic mechanisms without ever using the term information. Crick —, emphasis in original It is important not to confuse the genetic code and genetic information.
Brenner, letter to Perutz, Along with Brenner, in the late s and early s, many of the leading molecular biologists from the classical period redirected their research agendas, utilizing the newly developed molecular techniques to investigate unsolved problems in other fields. Concepts in Molecular Biology The concepts of mechanism , information , and gene all figured quite prominently in the history of molecular biology. Phyllis McKay Illari and Jon Williamson have more recently offered a characterization that draws on the essential features of all the earlier contributions: A mechanism for a phenomenon consists of entities and activities organized in such a way that they are responsible for the phenomenon.
Stephen Downes helpfully distinguishes three positions on the relation between information and the natural world: Information is present in DNA and other nucleotide sequences. Other cellular mechanisms contain no information. DNA and other nucleotide sequences do not contain information, nor do any other cellular mechanisms. Molecular Biology and General Philosophy of Science In addition to analyzing key concepts in the field, philosophers have employed case studies from molecular biology to address more general issues in the philosophy of science, such as reduction, explanation, extrapolation, and experimentation.
Rosenberg 4 Hence, the task of this explanatory reduction is to explain all functional biological phenomena via molecular biology. As Piotrowska explains, without the right context, even the complete lack of differences between two mechanisms cannot justify the inference that what is true of one mechanism will be true of another Piotrowska Conclusion An overview of the history of molecular biology revealed the original convergence of geneticists, physicists, and structural chemists on a common problem: the nature of inheritance.
Janis eds. Alberts, Bruce, A. Johnson, J. Lewis, M. Raff, K. Roberts, and P. New York: Garland. Ankeny, Rachel A. Austin, Christopher J. Baetu, Tudor M. Barrell, B. Air, and Clyde A. Bechtel, William and Robert C. Bickhard, Mark H. Philosophy of Complex Systems, Vol.
Bolker, Jessica A. Boogerd, Fred C. Hooker ed. Bruggeman, Jan-Hendrik S Hofmeyr. Wood and the Community of C. Burian, Richard M. Monod and P. Cairns, John, Gunther S. Stent; and James D.
Watson eds. New York: Zone Books. Cantor, Charles R. Carlson, Elof A. Clarke, Brendan, G. Gillies, P. Illari, F. Russo, and J. Cook, Thomas D. Craver, Carl F. Machamer and M.
Silberstein eds. Krishnamurthy ed. Crow, James F. Darden, Lindley and Carl F. Deichmann, U. It was then when it clicked that I had to major in molecular biology.
Over the course of my time at Pomona College, both in-person and virtually, majoring in molecular biology was one of the best decisions that I made.
The small joys of assays going smoothly, all the way through finding significant data that leads to more questions are what makes it worthwhile. Most importantly, it is the connections that are made with professors and classmates along the way that help build a solid foundation in my future career. Since I was little, I have always been enamored by science and attempting to understand how the body functions, one cell at a time. Our bodies are comprised of so many complex systems and interactions it would take centuries or millennia of research and experiments to understand every facet of ourselves.
Entering Pomona, I knew that I would major in either molecular biology or neuroscience. Unable to double major, I spoke with professors from both departments who guided me into choosing molecular biology as my major with a focus in neuroscience.
I love how this major provides a solid pathway to learn about molecular biology in the area of your choosing. No matter your focus, there are a variety of courses and electives that will satisfy your curiosity.
The project involved creating a novel system to engineer homing endonucleases to cut DNA sequences of our choosing, allowing us to target genes in a similar fashion to CRISPR. To accomplish this, we modified an established system known as PACE to work with I-CREI, a homing endonuclease, to allow it to go through several hundred rounds of evolution.
Creating this system would be invaluable in the future of gene editing as it could provide an alternative to CRISPR and help provide solutions to genetic diseases such as sickle cell anemia. This research was extremely fascinating to learn about and develop, and through my time in the lab, I gained valuable techniques and the experience to troubleshoot and figure out solutions to problems in the lab.
I hope to come back to Pomona one day as a professor and guide future students on their path. I want to leave a positive impact on the lives of students and offer the same support, generosity and kindness that my professors have given me. I chose to major in molecular biology because it is the major that is most in line with my career goal of becoming a practicing physician. I get to study how God designed life as we know it! This major allows students to interact with professors from both the Chemistry and Biology Departments!
Not only that, but we get exclusive access to three academic buildings The professors in this department and the Chemistry department make it all worth it! They are amazing! I chose to major in molecular biology because it was the perfect intersection between my love of biology and understanding the human body with my enjoyment of chemistry and understanding life on a cellular level. I really enjoy the major because it allows me to establish a strong foundation in biology and chemistry that can be applied to better understand a lot of upper division courses like immunology, for example, which I don't think I would be able to fully appreciate or understand had I not taken organic chemistry and biochemistry, which are required in the molecular biology major.
I hope to go into a career in healthcare and the major is perfect because it has allowed me to take many courses that have deepened my understanding of life and the processes that support it while exposing me to many people and professors who care deeply about me and my progress towards my career goals.
My project revolved around understanding the role of the NACC2 gene in cardiac neural crest development and what role it plays in the gene regulatory network. I used CRISPR-Cas9 to create a unilateral mutation in chick embryos, which allowed me to compare the left and right sides of the same specimen to determine if the NACC2 knockout was effective and if it caused any changes to the development on the knockout side of the embryo.
I was the first person in the lab to study this gene, so it was super exciting to know that all of my preliminary research truly was novel and would set the stage for further research on the gene when my results demonstrated it had influence on two important genes involved in neural crest development and differentiation.
It is a great way to get some experience and establish a relationship with great professors! The chemistry and molecular biology major students take many very difficult classes together and one of the things I love most about Pomona's science departments is the emphasis on camaraderie and collaboration.
When we go to mentor sessions, I know that everyone in that room is willing to help me understand the material I don't understand in the same way that I am willing to help others with material I do understand.
Professors work hard to foster a sense of community because they understand that the courses are difficult, and this mindset helps everyone in the long run. It has been especially important to me because it helps eradicate the detrimental overly-competitive nature of the sciences to encourage us to get better and smarter together rather than pushing others down for our own gain.
We all look out for each other and that collaboration is probably my favorite part of being a molecular biology major at Pomona. In the next section, we describe these methods and their relevance to clinical practice. Restriction enzymes provided a method by which scientists could specifically alter DNA. Restriction enzymes are bacterial proteins that recognize and cut DNA at specific nucleotide sequences, usually base pairs in length.
These DNA fragments can be separated by size, using the principle that small DNA fragments move through an electrical gradient on a gel faster than larger fragments; this process is called gel electrophoresis.
Gel electrophoresis can be accomplished with various media, two of the most common being agarose and acrylamide gels. Agarose gels separate fragments based mainly on charge density, whereas acrylamide gels are a porus media in which DNA fragments are separated by both charge and size. DNA fragments can then be visualized using ethidium bromide, a compound that intercalates into DNA, causing it to fluoresce when exposed to ultraviolet light.
Utilization of different restriction enzymes to cut the same DNA sequence results in a series of DNA fragment sizes, depending on the restriction enzyme used. Combining information from several enzymes produces a restriction map. A restriction map is analogous to a fingerprint, because each DNA sequence produces a unique pattern. There are many situations in which restriction enzymes are used in medicine.
One example is the identification of patients with sickle cell disease, where the substitution of valine for glutamic acid in the sixth amino acid of the beta-globin chain of hemoglobin alters the function of the protein in hypoxia. This mutation changes the restriction map, so restriction analysis can be used to easily differentiate between patients with normal hemoglobin and those with the disease.
This is possible because many restriction enzymes cut double-stranded DNA a few nucleotides apart, creating overhanging pieces known as "sticky ends" Figure 2. If the same restriction enzyme is used on two different pieces of DNA, complementary "sticky ends" align due to hydrogen bonding a process called annealing and are joined together by DNA ligase to form a new "designer DNA" sequence. Figure 2.
The restriction enzyme EcoRI makes staggered "sticky" ends. Determining the exact nucleotide sequence of a given fragment of DNA is essential in molecular biology.
Sequencing is usually performed by either the Maxam-Gilbert chemical modification method or, more commonly, by the Sanger method Figure 3.
The DNA strand to be sequenced is incubated with a short sequence of DNA called an oligonucleotide primer complementary to the end of the DNA of interest, an excess of all four nucleotides dideoxynucleotides; one of which is radiolabeled for convenient visualization of final DNA products , and a low concentration of a chosen dideoxynucleotide. In a sequencing reaction, thousands of DNA molecules are being synthesized simultaneously.
In this setting, dideoxynucleotides are incorporated randomly, resulting in a series of DNA fragments of increasing length, each truncated by incorporation of a dideoxynucleotide molecule, indicating the presence of a specific nucleotide at that point in the sequence.
Such reactions are performed with each of the four dideoxynucleotides. The resulting labeled DNA fragments are separated, by size, through an electric field on an acrylamide gel, and the pattern of bands on the gel reveals the DNA sequence.
DNA sequencing has, and continues to have, enormous importance in the laboratory and for clinical medicine. The Human Genome Project is a collaborative effort to determine the DNA sequence of the entire human genome approximately 3 billion base pairs. Already, sequences of genes known to be important in various diseases such as Huntington's disease, Alzheimer's disease, and cystic fibrosis as well as many others have been determined. Figure 3.
The Sanger DNA sequencing method. The reaction continues until a dideoxynucleotide is incorporated. This results in a series of labeled strands of various lengths.
The labeled fragments are separated by size on an acrylamide gel. The pattern of fragments gives the DNA sequence. Genes can be defined as discrete DNA sequences that, when transcribed into RNA, contain both regulatory regions as well as RNA sequences ultimately translated into protein.
The process of isolating a gene of interest from all other genes in the genome is called cloning Figure 4.
If part of the desired DNA sequence is already available, a portion of this sequence can be radiolabeled and allowed to anneal with complementary DNA sequences in the library a process called hybridization.
If the sequence of DNA is not known, protein products of the desired DNA may be able to be identified by using specific antibodies to the protein. Another method is to use a similar but not identical DNA sequence with less harsh experimental conditions than are normally used to identify related genes. These less stringent conditions allow the similar segments of DNA to hybridize without requiring perfect base pair matching.
Finally, another method of isolating important genes is called expression cloning. In expression cloning, a functional response for the encoded protein is tested and used as a guide to isolate specific DNA sequences that encode the protein of interest. Figure 4. Screening a DNA library. DNA is cut into fragments with restriction enzymes. These fragments are inserted into vectors, which are then replicated in bacteria.
Colonies each contain a single type of recombinant DNA fragment. The colonies are transferred and fixed to a nylon filter.
A radioactive probe for the desired sequence can then be hybridized to the filter, locating the colony with the desired DNA. N Engl J Med ; Once the gene is identified, multiple copies need to be produced; the process of producing multiple copies of DNA is called amplification.
One way a gene can be amplified is by using small circular pieces of DNA known as plasmids. Both the plasmid and gene are cut with the same restriction enzyme, enabling the foreign gene to be placed ligated into the plasmid. Many plasmids contain antibiotic resistant genes that make them easy to identify. The newly created plasmid that contains the foreign DNA of interest is incorporated into bacteria, a process known as transformation.
Only bacteria that have successfully incorporated the plasmid will grow in nutrients that contain antibiotic. Transformed bacteria then replicate.
Most plasmids used in molecular biology are "relax-control" plasmids, meaning that, in addition to replicating with each bacterial cell division, the plasmid also replicates many times within a single cell; the net result is rapid amplification of plasmid DNA. Messenger RNA and the encoded protein can be produced efficiently by using plasmid expression vectors that contain a highly active promoter region.
Bacterial, yeast, or mammalian cells are then transfected with the recombinant DNA, resulting in large quantities of the desired protein being produced. Cells are then lysed and the protein purified from other host cell proteins using various methods, one of which is chromatography.
Many clinical advances can be attributed to general methods used in cloning. Tissue plasminogen activator is one example of a gene whose encoded protein is now mass produced using recombinant techniques.
Genes for many human diseases have been identified and cloned, including those important in hemophilia, Duchenne's muscular dystrophy, and cystic fibrosis. Insertion of cloned DNA and subsequent amplification in bacterial cells is not the only method available to amplify segments of DNA. To specify the region to be amplified, it is necessary to synthesize two short oligonucleotides primers , each complementary to one strand of each of the ends of the DNA of interest.
Denatured DNA, primers, all four nucleotides, buffer, and the enzyme DNA polymerase are cooled to 42 degrees degrees Celsius, the temperature at which primers anneal to complementary DNA. The temperature is then raised to 72 degrees Celsius, the optimal temperature for DNA polymerase. The DNA polymerase used in PCR reactions is unique in that it is isolated from thermophilic bacterium and is stable at much higher temperatures than other polymerases.
The temperature is then elevated back to degrees Celsius, where the double-stranded DNA denatures and now forms four new templates for the next cycle in the reaction. The cycle of denaturing double-stranded DNA helices, hybridizing primers, and then incorporating nucleotides to growing templates is repeated times. Because this reaction is exponential, 30 cycles produce more than one million copies of the targeted DNA segment.
Figure 5. The polymerase chain reaction. Polymerase chain reaction is a very powerful technique, with wide applications. It can be used to provide ample amounts of DNA from a known gene. By modifying primer sequences slightly, mutations can be introduced into genes and the functional result studied. Clinically, PCR amplification of small quantities of DNA can detect infectious agents or identify residual cancer cells.
Polymerase chain reaction amplification of DNA followed by restriction enzyme analysis enables diagnosis of diseases such as sickle cell anemia from a single sample of blood. Recently, PCR followed by DNA analysis has begun to be used to determine parenthood in paternity battles and identify perpetrators in rape and murder cases.
Polymerase chain reaction is highly specific and can amplify a segment of DNA even if only one or two copies of the sequence are present in a sample, making it useful in many applications in medicine.
Polymerase chain reaction is not without difficulties, including its high sensitivity. Many genes have slightly different sequences that are of no clinical consequence. Such variations in the general population are called polymorphisms see section on Genetic Testing-Techniques for a more detailed discussion of polymorphisms. A further problem with PCR is the risk of contamination of the study sample; in this case, the resultant amplified DNA might be a contaminant rather than the targeted DNA, potentially leading to misdiagnosis.
However, PCR remains a valuable adjunct for molecular biologists and clinicians, being faster and easier than standard cloning methods. Whereas restriction enzyme analysis, cloning of genes, and PCR are used to study specific genes in detail, more general techniques such as Southern and Northern blotting can be applied to study DNA and RNA, respectively Figure 6. Southern blotting analyzes the structure and location of a gene.
Genomic DNA is cut with restriction enzymes and the resulting fragments are separated by size on an agarose gel. The fragments are then transferred to a solid support nitro-cellulose or nylon using an electric field or more slowly with a buffer gradient.
The presence and relative amount of a gene, as well as a physical map of the gene, can be produced by analyzing the resultant fragments.
This restriction map can be used to compare the DNA sample with others and detect difference in genes between individuals. Southern blotting is used to identify major gene rearrangements and deletions and can be used to detect genetically inherited gene abnormalities in a patient or their family. In the process of cloning a gene, Southern blotting provides a convenient method to identify a single gene within a larger-sized DNA fragment, and a method to compare genes between species.
Northern blotting analyzes the size and expression of specific mRNA. Northern analysis is frequently used to identify the size of mRNA message for a known gene in various tissues and cells. Northern blotting also can be used to identify an increase in the expression of specific mRNA in response to various stimuli.
Figure 6. Southern Blotting. DNA is cut with restriction enzymes and separated by size, using gel electrophoresis. The resulting fragments are transferred onto a nylon filter which is then hybridized with a DNA probe specific for the sequence of interest. Nonhybridized probe is washed away, and the filter is exposed to x-ray film. A DNA sequence complementary to the probe is seen as a dark band on the developed film.
N Engl J Med ; All of the molecular biology methods described thus far can be used with DNA or RNA isolated from a single tissue or cultured cells. However, none of these techniques maintains tissue architecture so that DNA or RNA can be localized to specific cells within a tissue. In situ hybridization determines RNA expression at a cellular level Figure 7.
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