Restriction enzymes are enzymes that cut DNA at specific recognition sequences. There are three main types of restriction enzymes - Type I, II, and III. Type II restriction enzymes are the most commonly used in molecular cloning and DNA manipulation. They recognize short palindromic DNA sequences of 4-8 base pairs and cleave DNA within or nearby these recognition sites. Restriction enzymes have many applications including DNA sequencing, DNA libraries, and recombinant DNA techniques which enable genetic engineering.

1. Restriction
Endonuclease
MCB5706:BIOMOLECULAR
ENGINEERING
Prof Tan Wen Siang

2. History and
Definition
Type I
Type II
Restriction Types
enzymes Type III
Artificial RE
Recombinant
DNA
genotype DNA
Application
DNA
sequencing
DNA storage –
libraries

5. DNA Palindromes
 The palindrome in which
the same forward and backwards are
on a single strand of DNA strand, as
in GTAATG
 The palindrome is
also a sequence that reads the same
forward and backwards, but the
forward and backward sequences are
found in complementary DNA strands
(GTATAC being complementary to
CATATG)
 Inverted repeat palindromes are
more common and have greater
biological importance than mirror-
like palindromes.

7. Back

9. HindIII
EcoRI
AluI
HaeIII

10. Back

12. Example: EcoR1
Genus: Escherichia
Species: coli
Strain: R
Order discovered: 1
Back

16.  Large enzymes
 Combination restriction-and-modification
 Cleave outside of their recognition
sequences
 Require two recognition sequences in
opposite orientations within the same
DNA molecule

17.  Cleave only normal and modified DNA
(methylated, hydroxymethylated and
glucosyl-hydroxymethylated bases).
 Recognition sequences have not been well
defined
 Cleavage takes place ~30 bp away from one
of the sites

18.  generated by fusing a natural or engineered DNA
binding domain to a nuclease domain
 can target large DNA sites (up to 36 bp)
 can be engineered to bind to desired DNA
sequences
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19.  Application in cut and paste DNA make genetic
engineering possible with coordination of Ligase Enzyme
 Rrestriction site could be added to primers
(forward&Reverse) that will be used for amplifying gene
 Both vector and amplified gene will be cut by RE to
produce sticky ends
 In ligation mixture the target gene will be inserted to vector
with the aid of sticky ends and DNA ligase

20. i. Designing of primer with
restriction site
ii. Amplifying target gene by
PCR and designed primer
iii. Double digestion of vector
and amplified gene by same
REs
iv. Ligation of gene and vector
v. Transformation
vi. Expression
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21. ( ) ingle ucleotide olymorphisms
distinguish gene alleles by specifically
recognizing single base changes in DNA
 By use of SNP the restriction enzyme can
be used to
without the need for expensive gene
sequencing

22. DNA molecule 1 differs from DNA
molecule 2 at a single base-pair Back
location (a C/T polymorphism)

23.  (RFLP) Restriction Fragment Length
Polymorphism
 Molecular biologists have identified
regions of the human genome where
restriction fragment lengths are highly
variable between individuals
 Electrophoresis of these RFLP’s produce
different patterns of DNA bands

25. Restriction Enzymes for RFLP
_
DNA is negatively
charged from the
phosphate backbone
+
Visualize DNA with ethidium
bromide – fluoresces ONLY
when bound to DNA