PCR is used to amplify a DNA region to be analyzed. The PCR products are diluted in a loading solution that contains formamide and indicator dyes (e.g., bromophenol blue and xylene cyanol FF). The diluted PCR products are heated to over 90°C for a few minutes to denature the products into single strands and then cooled immediately in ice water.
High concentration of formamide (a chemical denaturant) in the loading solution and immediate cooling are required to keep a sizable proportion of the products in single strands. The denatured PCR products are then loaded onto a nondenaturing polyacrylamide gel (i.e., without chemical denaturants). Samples diluted in formamide are denser than the buffer and will sink to the bottom of the wells. Separation of the single strands is achieved by electrophoresis.
The duration of electrophoresis depends on the gel composition, voltage applied, buffer/gel temperature, and the size and base composition of the PCR products. After electrophoresis, the DNA bands are visualized by silver staining or, less commonly, SYBR Green II. Though less popular now, PCR products can also be radioactively labeled and the bands detected by autoradiography. Banding patterns of samples are compared.
The presence of different banding patterns among samples indicates that sequence variations exist in the DNA sequence amplified by the two PCR primers. Silver-stained gels can be dried in a gel dryer and the dried gels kept for permanent records if so desired.
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