Two-Step Gradient PCR

Introduction
Science underwent revolutionary changes during the 1980's, particularly in the field of genetics. One of the most significant changes was inspired by the 1985 article by Saiki, et al, in which the amplification of specific beta-globin sequences and the subsequent restriction analysis for diagnosis of sickle cell anemia is described (1). The technique referenced utilized biological and chemical components to orchestrate an enzymatic amplification reaction conducted by a DNA polymerase. This technique was named the Polymerase Chain Reaction (PCR).

This article describes an important extension of the PCR technique—Two-step Gradient PCR. This method offers significant time-savings and minimizes reagent use, relative to a standard PCR optimization protocol. The method is applied to the standardizing of the amplification of a segment of mouse tissue plasminogen activator (tPA) cDNA, using an oligonucleotide set. The calculated melting temperatures for this amplification were 57.3°C for K2-RC, and 57.8°C for GF-ATG (2). The standardization of the reactions was performed using the Eppendorf® Mastercycler® gradient (3), which provided excellent, reproducible, and rapid results.

Objective
The presentation of a practical method that reduces the time devoted to uncovering optimal annealing temperatures. This method incorporates a two-step protocol (combining the annealing and elongation steps), which is designed for significant time-savings and a reduction in reagent use during optimization and standard PCR experiments.

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