Molecular Genetics Molecular genetics is the field of biology and genetics that studies the structure and

function of genes at a molecular level. The field studies how the genes are transferred from generation to generation. Molecular genetics employs the methods of genetics and molecular biology. It is so-called to differentiate it from other sub fields of genetics such as ecological genetics and population genetics. An important area within molecular genetics is the use of molecular information to determine the patterns of descent, and therefore the correct scientific classification of organisms: this is called molecular systematic. Forward genetics One of the first tools available to molecular geneticists is the forward genetic screen. The aim of this technique is to identify mutations that produce a certain phenotype. A mutagen is very often used to accelerate this process. Once mutants have been isolated, the mutated gene can be molecularly identified. Reverse genetics While forward genetic screens are productive, a more straightforward approach is to simply determine the phenotype that results from mutating a given gene. This is called reverse genetics. In some organisms, such as yeast and mice, it is possible to induce the deletion of a particular gene, creating what's known as a gene "knockout" - the laboratory origin of socalled "knockout mice" for further study. In other words this process involves the creation of transgenic organisms that do not express a gene of interest. Alternative methods of reverse genetic research include the random induction of DNA deletions and subsequent selection for deletions in a gene of interest, as well as the application of RNA interference.

Gene therapy
A mutation in a gene can result in a severe medical condition. A protein encoded by a mutated gene may malfunction and cells that rely on the protein might therefore fail to function properly. This can cause problems for specific tissues or organs, or for the entire body. This might manifest through the course of development (like a cleft palate) or as an abnormal response to stimuli (like a peanut allergy). Conditions related to gene mutations are called genetic disorders. One way to fix such a physiological problem is gene therapy. By adding a corrected copy of the gene, a functional form of the protein can be produced, and affected cells, tissues, and organs may work properly. As opposed to drug-based approaches, gene therapy repairs the underlying genetic defect. a.) Classical gene therapy Classical gene therapy is the approach which delivers genes, via a modified virus or "vector" to the appropriate target cells with a goal of attaining optimal expression of the new, introduced gene. Once inside the patient, the expressed genes are intended to produce

a product that the patient lacks, kill diseased cells directly by producing a toxin, or activate the immune system to help the killing of diseased cells. b.) Non classical gene therapy Non classical gene therapy inhibits the expression of genes related to pathogenesis, or corrects a genetic defect and restores normal gene expression.

Principles for gene transfer

Classical gene therapies usually require efficient transfer of cloned genes into the disease cells so that the introduced genes are expressed at sufficiently high levels to change the patient's physiology. There are several different physicochemical and biological methods that can be used to transfer genes into human cells. The size of the DNA fragments that can be transferred is very limited, and often the transferred gene is not a conventional gene. Horizontal gene transfer is the transfer of genetic material from one cell to another that is not its offspring. Artificial horizontal gene transfer is a form of genetic engineering. a.) In vivo gene transfer During In vivo gene transfer, the genes are transferred directly into the tissue of the patient and this can be the only possible option in patients with tissues where individual cells cannot be cultured in vitro in sufficient numbers (e.g. brain cells). Also, in vivo gene transfer is necessary when cultured cells cannot be re-implanted in patients effectively. b.) Ex vivo gene transfer During ex vivo gene transfer the cells are cultured outside the body and then the genes are transferred into the cells grown in culture. The cells that have been transformed successfully are expanded by cell culture and then introduced into the patient.

Techniques in molecular genetics

There are three general techniques used for molecular genetics: amplification, separation and detection, and expression. Specifically used for amplification is polymerase chain reaction, which is an indispensable tool in a great variety of applications. In the separation and detection technique DNA and mRNA are isolated from their cells. Gene expression in cells or organisms is done in a place or time that is not normal for that specific gene.

Amplification

There are other methods for amplification besides polymerase chain reaction. Cloning DNA in bacteria is also a way to amplify DNA in genes. Polymerase chain reaction The main materials used in polymerase chain reaction are DNA nucleotides, template DNA, primers and Taq polymerase. Cloning DNA in bacteria The word cloning for this type of amplification entails making multiple identical copies of a sequence of DNA. The target DNA sequence is then inserted into a cloning vector. Because this vector originates from a self-replicating virus, plasmid, or higher organism cell when the appropriate size DNA is inserted the target and vector DNA fragments are then ligated and create a recombinant DNA molecule. The recombinant DNA molecules are then put into a bacteria strain (usually E. coli) which produces several identical copies by transformation. Transformation is the DNA uptake mechanism possessed by bacteria. However, only one recombinant DNA molecule can be cloned within a single bacteria cell, so each clone is of just one DNA insert.

Separation and detection

In separation and detection DNA and mRNA are isolated from cells (the separation) and then detected simply by the isolation. Cell cultures are also grown to provide a constant supply of cells ready for isolation. Cell cultures A cell culture for molecular genetics is a culture that is grown in artificial conditions. DNA isolation DNA isolation extracts DNA from a cell in a pure form. First, the DNA is separated from cellular components such as proteins, RNA, and lipids. mRNA isolation Expressed DNA that codes for the synthesis of a protein is the final goal for scientists and this expressed DNA is obtained by isolating mRNA (Messenger RNA).

Basic Theory

2.1 The basic theory of classical genetics The basic theory associated with classical genetics provided explanations of the transmission of traits from parents to offspring. Morgan and his collaborators drew upon a conceptual division between the genetic makeup of an organism, termed its genotype, and its observed manifestation called its phenotype (see the entry on the genotype/phenotype distinction). The relation between the two was treated as causal: genotype in conjunction with environment produces phenotype. The theory explained the transmission of phenotypic differences from parents to offspring by following the transmission of gene differences from generation to generation and attributing the presence of alternative traits to the presence of alternative forms of genes.

2.2 Molecular-level answers to questions left behind by classical genetics

Research in molecular biology and genetics has yielded answers to the basic questions left unanswered by classical genetics about the make-up of genes, the mechanism of gene replication, what genes do, and the way that gene differences bring about phenotypic differences. These answers are couched in terms of molecular level phenomena and they provide much of the basic theory associated with molecular genetics.

2.3 Distinguishing between basic and fundamental theories of molecular genetics

The modest answer to the question What do genes do? is that they code for or determine the linear sequences in RNA molecules and polypeptides synthesized in the cell. (Even this modest answer needs to be qualified because RNA molecules are often spliced and edited in ways that affect the linear sequence of amino acids in the eventual polypeptide product.) But biologists have offered a far less modest answer as well. The bolder answer is part of a sweeping, fundamental theory. According to this theory, genes are fundamental entities that direct the development and functioning of organisms by producing proteins that in turn regulate all the important cellular processes. It is often claimed that genes provide the information, the blueprint, or the program for an organism. It is useful to distinguish this sweeping, fundamental theory about the allegedly fundamental role of genes from the modest, basic theory about what genes do with respect to the synthesis of RNA and polypeptides.

Was classical genetics reduced to molecular genetics?

Philosophers of science have been intrigued by ideals of reductionism and the grand scheme that all science will one day be reduced to a universal science of fundamental physics (see the entry on inter-theory relations in physics) for philosophical and scientific concepts of reductionism in the context of physical science). Philosophical reductionists believe that scientific knowledge progresses when higher-level sciences (e.g., chemistry) are reduced to lower-level sciences (e.g., physics). The so-called received view of scientific knowledge, codified in Nagel (1961) and Hempel (1966), promoted reductionism as a central ideal for science, and confidently asserted that much progress had been made in the

reduction of chemistry to physics. Nagel constructed a formal model of reduction and applied it to illuminate how the science of thermodynamics, which was couched in terms of higher-level concepts such as pressure and temperature, was allegedly reduced to statistical mechanics, couched in terms of the lower-level concepts of Newtonian dynamics such as force and mean kinetic energy. In 1969, Schaffner claimed that the same kind of advance was now taking place in genetics, and that the science of classical genetics was being reduced to an emerging science of molecular genetics. Schaffner's claim, however, was quickly challenged by Hull. Other philosophers of biology developed Hull's antireductionist arguments and a near consensus developed that classical genetics was not and would not be reduced to molecular genetics. Although the philosophical case for antireductionism has been challenged, many philosophers still assume that the anti-reductionist account of genetics provides an exemplar for anti-reductionist analyses of other sciences.

What is a gene?
A common claim in the philosophical literature about molecular genetics is that genes cannot be conceived at the molecular level. Of course, philosophers do not deny that biologists use the term gene, but many philosophers believe gene is a dummy term, a placeholder for many different concepts. Different responses to gene skepticism illustrate a variety of philosophical aims and approaches. One kind of response is to analyze explanations closely tied to experimental practice (rather than sweeping generalizations of a fundamental theory) in order to determine whether there are uniform patterns of reasoning about genes that could (a) be codified into clear concepts, and/or (b) used to establish the reference of the term. Another kind of response is to propose new gene concepts that will better serve the expressed aims of practicing biologists. A third kind of response is to implement survey analysis, rather than conduct traditional methods of philosophical analysis. A fourth kind of response is to embrace the (allegedly) necessary vagueness of the gene concept(s) and to examine why use of the term gene is so useful.

What do genes and DNA do?

The science called molecular genetics is associated with a fundamental theory according to which genes and DNA direct all basic life processes by providing the information specifying the development and functioning of organisms. The genome is said to specify the developmental program, master plan, or blue print for development while other elements provide the materials (e.g., Bonner 1965, Jacob and Monod 1961, Mayr 1961, Maynard Smith 2000, Rosenberg 2006). Although the idea that the chromosomes contain a code-script for the development and functioning of an organism was famously expressed by Schrodinger (1944) before the era of molecular genetics, today it is often expressed in explicitly molecular terms. The information of development and function, which is passed down from one generation to the next, is allegedly encoded in the nucleotide sequences comprising genes and DNA. This so-called genetic information is first transcribed into RNA, then translated into proteins, and finally expressed in the development and functioning of organisms.

6. Why is so much biological research centered on genes and DNA?

In official and public contexts, scientists appeal to the fundamental theory associated with molecular genetics to justify centering research on genes and DNA (e.g., see the websites of funding agencies such as National Center for Biotechnology Information). Genes are typically referred to as the fundamental units that are responsible for guiding all basic life processes. Usually a combination of causal and information metaphors is invoked to explain the role of genes. Genes are said to produce RNA and polypeptides, to provide instructions, or direct processes. But philosophical investigation has shown that these kinds of sweeping claims cannot withstand careful scrutiny. Why, then, is so much research centered on genes and DNA? One answer to this question is that biologists are blinded by an ideology of genetic determinism. But Wagner's defense of gene centrism suggests another answer, an answer that resonates with Keller's explanation (2000) of why gene talk is useful.

MOLECULAR GENETICS: PIECING IT TOGETHER

Molecular genetics is the study of the agents that pass information from generation to generation. These molecules, our genes, are long polymers of deoxyribonucleic acid, or DNA. Just four chemical building blocksguanine (G), adenine (A), thymine (T), and cytosine (C)are placed in a unique order to code for all of the genes in all living organisms.

Genes determine hereditary traits, such as the color of our hair or our eyes. They do this by providing instructions for how every activity in every cell of our body should be carried out. For example, a gene may tell a liver cell to remove excess cholesterol from our bloodstream. How does a gene do this? It will instruct the cell to make a particular protein. It is this protein that then carries out the actual work. In the case of excess blood cholesterol, it is the receptor proteins on the outside of a liver cell that bind to and remove cholesterol from the blood. The cholesterol molecules can then be transported into the cell, where they are further processed by other proteins. Many diseases are caused by mutations or changes in the DNA sequence of a gene. When the information coded for by a gene changes, the resulting protein may not function properly or may not even be made at all. In either case, the cells containing that genetic change may no longer perform as expected. We now know that mutations in genes code for the cholesterol receptor protein associated with a disease called familial hypercholesterolemia. The cells of an individual with this disease end up having reduced receptor function and cannot remove a sufficient amount of low density lipoprotein (LDL),

or bad cholesterol, from their bloodstream. A person may then develop dangerously high levels of cholesterol, putting them at increased risk for both heart attack and stroke. How do scientists study and find these genetic mutations? They have available to them a variety of tools and technologies to compare a DNA sequence isolated from a healthy person to the same DNA sequence extracted from an afflicted person. Advanced computer technologies, combined with the explosion of genetic data generated from the various whole genome sequencing projects, enable scientists to use these molecular genetic tools to diagnose disease and to design new drugs and therapies. Below is a review of some common laboratory methods that geneticists scientists who study the inheritance pattern of specific traitscan use to obtain and work with DNA, followed by a discussion of some applications.

Isolating DNA and mRNA from Cells

Cell Culture Cell culture involves growing cells under artificial conditions, such as in the laboratory, either attached to some type of artificial surface or suspended in a special solution.

DNA Isolation DNA isolation refers to the process of extracting DNA from a cell in a relatively pure form. It involves separating DNA from other cellular components, such as proteins, RNA, and lipids.

mRNA Isolation Many researchers want to work with what is called expressed DNA, or DNA that codes directly for the synthesis of a protein. This special type of DNA is obtained by first isolating messenger RNA (mRNA), an intermediate between the expressed portions of DNA and the protein product.

This drawing demonstrates how poly(A) RNA can be isolated from other RNAs by separation on a special solid support material. In this example, the material is made up of

glass beads to which thymine molecules are attached. Because adenine and thymine molecules readily bind to each other, mRNAs with poly(A) tails will be selectively retained on the beads. As seen on the left-hand side of the diagram, a solution containing various RNA populations, including mRNAs with poly(A) tails (red) as well as other RNAs and cellular material (purple), is applied to the separation column. Only the poly(A) RNA is retained, because it is immobilized on the solid support material. The other RNAs and cellular material pass through the column. On the right, the bound poly(A) mRNA is retrieved by treating the column with a special buffer solution that breaks the thymine nucleotideAAA bond. The mRNA can be collected in a tube for further experimentation. Because adenine and thymine readily bind to each other, poly(A) mRNA is selectively retained on the beads while the other cellular components are washed away. Once isolated, purified mRNA is converted to single-stranded DNA using the enzyme reverse transcriptase and is then made into a stable double-stranded DNA using the enzyme DNA polymerase. DNA produced in this way is called complementary DNA (cDNA) because its sequence, at least the first strand, is complementary to that of the mRNA from which it was made. Why do researchers go to the trouble of making cDNA? cDNA is a much more stable compound than mRNA and, more importantly, because it was generated from an mRNA in which the non-coding regions have been removed, cDNA represents only expressed DNA sequence.

Chain

termination

DNA

sequencing.

Chain termination sequencing involves the synthesis of new strands of DNA complementary to a single-stranded template (step I). The template DNA is supplied with a mixture of all four deoxynucleotides, four dideoxynucleotides (each labelled with a different colored fluorescent tag), and DNA polymerase (step II). Because all four deoxynucleotides are present, chain elongation proceeds until, by chance, DNA polymerase inserts a dideoxynucleotide. The result is a new set of DNA chains, all of different lengths (step III). The fragments are then separated by size using gel electrophoresis (step IV). As each labeled DNA fragment passes a detector at the bottom of the gel, the color is recorded. The DNA sequence is then reconstructed from the pattern of colors representing each nucleotide sequence (step V). Variations of this method have been developed for automated sequencing machines. In one method, called cycle sequencing, the dideoxynucleotides, not the primers, are tagged with

different colored fluorescent dyes; thus, all four reactions occur in the same tube and are separated in the same lane on the gel. As each labeled DNA fragment passes a detector at the bottom of the gel, the color is recorded, and the sequence is reconstructed from the pattern of colors representing each nucleotide in the sequence. Chromosome Analysis Cytogenetics is the field of science that deals with the relationship between human cells and their chemical building blocksand heredity. Fluorescence in Situ Hybridization Fluorescence in situ hybridization (FISH), a newer method for analyzing chromosomes, uses fluorescent molecules, called dyes, to "paint" genes on a chromosome. This technique is particularly useful for gene mapping and for detecting various chromosomal abnormalities. Somatic Cell Hybridization The term "somatic" cell refers to all the cells in an organism that have differentiated into a specific cell type, excluding germ cells, stem cells, and gametes. Somatic cell hybridization is the technique of combining two cells from different tissues or species in a cell culture, typically human and rodent, with the intent of deriving various cell lines, each with a different combination of chromosomes. Human cells (left) and mouse cells (right), grown in culture, can be fused by treatment with a virus or chemical agent, yielding what is called a hybrid cell (bottom) containing both human and mouse chromosomes. In this example, three human chromosomes (green, purple, and blue, with outlines) and two mouse chromosomes (red and yellow, without outlines) are shown, with the hybrid cell having a mix of the five chromosomes.

Impact of Molecular Genetics

Most sequencing and analysis technologies were developed from studies of nonhuman genomes, notably those of the bacterium Escherichia coli, the yeast Saccharomyces cerevisiae, the fruit fly Drosophila melanogaster, the roundworm Caenorhabditis elegans, and the laboratory mouse Mus musculus. These simpler systems provide excellent models for developing and testing the procedures needed for studying the much more complex human genome.

A large amount of genetic information has already been derived from these organisms, providing valuable data for the analysis of normal human gene regulation, genetic diseases, and evolutionary processes. For example, researchers have already identified single genes associated with a number of diseases, such as cystic fibrosis. As research progresses, investigators will also uncover the mechanisms for diseases caused by several genes or by single genes interacting with environmental factors. Genetic susceptibilities have been implicated in many major disabling and fatal diseases including heart disease, stroke, diabetes, and several kinds of cancer. The identification of these genes and their proteins will pave the way to more effective therapies and preventive measures. Investigators determining the underlying biology of genome organization and gene regulation will also begin to understand how humans develop, why this process sometimes goes awry, and what changes take place as people age.