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									                  DNA Probe

• One of the most powerful methods
  in molecular genetics is the detection
  of DNA molecules by a procedure
  called hybridisation
• As DNA molecules are not visible to
  the naked eye, they are labelled with
  a reporter group radioactive or non-
  radioactive).
• A labelled DNA molecule is called a
  probe.
   DNA Labelling Techniques

• Two general approaches;
  – Incorporation of reporter
    molecule into the DNA either
    directly or indirectly, synthesis
    using a DNA polymerase
     • Primer extension
     • Nick Translation
     • Random Priming
  – End labelling
     • T4 polynucleotide kinase
     • Terminal transferase
     • End fill with DNA polymerase
Nucleotide Phosphate
   nomenclature
         Types of Reporter Molecules


                   Radio labels


Label          Detail                Application




32
     P         Strong beta emitter   Southern
                                     hybridisation,
                                     colony/plaque
                                     hybridisation
35
     S         Weak beta emitter     DNA sequencing




3
 H             Gamma emitter         In situ hybridisation
           Random Priming

• Double stranded target DNA is
  denatured
• Random sequence hexamers (6) are
  added
• Klenow fragment of DNA
  polymerase I is added together with
  buffer, Mg2+, unlabelled dNTP’s
  and a labelled dNTP (usually dCTP)
• The reaction is incubated at 25-37oC
• Upon completion the DNA is
  denatured by heat and used
Random Priming
Nick Translation
  End Labelling
(Polynucleotide kinase)
  End labelling
(Terminal transferase)
                   End Labelling
                         (3’ overhang)


•   3’ overhang

    eg. Pst I

                  5’-CGAT-3’

                  3’-GC       5’


                              32
                                   PadGTP + T4 DNA polymerase

                              (uses the 3’- 5’ exonuclease)



                  5’-C    -3’

                  3’-GC   -5’



                  5’-CG -3’

                  3’-GC -5’
                   End Labelling
                       (5’ overhang)


•   5’ overhang

    eg. BamH1

                  5’-GATCC-3’

                  3’     G-5’



                          dATP/dTTP/dGTP or dCTP

                          + klenow DNA polymerase)

                          or T4 DNA polymerase



                  5’-GATCC-3’

                  3’-CTAGG-5’
           Non Radioactive labelling
•   Amersham Int
 ECL reaction
(Chemiluminesence)

								
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