DETECTION AND ANALYSIS OF NUCLEIC ACIDS - A Guide to Methods in the Biomedical Sciences - page 30

enzyme EcoRI (isolated from the bacterium Escherichia coli) recog-
nizes the DNA base sequence GAATTC and cuts between the G and A
(on the opposite strand the sequence is CTTAAG, and the cut occurs
between the A and G on that strand). Restriction enzymes are often
classified by the number of nucleotides they recognize. Because RI

recognizes a 6 base pair sequence, it is called a “6-cutter”. Most restric-
tion enzymes recognize 4, 6 or 8 bp sequences, although some have
longer recognition sites. The longer and more specific the sequence
recognized by a restriction enzyme, the fewer of these sites that are
expected to be present in any given stretch of DNA. While the majority
of restriction enzymes recognize DNA sequences irrespective of their
methylation status, some restriction enzymes recognize a sequence mo-
tif in unmethylated but not methylated DNA.
Restriction enzymes are a basic tool in the analysis of DNA (restric-
tion mapping) and recombinant DNA technology. Restriction enzymes
are used to enzymatically digest genomic DNA into fragments before
electrophoretic separation and Southern blotting, to generate fragments
of DNA for cloning into vectors, and production of small fragments from
a gene for making probes for Southern or Northern blots. DNA can be
digested to completion using restriction enzymes or, for the production
of genomic libraries where large stretches of DNA are desired, partial
digests can be carried out. Partial digestion means that the enzyme is
used in sub-optimal concentrations and that the DNA is incubated with
the enzyme for a limited period of time. In this way, large pieces of DNA
with internal sites that have not been cut by the restriction enzyme are
generated and can be used for cloning purposes.

Eco

Restriction fragment length polymorphism (RFLP)

RFLPs are a type of length polymorphism in the genomes of homolo-
gous segments of DNA of different individuals that have been digested
with a restriction enzyme. The digested DNA is resolved on agarose
gels, and specific probes are used to identify regions of the DNA that
exhibit length polymorphisms. The use of RFLPs to help map genes
involved in human diseases was first suggested by David Botstein (13),
and is now widely used not only to help map genes that contribute to
diseases, but in forensics for the precise identification of DNA samples
(see below).

Single nucleotide polymorphisms (SNP)
SNPs are single base pair variations in the DNA sequence of an in-
dividual gene. They occur in the human population at a relatively high