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.
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 products. 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