Ready to use agarose encapsulated PCR reagents.

Many variations of the polymerase chain reaction (PCR) have recently evolved, enhancing this powerful tool for use in cloning, nucleic acid sequencing and diagnostic testing. Construction of a large number of PCR reactions typical for diagnostic applications (HLA typing, genetic screening, microbial identification) requires time-consuming and repetitive pipeting. Even a master mix made in the laboratory or supplied with kits requires assembly of completed PCR reactions. We have developed a technique for encapsulating all of the components of a PCR reaction, including polymerase as well as gel loading buffer and dye, in an agarose matrix that can be easily shipped in pre-aliquoted tubes and stored for months at –20 C. To complete the PCR reaction the researcher need only add DNA template to the matrix and commence thermocycling. After thermocycling the reaction can be loaded directly onto an agarose gel for analysis. This technique enables one to make identical sets of reactions at one time which can be distributed into 96-well plates, strip tubes or single tubes in a form suitable for storage, shipping and room temperature assembly of reactions. To determine the appropriate agarose with which to encapsulate the PCR reactants, various agarose types and concentrations were tested to ascertain whether there was any inhibitory effect on PCR reaction yield. A comparison of conventional PCR reactions with those containing 0.1–0.5% agarose (Fisher Biotech agarose, cat.#BP1356-100, Fisher Biotech, Fairlawn, NJ; NuSieve 3:1, cat.#50092, FMC, Rockland, ME; low-melting point agarose, cat.#A-9414, Sigma Chemical Co., St Louis, MO and Trevigel 500 cat.#9804-25-P, Trevigen, Gaithersburg, MD) revealed that agarose has no inhibitory effect on the yield of PCR products even at low template concentration (100 copies in a 50 μl reaction volume). Although the total yield in each reaction was not quantitated, visual inspection indicated no difference in product yield, spurious band formation or primer–dimer formation between conventional PCR and agarose-containing PCR. For subsequent testing purposes, Trevigel 500 was selected because: (i) it has the highest reported melting temperature (good hot start quality), (ii) it is the only agarose reported to be DNAse and RNAse free and (iii) it has high gel strength (important in shipping). An additional consideration when formulating an appropriate agarose matrix is to determine the concentration which yields good mechanical properties without interfering with post-reaction pipeting due to increased viscosity. The limits which appear to fit this criteria is an encapsulation matrix of 0.5% which yields a post-reaction agarose concentration of <0.07%. To demonstrate this technique, all the reagents available in the Perkin-Elmer Gene Amplimer kit (Perkin-Elmer, Foster City, CA) for the detection of HIV-1 gag gene, except for DNA t mplate, were encapsulated in an agarose matrix. Table 1 lists the concentration of the components of the encapsulations and the inal concentrations of the components for a 25 μl reaction volume. Assembly of 10 encapsulated reagents was performed in the following manner. Appropriate amounts of dry reaction components 1–7 (including a set of primers for HIV-1 gag gene) were resuspended in 25 μl of a 0.5% agarose/50% glycerol so ution (58 C), after which 5.0 U Taq polymerase was added to the mixture. An aliquot of 2.5 μl of this molten encapsulation olution was then added to each of 10 × 0.2 ml thin-walled PCR tubes, allowed to gel and placed in a –20 C freezer for 2 weeks. Three encapsulated reactions were tested against freshly prepared PCR reactions incorporating the reactant without agarose matrix. A solution containing 5 000, 1000 or 500 copies of HIV-1 template was added to the encapsulated reactions and freshly made controls. The amplification was performed according to the Gene Amplimer protocol by cycling the samples through a 1 min denaturing step (94 C), followed by 30 cycles of alternating 60 C for 60 s and 94 C for 60 s. The reactions were cooled to room temperature and 10 μl was loaded onto a 4.0% NuSieve (FMC BioProducts) agarose gel for analysis. According to the pr otocol, a 115 bp product is generated from the amplification of the gag gene from HIV-1. As can be seen in Figure 1, the control PCR reactions and the stored encapsulated reactions appear identical, even at low copy number ( ∼500).