Role of restriction enzymes Examples of restriction enzymes Cutting of DNA by restriction enzymes restriction mapping

1. R E S T R I C T I O
N E N Z Y M E S
A N D
M A P P I N G
H A D I A K H A D I J A
R O L L # 1 2
C O U R S E : G E N O M I C S
P R E S E N T E D T O D R . S A D A F N A Z
S C H O O L O F B I O L O G I C A L S C I E N C E S
U N I V E R S I T Y O F T H E P U N J A B
L A H O R E
Dated : September 15,
2021

2. Restriction Enzymes
• Enzymes (Endonucleases)
that recognize specific
sequence within the DNA and
cut the DNA at restriction sites
• Also called as Molecular
Scissors
• Present in bacteria as a
natural defense mechanism
against foreign DNA
• Bacterial DNA is not cut by
these enzymes as their DNA is
methylated

3. T H R E E
S C I E N T I S T S
W E R E
A W A R D E D
W I T H N O B E L
P R I Z E F O R
D I S C O V E R Y
O F
R E S T R I C T I O
N E N Z Y M E S

4. Application
• Recombinant DNA Technology
• Restriction Endonucleases

5. Restriction
Endonucleases
• Make cuts at internal phosphodiester
bonds
• Three main types of restriction
endonucleases (Type I, II & III)
• Type I and III cut the DNA at different
positions relative to the recognition
sequence
• Type II enzymes cut DNA always at the
same place, either within the recognition
sequence or very close to it

6. Type II Restriction Endonucleases
• Digestion of DNA with a type II enzyme gives a reproducible set
of fragments whose sequences are predictable if the sequence
of the target DNA molecule is known.
• For example, Type II enzyme called EcoRI cuts DNA only at the
hexanucleotide 5ʹGAATTC3ʹ.
• Many enzymes have hexanucleotide target sites, but others
recognize shorter or longer sequences.
• Most enzymes cut within the recognition sequence, but a few,
such as BsrBI, cut at a specified position outside of this
sequence.

8. DNA cutting by Restriction Enzymes
• Restriction enzymes cut DNA in two different ways.
• Many make a simple double-stranded cut, giving a blunt or
flush end (symmetric cuts)
• others cut the two DNA strands at different positions, so that the
resulting DNA fragments have short, single-stranded overhangs
at each end giving sticky or cohesive ends (asymmetric
cuts)

9. DNA cutting by Restriction Enzymes
• Some sticky end cutters give 5ʹ-overhangs (for example,
Sau3AI, HinfI), whereas others leave 3ʹ-overhangs (for
example, PstI)

10. DNA cutting by Restriction Enzymes
• Some pairs of restriction enzymes have different recognition
sequences but give the same sticky ends (Isoschizomers)
• For example, Sau3AI and BamHI both give a 5ʹ-GATC-3ʹ sticky
end, even though Sau3AI has a four-base-pair recognition
sequence and BamHI recognizes a six-base-pair sequence

11. Restriction
Mapping
• Physical mapping by direct
examination of DNA
molecules or chromosomes
• The simplest direct
examination methods are
those that enable the
positions of restriction sites to
be located in a DNA molecule.
This process is called
restriction mapping.

12. Construction of Restriction Maps
• Partial digestion-a single enzyme is used but the digestion does
not go to completion,
• because the reaction is incubated only for a short time or a
suboptimal incubation temperature is used.
• DNA molecule is digested with two different restriction enzymes
(double digestion) that recognize different target sequences
• Restriction map is drawn by comparing the sizes of fragments
produced by digestion of DNA molecules.
• Example of restriction enzymes EcoRI and BamHI

15. https://doi.org/10.1038/s41598-021-91264-6

16. Reference
• BOOK: GENOMES 4 BY T. A. BROWN

17. T H A N K Y O U