生物化学原理教学（杨荣武）Chapter7 Properties of nucleic acids and research methods.pptVIP

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生物化学原理教学（杨荣武）Chapter7 Properties of nucleic acids and research methods.ppt

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If one fellows the absorbance as a function of temperature, the midpoint temperature of the absorbance curve is termed melting temperature, Tm. DNA of different sequences have different Tm. Tm is higher for DNA that contain more GC pairs; Tm is also directly proportional to the ionic strength of the solution. Chain termination method (dideoxy method), developed by F. Sanger Base-specific chemical cleavage, developed by Maxam and Gilbert Both use autoradiography - X-ray film develops in response to presence of radioactive isotopes in nucleic acid molecules Pyrosequencing and deep sequencing Summary for DNA Replication DNA is a double-helical molecule Each strand of the helix must be copied in complementary fashion by DNA polymerase Each strand is a template for copying DNA polymerase requires template and primer Primer: an oligonucleotide that pairs with the end of the template molecule to form dsDNA DNA polymerases add nucleotides in 5-3 direction Based on DNA polymerase reaction Run four separate reactions Each reaction mixture contains dATP, dGTP, dCTP and dTTP, one of which is P-32-labelled Each reaction also contains a small amount of one dideoxynucleotide: either ddATP, ddGTP, ddCTP or ddTTP Most of the time, the polymerase uses normal nucleotides and DNA molecules grow normally Occasionally, the polymerase uses a dideoxynucleotide, which adds to the chain and then prevents further growth in that molecule Random insertion of dd-nucleotides leaves (optimally) at least a few chains terminated at every occurrence of a given nucleotide Chain Termination Method Run each reaction mixture on electrophoresis gel Short fragments go to bottom, long fragments on top Read the sequence from bottom of gel to top Convert this sequence to the complementary sequence Now read from the other end and you have the sequence you wanted - read 5 to 3 Not used as frequently as Sangers Start with ssDNA labelled with 32P at one end Strand is cleaved by chemical re