in three bands (Figure 5-23). The 700bp and 300bp bands were cloned and sequenced

and were found to be identical except for the fact that the 300bp product had a

polyadenylated tail 400 bases upstream (in an adenine-rich region) of the polyadenylated

tail belonging to the larger product. Cloning of the 1,200bp product was unsuccessful.






 O 1500



 S600

 S300








Figure 5-23. Results of 3' RACE performed on liver, showing multiple products obtained.

 In addition, it was also observed that further amplifying a PCR product obtained

via RACE in order to increase the yield of this product, often resulted in the formation of

smaller products. For example, the 3' RACE performed in order to extend I4_degenerate

resulted in two products which showed up as an intense 300bp band and a faint 700bp

band (which was the expected product size) (Figure 5-24A). The gel piece containing the

larger amplicon was purified and then subj ected to an additional round of PCR (Figure 5-

24B). The result were three bands: an expected one at 700bp (which was sequenced and

corresponded to the sequence containing a full-length 3' end (I4_3R)), and two smaller

bands approximately 300 and 200bp in size. This could mean that there is some form of