Ready to use agarose encapsulated PCR reagents

• Published in: Nucleic acids research Vol. 24; no. 8; pp. 1580 - 1581
• Main Authors: Setterquist, R.A, Smith, G.K
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 15.04.1996
• Subjects: agarose; AIDS/HIV; biochemical techniques; encapsulation; Genes, gag; HIV-1 - genetics; Humans; Indicators and Reagents; Polymerase Chain Reaction; Sepharose
• ISSN: 0305-1048; 1362-4962; 1362-4962
• DOI: 10.1093/nar/24.8.1580

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 degree 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 mu l reaction volume). Although the total yield in each reaction was not quantitated, visual inspection indicated no difference in product yield, spurious band formation on 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).