9. Write short notes on: 

                                 a) the polymerase chain reaction  
                                 b) immunohistochemistry  
                                 c) in situ hybridization. 

a) the polymerase chain reaction
  • Polymerase chain reaction (PCR), is a common method of creating copies of specific fragments of DNA. PCR rapidly amplifies a single DNA molecule into many billions of molecules. 
  • There are three basic steps in PCR. First, the target genetic material must be denatured-that is, the strands of its helix must be unwound and separated-by heating to 90-96°C. 
  • The second step is hybridization or annealing, in which the primers bind to their complementary bases on the now single-stranded DNA. 
  • The third is DNA synthesis by a polymerase. Starting from the primer, the polymerase can read a template strand and match it with complementary nucleotides very quickly. The result is two new helixes in place of the first, each composed of one of the original strands plus its newly assembled complementary strand.
  • All PCR really requires in the way of equipment is a reaction tube, reagents, and a source of heat. But different temperatures are optimal for each of the three steps, so machines now control these temperature variations automatically.
  • it can be used to detect genetic abnormality, the presence of microbes and cancer cells. 
b) immunohistochemistry  
  • The use of antibodies or antisera as histological tools for identifying patterns of antigen distribution within a tissue or an organism. 
  • An antibody (or mixture of antibodies) that binds to a specific protein or other antigen is tagged with a fluorescent chemical or an enzyme that can convert a substrate to a visible dye. 
  • The tagged antibody is incubated with the tissue and after washing unbound antibody away the bound antibody distribution is revealed by fluorescence microscopy or incubation with a chromogenic substrate. 
  • It is assumed that antibody distribution reflects antigen distribution. 
c) in situ hybridization. 
  • In situ hybridization, as the name suggests, is a method of localizing, either mRNA within the cytoplasm or DNA within the chromosomes of the nucleus, by hybridizing the sequence of interest to a complimentary strand of a nucleotide probe. 
  • Normal hybridization requires the isolation of DNA or RNA, separating it on a gel, blotting it onto nitrocellulose and probing it with a complimentary sequence. 
  • The basic principles for in situ hybridization are the same, except one is utilizing the probe to detect specific nucleotide sequences within cells and tissues. The sensitivity of the technique is such that threshold levels of detection are in the region of 10-20 copies of mRNA or DNA per cell. 
  • In situ hybridization presents a unique set of problems as the sequence to be detected will be at a lower concentration, be masked because of associated protein, or protected within a cell or cellular structure. Therefore, in order to probe the tissue or cells of interest one has to increase the permeability of the cell and the visibility of he nucleotide sequence to the probe without destroying the structural integrity of the cell or tissue. 
The tissues can be prepared in the following way:
    • fixed in formalin
    • snap frozen, and embedded in a special support medium for cryosectioning. 
    • cells in suspension can be cytospun onto glass slides and fixed with methanol
    • preparation of metaphase chromosmal spreads, normally fixed with a mixture of methanol and acetic acid.

    There is essentially four types of probe used
         a. Oligonucleotide probes 
         b. Single stranded DNA probes. 
         c. Double stranded DNA probes 
         d. RNA probes or Riboprobes

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