Restriction Enzymes
Table 7
Isoschizomer Pairs that Can Be Used in the Analysis
of the Methylation State of DNA175Methylated sequence Cut by Not cut byMethylation at CG
Methylation at GATC
Methylation at CCWGG
Methylation at CNG
cmsCGG MspI HpalI or HaplI
ccmsCGGG XmaI or Cfr9I SmaI
TcmsCGGA AcclII BspMII
GATmsC MboI Sau3AI
GGTACmsC KpnI Asp718
GGWCmSC AflI AvalI or Eco47I
Gm6ATC Sau3AI MboI or NdelI
RGm6ATCY XholI or BstYI MflI
TCCGGm6A BspMII or MroI AcclII
cmSCWGG BstNI or MvaI EcoRII
cmSCWGG BstNI or MvaI EcoRII
GGTAm5CC KpnI Asp718
Tm5CCGGA AcclII BspMIItions that preserve the native structure of DNA are used to separate
double-stranded DNA, denaturing conditions (e.g., electrophoresis at
alkaline pH or in the presence of chaotropic agents) are used to analyze
single-stranded DNA fragments. Depending on the size of the frag-
ments to be separated, different gel systems are used (Table 4).
- Analytical Applications
Tables 8-11 summarize the recognition sequences and sites of cleav-
age of commercially available restriction enzymes. Main suppliers of
restriction and modification enzymes are listed in Table 12.
4.1. Restriction Mapping of DNA Molecules
A set of different restriction endonucleases can be used to produce
a map of their recognition sequences in a given DNA. Such a map allows
the positioning of functional sites and the location of hybridization
probes, as well as the unambiguous identification of this DNA. In rare
cases, for example with some hemoglobinopathias, point mutations
can be identified by restriction mapping if they impair the-cleavage of
one of these enzymes. To obtain an informative map, one should choose
restriction enzymes that cut the DNA at least two and up to about ten
times within the stretch of interest. An unequivocal assignment of the