The Action of Endonucleases and Methylases Electrophoretic Analysis of DNA Restriction Fragments Kotam J. Kaneko, John M. Burke, and Lawrence J. ~ a ~ l a n ' Williams College, Williamstown, MA 01267 The restriction endonucleases have played an essential role in the development of the field of recombinant DNA technology. These enzymes catalyze the hydrolysis of double-stranded DNA with a high level of specificity to generate a well-defined series of polynucleotides called restriction fragments. The sequence specificity has been employed to generate fragments for the construction of recombinant DNA molecules and for DNA sequence analysis. This paper presents the procedures used in endonuclease dieestion and the electrophoretic analysis of the restriction fragments. In fact, two related experiments are outlined. One involves the use of a restriction~endonucleaseto generate a set of welldefined fragments which then may be used as molecular weight standards for gel electrophoresis. With this as an aid, the cleavage pattern of the DNA with other endonucleases can be analyzed. The second experiment employs endonucleases to monitor the protection afforded the DNA upon methylation and illustrates the differences obtained upon in vitro and i n viuo methylation.
ties, in the most common type, type 11, the restriction and methylation activities are actually the result of two different enzymes (8,9). The EcoRI system is the best characterized one, where the restriction enzyme (10, 11) is a dimer of identical subunits and the methylase (12,131 is a monomer. Specifically, the methylase catalyzes the transfer of methyl groups from S-adenosylmethionine (SAM) to the adenines in the EcoRI recognition sequence indicated with an asterisk in the structure ahove. Since the recognition sequence is a palindrome, the methylase can react with bases in both strands of the DNA. However, the methylase adds methyl groups in a progressive fashion. The target DNA then may he fully methylated (both strands are modified and neither restriction nor methylation can occur), hemimethylated (one strand is methylated, which is the result of replication of fully methylated DNA, and is the usual substrate for the methylase) or unmethylated (which is most likely to be the substrate of the restriction enzyme since this enzyme does not bind to methylated sites).
Restrlctlon Endonucleases Restriction endonucleases are enzvmes that recoenize specific rrquences within double-stranded DNA and h?;drolyze the ph~~rphodiester bonds within or near their ~ a r t i c u . lar recognition sequences (1, 2). These sequences
