lapcrchapter - Tips for Long and Accurate PCR Wayne M....

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Tips for Long and Accurate PCR Wayne M. Barnes Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri USA and DNA Polymerase Technology, Inc., St. Louis, Missouri wayne@barnes1.wustl.edu and barnes@klentaq.com In 1992 I, and I assume others, were working on mixing and fusing various domains of different DNA polymerases, in order to have the advantage of long reads and/or high fidelity, combined with the robust reliability of Taq DNA polymerase. It was assumed that processivity (long "hang time", staying on the DNA for long periods and long distances) was key to long PCR. That may yet come true some day, but so far, the opposite is true. The Klentaq1, an N-terminal deletion of Taq DNA polymerase, is even less processive than the full-length Taq, yet it was used to set the record of 35 kb in 1993. Ironically, less hang time was found to allow other components of an enzyme mixture to access the growing chain to repair or edit problem DNA molecules that were limiting extension -- those with mismatched bases at the 3'-end. In this chapter I will use the acronym LA to mean Long and Accurate. Long being more than the ca. 3 kb limit of efficient PCR without a mixture of enzymes, and accurate to mean high fidelity, since the long PCR mixtures were found to simultaneously provide some 10-fold fewer mutations in the PCR product (Barnes, 1994). I will also use LA as a suffix to mean a mixture of DNA polymerases, the major one usually being Taq or Klentaq1 (which have no 3'-exonuclease proofreading activity) and the minor one is an archaebacterial DNA polymerase such as Deep Vent, Vent, or Pfu (Barnes, 1994). TaqLA means Taq DNA polymerase mixed with a small amount of Deep Vent or Pfu. TaqLA is sold under various names, such as Expand, Elongase, ExTaq, TaqPlus, Accutaq, etc. KlentaqLA means Klentaq1 with a similar low- level partner of proofreading DNA polymerase. Other components are being introduced to improve LA PCR. Among these I mention one enzyme component and one chemical component. (1) dUTPase. Stratagene (Hogrefe et al., 2002) has discovered that a buildup of deaminated dCTP , namely dUTP was causing the incorporation of uracil into DNA. Uracil in DNA is a disaster for the archaebacterial component of LA PCR mixtures of DNA polymerase, since the archeabacterial DNA polymerases (such as Pfu) bind almost irreversiby to dU-containing-DNA under in vitro conditions (Lasken et al, 1996). A cure introduced by Stratagene is thermostable dUTPase . This enzyme is now included in some of their PCR enzyme mixtures, in addition to the DNA polymerase components with low and high 3'- exonuclease. (2) Betaine. Although introduced for high GC targets, I find that betaine never hurts, and usually helps, even for long PCR up to at least 20 kb. It is included at surprisingly high levels, such as 2 molar final (Baskaran et al., 1996; see tip #4 below). Since the introduction of mixtures of DNA polymerases
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lapcrchapter - Tips for Long and Accurate PCR Wayne M....

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