Microbiological Techniques Which Deal with Chromosomal Damage - Essay Example

A chromosome (in Greek chroma = color and soma = body) is, minimally, a very long, continuous piece of DNA, which contains many genes, regulatory elements and other intervening nucleotide sequences. In the chromosomes of eukaryotes, the uncondensed DNA exists in a quasi-ordered structure inside the nucleus, where it wraps around histones, and where this composite material is called chromatin. Changes that affect entire chromosomes or segments of chromosomes can cause problems with growth, development, and function of the body's systems. These changes can affect many genes along the chromosome and alter the proteins made by those genes. Conditions caused by a change in the number or structure of chromosomes are known as chromosomal disorders (Lewontin, 1974). Some chromosomal conditions are caused by changes in the number of chromosomes, called aneuploidy. These changes are not inherited, but occur as random events during the formation of reproductive cells (ova and sperm cells). An error in cell division called nondisjunction results in reproductive cells with an abnormal number of chromosomes. For example, a reproductive cell may accidentally gain or lose one copy of a chromosome. If one of these atypical reproductive cells contributes to the genetic makeup of a child, the child will have an extra (trisomy) or missing chromosome (monosomy) in each of the body's cells. The formation of ring chromosomes following fertilization also cause genetic disorders. Chromosomal disorders can also be caused by changes in chromosome structure. These changes are caused by the breakage and reunion of chromosome segments when an egg or sperm cell is formed or in early fetal development. Pieces of DNA can be rearranged within one chromosome, or transferred between two or more chromosomes. The effects of structural changes depend on their size and location. Many different structural changes are possible; some cause medical problems, while others may have no effect on a person's health (Roberts et al. 2002; James & Motoo 1974). Since the late 1950s and early 1960s, molecular biologists have learned to characterise, isolate, and manipulate the molecular components of cells and organisms. These components include DNA, the repository of genetic information; RNA, a close relative of DNA whose functions range from serving as a temporary working copy of DNA to actual structural and enzymatic functions as well as a functional and structural part of the translational apparatus; and proteins, the major structural and enzymatic type of molecule in cells (Beatty, 1986; Derrington & Grey, 1996). One of the most basic techniques of molecular biology to study protein function and prevent possible chromosomal damage is expression cloning. In this technique, DNA coding for a protein of interest is cloned (using PCR and/or restriction enzymes) into a plasmid (known as an expression vector). This plasmid may have special promoter elements to drive production of the protein of interest, and may also have antibiotic resistance markers to help follow the plasmid. This plasmid can be inserted into either bacterial or animal cells. Introducing DNA into bacterial cells is called transformation, and can be completed with several methods, including electroporation, microinjection, passive uptake and conjugation. Introducing DNA into eukaryotic cells, such as animal cells, is called transfection. Several different transfection techniques are available, including calcium phosphate transfection, liposome transfection, and proprietary transfection reagents such as Fugene. DNA can also be introduced into cells using viruses or pathenogenic bacteria as carriers. In such cases, the technique is called viral/bacterial transduction, and the cells are said to be transduced (Molecular cloning [A lab manual], 2001). In either case, DNA coding for a protein of interest is now inside a cell, and the protein can now be expressed. A variety of systems, such as inducible promoters and specific cell-signaling factors, are available to help express the protein of interest at high levels. Large quantities of a protein can then be extracted from the bacterial or eukaryotic cell. The protein can be tested for enzymatic activity under a variety of situations, the protein may be crystallized so its tertiary structure can be studied, or, in the pharmaceutical industry, the activity of new drugs against the protein can be studied. The detection of hereditary diseases in a given genome is a long and difficult process, which can be shortened significantly by using PCR. Each gene in question can easily be amplified through PCR by using the appropriate primers and then sequenced to detect mutations. Polymerase chain reaction (PCR) is a molecular biological technique for amplifying (creating multiple copies of) DNA without using a living organism, such as E. coli or yeast. The technique allows a small sample of DNA to be copied multiple times so it can be used for analysis (Cohen et al. 1973). PCR is used to amplify a short, well-defined part of a DNA strand. This can be a single gene, or just a part of a gene. As opposed to living organisms, the PCR process can copy only short DNA fragments, usually up to 10 kb (kb=kilo base pairs=1000 base pairs). DNA is double-stranded, and therefore, it is measured in complementary DNA building blocks (nucleic acids) called base pairs. Certain methods can copy fragments up to 40 kb in size, which is still much less than the chromosomal DNA of a eukaryotic cell--for example, a human cell contains about three billion base pairs. PCR, as currently practiced, requires several basic components. These components are: DNA template, which contains the region of the DNA fragment to be amplified Two primers, which determine the beginning and end of the region to be amplified (see following section on primers) DNA-Polymerase, which copies the region to be amplified Nucleotides, from which the DNA-Polymerase builds the new DNA Buffer, which provides a suitable chemical environment for the DNA-Polymerase The PCR reaction is carried out in a thermal cycler. This is a machine that heats and cools the reaction tubes within it to the precise temperature required for each step of the reaction. To prevent evaporation of the reaction mixture, a heated lid is placed on top of the reaction tubes or a layer of oil is put on the surface of the reaction mixture (Hartl, 2000). The PCR process consists of a series of twenty to thirty cycles. Each cycle consists of three steps. (1) The double-stranded DNA has to be heated to 94-96C in order to separate the strands. This step is called denaturing; it breaks apart the hydrogen bonds that connect the two DNA strands. Prior to the first cycle, the DNA is often denatured for an extended time to ensure that both the template DNA and the primers have completely separated and are now single-strand only. Time: 1-2 minutes. (2) After separating the DNA strands, the temperature is lowered so the primers can attach themselves to the single DNA strands. This step is called annealing. The temperature of this stage depends on the primers and is usually 5C below their melting temperature (45-60C). A wrong temperature during the annealing step can result in primers not binding to the template DNA at all, or binding at random. Time: 1-2 minutes. (3) Finally, the DNA-Polymerase has to fill in the missing strands. It starts at the annealed primer and works its way along the DNA strand. This step is called elongation. The elongation temperature depends on the DNA-Polymerase. The time for this step depends both on the DNA-Polymerase itself and on the length of the DNA fragment to be amplified. As a rule-of-thumb, 1 minute per 1000bp (Franklin, 2002). Gel electrophoresis is another group of techniques used by scientists to prevent chromosome disorder while separating molecules based on physical characteristics such as size, shape, or isoelectric point. Gel electrophoresis may be used as a preparative technique to partially purify molecules prior to use of other methods such as mass spectrometry, PCR, cloning, DNA sequencing, or immuno-blotting for further characterization. The first part, "gel", refers to the matrix used to separate the molecules. In most cases the gel is a crosslinked polymer whose porosity can be controlled by the scientist. The gel forms a solid but porous matrix that looks and feels like clear jello. The second part, "electrophoresis", refers to the electromotive force (EMF) that is used to push or pull the molecules through the gel matrix; by placing the molecules in wells in the gel and applying an electric current, the molecules will move through the matrix at different rates, towards the anode if negatively charged or towards the cathode if positively charged (note that gel electrophoresis operates as an electrolytic cell; the anode is positive and the cathode is negative). Double-stranded DNA fragments natually behave as long rods, so their migration through the gel is relative to their radius of gyration, or, roughly, size. Single-stranded DNA or RNA tend to fold up into molecules with complex shapes and migrate through the gel in a complicated manner based on their tertiary structure. After the electrophoresis run, when the smallest molecules have almost reached the anode, the molecules in the gel can be stained to make them visible. Ethidium bromide, Silver, or Coomassie blue dye can be used. Other methods can also be used to visualize the separation of the mixture's components on the gel. If the analyte molecules luminesce under ultraviolet light, a photograph can be taken of the gel under ultraviolet light. If the molecules to be separated contain radioactive atoms, an autoradiogram can be recorded of the gel. Bands in different lanes that end up at the same distance from the top contain molecules that passed through the gel with the same speed, which usually means they are approximately the same size. There are special markers available - ladders - which contain a mixture of molecules of known sizes. If such a marker was run on one lane in the gel parallel to the unknown samples, the bands observed can be compared to those of the unknown in order to determine their size. The distance a band travels is approximately inversely proportional to the logarithm of the size of the molecule (Gillespie, 1998). There are also other microbiological techniques which deal with chromosomal damage, including southern blotting, northern blotting and immunochemistry. Reference: 1. Beatty, James (1986) "The synthesis and the synthetic theory" in Integrating Scientific Disciplines. 2. Cohen, S.N., Chang, A.C.Y., Boyer, H. & Heling, R.B. (1973) Construction of biologically functional bacterial plasmids in vitro. Proc. Natl. Acad. Sci. USA. 3. Derrington, Kevin and Grey, Thomas (1996) History of Molecular Biology. New York. 4. Franklin, Robert (2002). Stimulation of chromosome deviations in un-irradiated chromatin after incomplete irradiation of a cell nucleus. 5. Gillespie, John (1998) Population Genetics: A Concise Guide, Johns Hopkins Press. 6. Hartl, Daniel (2000) Primer of Population Genetics, 3rd edition, Sinauer. 7. James F. Crow and Motoo Kimura (1972) Introduction to Population Genetics Theory. Harper & Row. 8. Lewontin, Richard (1974). The Genetic Basis of Evolutionary Change. Columbia University Press. New York. 9. Molecular cloning [A lab manual] Sambrook and Russell, 3rd edition. Cold Spring Harbor Laboratory Press. 2001. 10. Roberts, K., Raff, M. et al. (2002) Molecular Biology of the Cell 4th Edition, Routledge. Read More