Worm Breeder's Gazette 13(4): 26 (October 1, 1994)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

C. elegans Molecular Genetics and Long PCR.

Scott R. Townsend, Cathy Savage, Alyce L. Finelli, Ting Xie, Richard W. Padgett

Waksman Institute and Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08855

  Until recently, it was difficult to synthesize fragments larger than 2 kb with any
regularity using PCR. Employing a modification of the Barnes technique (Barnes,
PNAS 91:2216), we have reliably synthesized fragments of up to 14 kb from plasmid
DNA and up to 4.5 kb from C. elegans genomic DNA. We believe that "long PCR" may
be a useful tool for many molecular genetic techniques.
  We used a mixture of taq polymerase and pfu polymerase, a thermostable DNA
polymerase that has an exonuclease (proofreading) function. The exonuclease
function of pfu polymerase corrects the errors that are characteristic of synthesis by taq
polymerase alone, thereby allowing further extension than was possible using older
methods. We used unit ratios of taq:pfu between 4:1 and 16:1, and we added
approximately one unit of taq per kb of the fragment to be amplified. An alternative to
pfu is Stratagene' s Taq Extender (which, incidentally, contains pfu according to
Stratagene). When using pfu or Taq Extender, standard taq buffers should not be used
because they inhibit the activity of pfu. We have had success using Stratagene' s 10X
Taq Extender buffer. Other parameters of "long PCR" include 30-35 cycles and
extension times (72260C) of about one minute per kb of fragment to be amplified. We have
found that when amplifying plasmid or cosmid DNA, annealing temperatures around
42260C worked well, but that when amplifying genomic DNA, annealing temperatures of
55-60260C were necessary to minimize background and small molecular weight
  Until now, the molecular genetic analysis of genes required the subcloning of
YACs, cosmids, or lambda clones. Such analysis often included using large pieces of
DNA for gene rescue or for lacZ fusion to examine expression patterns. If the
fortuitous placement of restriction sites was not convenient, the molecular
manipulations could be difficult and time consuming. We envision "long PCR" as an
aid in many molecular genetic techniques. Using PCR primers with unique
restriction sites engineered in, one can easily subclone the PCR product. We have
successfully subcloned and sequenced "long PCR" products from mutants as a method
of cloning a gene. "long PCR" could also prove useful as an aid in gene rescue. After
achieving rescue with a large piece of DNA, one could employ PCR to narrow down the
rescuing fragment. Other applications might include lacZ or GFP fusions, which
now would be easier to fuse in frame.
  10X Taq Extender buffer (Stratagene)
  200mN Tris-HCl (pH 8.8)
  100mM (NH4)2.M gSO4
  100mM KCl
  20mM M gSO4
  1% Triton X-100
  1 mg/ml nuclease-free BSA