Novel 2 to 4 Base Pair Specific Enzymes for PCR Fingerprinting

A large number of RFLP variants in plant and animal diseases can be detected using the polymerase chain reaction (PCR) followed by restriction endonuclease cleavage. However, the resolution of PCR fingerprinting techniques is limited by rather long (4 to 6 base pair) DNA sequence specificities of most bacterial restriction enzymes. Bacterail restriction sites occur, on average, once every 4^4 to 4^6 b.p.; and most such sites are not polymophic. In contrast, Chlorella virus (Cvi) endonucleases have short (2 to 4 base pair) sequence specificities and therefore ideal for find structure gene mapping applications such as PCR-coupled mapping. Cvi enzymes cut once every 4^2 to 4^4 b.p. provide a simple, inexpensive means of rapidly fingerprinting genetic variants of economically important plant and animal diseases. Phase I studies are proposed to purify five different Cvi endonucleases which have short (2 to 3 base pair) sequence specificities: CviJI (RG CY), CviNYSI (CC), Cvi109I (YC GR), CviNY2AI (R AG), and CviGI (CGR). These frequently cutting Cvi endonucleases will be tested in PCR fingerprinting of an agriculturally important viral plant pathogen, Barley Yellow Dwarf Virus (BYDV)Cvi endonucleases represent a major new class of fine structure gene mapping tool.