Analysis of PCR Products

Agarose Gel Electrophoresis Analysis of PCR Products

To quickly and easily analyze and resolve PCR products, a 3% NuSieve can be used GTG agarose (FMC Bioproducts, Rockland, ME) and 1% Seakem GTG agarose (FMC Bioproducts) gel run in either TBE (89 mM Tris-borate, 2 mM EDTA) or TAE (40 mM Tris-acetate, 2 mM EDTA, pH approx 8.5). The resolved DNA bands are detected by staining the gels with either approx 0.5 μg/mL of ethidium bromide, followed by destaining with water or SYBR® Green 1 (Molecular Probes Inc., Eugene, OR) and finally photographed under UV illumination. Use a 123-basepair (bp) or 1-kilobasepair (kbp) ladder as a convenient marker for size estimates of the products.

Other Analytical Methods to Analyze PCR Products

There are a variety of other detection methods available for PCR product analysis, such as ethidium bromide-stained 8–10% polyacrylamide gels run in TBE buffer, Southern gels or dot/blots, subcloning and direct sequencing, HPLC analysis, and the use of 96-well microplates, to name a few. The reverse dot-blot method combines PCR amplification with nonradioactive detection. The introduction of fluorescent dyes to PCR, together with a suitable instrument for real-time, online quantification of PCR products during amplification has led to the development of kinetic PCR or quantitative PCR. Quantitative PCR (QPC) measures PCR product accumulation during the exponential phase of the reaction and before amplification becomes vulnerable, i.e., when reagents become limited. The ABI Prism 7700 (Applied Biosystems) and the LightCycler (Roche Molecular Biochemicals, Mannheim, Germany) are integrated fluorescent detection devices that allow fluorescence monitoring either continuously or once per cycle. These instruments can also characterize PCR products by their melting characteristics, e.g., to discriminate singlebase mutations from a wild-type sequence. The recently designed Mx4000™ Multiplex Quantitative PCR System (Strategene, La Jolla, CA) can generate and analyze data for multiple fluorescent real-time QPCR assays.