Restriction enzymes cut DNA molecules at specific recognition sites. Restriction mapping involves digesting an unknown DNA segment with restriction enzymes and analyzing the fragment sizes to determine the locations of restriction sites. One method involves single and double digestions with two enzymes followed by gel electrophoresis to separate the fragments by size. By comparing the fragment patterns between single and double digestions, the positions of each restriction site can be mapped, generating a restriction map of the DNA segment. Restriction mapping was previously important for characterizing cloned DNA but is now easier using DNA sequencing, though analysis of restriction sites remains useful for comparing chromosomal organization between strains.

1. Restriction Mapping
SUNIL BHANDARI
BALKUMARI COLLEGE

2. Restriction enzymes:
• Restriction enzymes are one of the most important tools in
the recombinant DNA technology.
• These are DNA cutting enzyme present in bacteria
(prokaryotes) that recognizes the specific sites in the DNA,
called restriction sites and make a DNA double strand break
at or near recognition sites.
• After cutting, the newly produced DNA ends will have either a
blunt ended or sticky (staggered) end.

3. Restriction Enzyme/Endonuclease
“Also known as molecular scissor”
• Type I enzymes: They are complex multi subunit combination
restriction and modification enzymes that cut DNA at random far
from their recognition sequences
• Type II enzymes: Cut DNA at defined positions close to or within
their recognition sequences
• Type III enzymes: They cleave outside of their recognition
sequences and require two such sequences in opposite
orientations within the same DNA molecule to accomplish
cleavage they rarely give complete digests
• Type IV enzymes: Recognize modified, typically methylated DNA
and cleave at that region

4. Example of restriction enzymes and their corresponding restriction sits

5. Restriction Mapping
• Restriction mapping is a method used to map an unknown segment of DNA
by breaking it into pieces and then identifying the locations of the
breakpoints. This method relies upon the use of proteins called restriction
enzymes, which can cut, or digest, DNA molecules at short, specific sequences
called restriction sites After a DNA segment has been digested using a
restriction enzyme, the resulting fragments can be examined using a laboratory
method called gel electrophoresis, which is used to separate pieces of DNA
according to their size.
• One common method for constructing a restriction map involves digesting the
unknown DNA sample in three ways. Here, two portions of the DNA sample
are individually digested with different restriction enzymes, and a third
portion of the DNA sample is double digested with both restriction enzymes
at the same time
• Next, each digestion sample is separated using gel electrophoresis, and the
sizes of the DNA fragments are recorded. The total length of the fragments in
each digestion will be equal However, because the length of each individual
DNA fragment depends upon the positions of its restriction sites, each
restriction site can be mapped according to the lengths of the fragments.

6. • The information from the double digestion is particularly
useful for correctly mapping the sites. The final drawing of the
DNA segment that shows the positions of the restriction sites is
called a restriction map
• It is possible to determine the position of restriction sites on a
DNA fragment by digesting it with various restriction enzymes,
singly and in combination, and analyzing the fragment sizes
obtained.
• Restriction mapping used to be a key procedure for characterizing
a cloned fragment of DNA, but this is now more easily done by
DNA sequencing.
• Nevertheless, analysis of restriction sites (or fragment sizes) is still
useful, for example in comparing the chromosomal organization of
different strains.

9. Assignment:01
Consider, a 4kb DNA is digested with two restriction enzymes (HindIII and BamHI).The
digested product is run through gel electrophoresis and DNA bands of following size is
obtained. Draw the restriction map representation following digestion.
Ans:
See
last
page

10. Application of
Restriction Mapping:
1. Identification of
restriction sites
2. Insertion and deletion
study of a gene
3. Insert analysis during
cloning
4. Verification of the size
of the insert during
cloning
5. Mutation studies

11. Thank you…