Nucleotide analysis of restricted fragment DNA extremities for characterizing the restriction enzyme cutting activity

ABSTRACT

Nucleotide analysis of restricted fragment DNA extremities at definite cut sizes identified there were 3 extremities flat; cross with DNA 5’; cross with DNA 5’. This study presented a method which able to characterize unknown restriction enzymes by analyzing the cutting extremities of restricted fragments. The method is based on Sanger sequencing of DNA around the cut site and non-radioactive chemical. Klenow reaction had two active characters analyzing DNA 5’3’ and exonucleasa 3’5’, allowing to determine the type of cutting of restrictive enzymes. This results allowed to identify the cut sizes of two new enzymes for Sml1, the cut sites occurs within the recognition nucleotide sequence; BciVI recognizing a non-palindrome sequence and cuts outside this sequence.