Polyacrylamide gels are defined by two parameters: %T and %C. The %T refers to the total amount in grams of acrylamide and N,N'-methylene-bisacrylamide (a common crosslinker, usually abbreviated as bis) in 100 mL solution. The %C refers to the proportion of the total monomers (acrylamide plus bis) that is the crosslinker (bis). The %C can also be expressed in another format as the ratio of acrylamide to bis; for example, 2%C is equivalent to an acrylamide:bis ratio of 49:1.
The probability of detecting sequence variations in a PCR product is higher if the %C is lower (9). Low levels of crosslinking produce large pores in the gels, and thus allow efficient separation of bulky single strand conformers. We use a nondenaturing gel of 10%T/1%C as a starting point in conjunction with a conventional vertical electrophoresis system (e.g., SE600 from Hoefer) and a medium-sized gel (e.g., 16 × 14 cm).
Large-sized gels of 5%T and 1−2%C are also commonly used together with conventional electrophoresis systems for manual sequencing. Another commercially available gel matrix called Mutation Detection Enhancement (MDE) Gel is also widely used for SSCP analysis. It is a polyacrylamide-like matrix and is claimed to be very sensitive to DNA conformational changes. The buffer most commonly used in SSCP analysis is Tris-borate-EDTA buffer with an alkaline pH.
However, low pH buffer (e.g., Tris-MES-EDTA, pH 6.3) can still maintain very high sensitivity of detecting sequence variations for PCR fragments up to 800 bp in length.
No comments:
Post a Comment