T7 DNA polymerase
Process for amplifying, detecting, and/or cloning nucleic acid sequences using a thermostable enzyme
Use of grease or wax in the polymerase chain reaction Patent #: 5411876
ApplicationNo. 437318 filed on 05/09/1995
US Classes:435/91.1, Polynucleotide (e.g., nucleic acid, oligonucleotide, etc.)435/91.2Acellular exponential or geometric amplification (e.g., PCR, etc.)
ExaminersPrimary: Zitomer, Stephanie W.
Attorney, Agent or Firm
International ClassC12P 019/34
AbstractAmplified DNA is prepared for sequencing by contacting with exonuclease I and alkaline phosphatase which degrade undesirable excess primers dNTPs and non-specifically amplified single stranded DNA therein. The enzymes are provided in a kit.The method for sequencing DNA includes the following steps: providing a polynucleotide primer complementary to a region of the DNA, providing the DNA to be sequenced, and contacting that primer and DNA together in the presence of a DNA polymerase and between 1 and 3 dNTPs, at least one of the dNTP being labelled. The primer and DNA are contacted under conditions which allow extension of the primer by addition of one or more of the dNTPs to the primer to form an extended primer. The primer and DNA are then dissociated, generally by heating, and the contacting and dissociating steps repeated a plurality of times (usually 10-200 times). Finally, the extended primer is contacted with the DNA in the presence of a DNA polymerase (which is generally the same polymerase as used in the initial labelling step) all four dNTPs and a chain terminating agent.