Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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Long and Accurate PCR (LA PCR)

(Protocol summary only for purposes of this preview site)

Standard PCR is easily capable of amplifying segments of DNA smaller than 3 kb in lengthsufficient for most purposes, but not enough to amplify an entire mammalian gene, nor even a cDNA of average dimensions. Instead of full-length products, standard PCR amplification of longer templates generates variously sized truncated molecules that appear as unattractive smears on a gel. Explanations to account for the failure of standard PCR to amplify long templates include:

  • damage to the template and product DNAs during exposure to high temperature in buffers that may not adequately maintain control over pH
  • the presence of stray divalent cations (Mn2 is always the prime suspect) that may promote cleavage of DNA at high temperature
  • difficulties in denaturing very long DNA molecules during the heating step of the PCR cycle
  • the high rate of incorporation of incorrect bases by thermostable polymerases such as Taq that lack an editing function (the incorporation of a mismatched base at the 3 end of a growing strand may cause the enzyme to stall and may limit the size of the PCR product)


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(Limited time special offer.) Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook
Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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