Analysis of 5' RACE Results: Follow

Analysis of 5' RACE Results: Following PCR, products may be analyzed by agarosegel electrophoresis (1% to 2%) and ethidium bromide staining. Band intensity and sizedistribution of resulting products depends on the specificity of GSPs used for cDNA synthesisand PCR, the complexity and relative abundance of target cDNA, and the PCRconditions used. Amplification products may vary from a single specific band to multiplediscrete products to a broad diffuse smear. Incomplete cDNA synthesis, aberrant primingof GSPs during first strand synthesis or PCR, mispriming by the anchor primer, as wellas primer-dimer and other PCR artifacts may contribute to the complexity of productsobtained by 5' RACE. Identification of specific product bands may be complicated by thepresence of nonspecific products that are dependent on both reverse transcription anddC-tailing (36). If sequences are available for use as internal probes, it is strongly recommendedthat Southern blot analysis be used to identify specific product bands.Specific products can also be identified using a diagnostic restriction endonucleasedigestion if the amplified cDNA sequence contains a known restriction site.