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 can any1 explain this plz..  



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Author3 Posts
  #1

can any1 plz explain y we do this sequencing n what do we expect out of this...........

a. Sequencing – The major method of sequencing is the Sanger dideoxy nucleotide method. An elongation reaction is carried out using a primer just upstream of the portion to be sequenced. The mixture includes radioactive nucleotides except one of the nucleotides (A,T,G,or C) is dideoxy. That is, it does not have an oxygen at the 2 or 3 position of the ribose sugar. When one of the dideoxy bases is incorporated into the growing chain, elongation is stopped. A reaction is run containing dideoxy nucleotides of each base. For instance, one reaction contains all of the nucleotides but the adenosines are dideoxy. The results are then run on a gel. The sequence can be read by observing which bases were the terminating base at each position on the sequence.



thanku......<?xml:namespace prefix = o ns = "urn:schemas-microsoft-com:office:office" />





  #2

to map out the genetic code i think i.e sequence of bases


  #3

Sequencing DNA: The Sanger Method
Most DNA sequencing is currently done using the "chain termination" technique developed initially by Frederick Sanger, for which he earned his second Nobel Prize (figure 12). (1) A short single-stranded primer is added to the end of a single-stranded DNA fragment of unknown sequence. The primer provides a 3´ end for DNA polymerase. (2) The primed fragment is added, along with DNA polymerase and a supply of all four deoxynucleotides (d-nucleotides), to four synthesis tubes. Each contains a different dideoxynucleotide (dd-nucleotide); such nucleotides lack both the 2´ and the 3´ OH groups and are thus chain-terminating. The first tube, for example, contains ddATP and stops synthesis whenever ddA is incorporated into DNA instead of dATP. Because of the relatively low concentration of ddATP compared to dATP, ddA will not
necessarily be added to the first A site; this tube will contain a series of fragments of different lengths, corresponding to the different distances the polymerase traveled from the primer before a ddA was incorporated. (3) These fragments can be separated according to size by electrophoresis. (4) A radioactive label (here dATP*) allows the fragments to be visualized on X-ray film, and the newly made sequence can be read directly from the film. Try it. (5) The original fragment has the complementary sequence.
Techniques such as Southern blotting and PCR enable investigators to identify specific genes and produce them in large quantities, while RFLP analysis and the Sanger method identify individuals and unknown gene sequences.










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