Gel Electrophoresis:
A tool for estimating RT/PCR fragment sizes

RT/PCR

RT/PCR is used to amplify and examine a 743 base pair (bp) region of VP2.

RT/PCR Products

The products of RT/PCR are identified by differential migration due to fragment size with electrophoresis.  10 ul of RT/PCR product is added to 5 ul Orange G dye and placed in a 1% agarose gel. The RT/PCR products are stained with ethidium bromide (10mg/ml).  The gel is viewed under UV light.  A 100 bp ladder was used as a marker.

The RT/PCR products are digested with the use of the restriction enzymes BstN I and Mbo I.  The RT/PCR - RFLP assay examines the 743 bp fragment for the presence and location of multiple BstN I or Mbo I restriction sites by comparing the sizes of resulting restriction fragments.

RT/PCR-RFLP Products

The restriction fragments are examined on a 2.5% agarose gel followed by staining with a compound that will fluoresce under ultra-violet light. 

Five different RFLP's were detected for serotype 1 vaccine viruses and previously described strains of IBDV (Table).  We have observed greater genetic heterogeneity in the 743 bp fragment of VP2 examined among field strains when compared to vaccine strains.