Restriction endonucleases are categorized into various types based on their composition, cofactor requirements, target sequence nature, and cleavage positions. Sticky ends, linkers, and adaptors are important tools for creating recombinant DNA, while different enzyme types, such as Type I, II, and III, exhibit distinct characteristics in recognition and cleavage of DNA. The document also details the nomenclature of restriction enzymes based on their bacterial origins and provides examples of each type.

1. Restriction endonuclease:
Their types and restriction digestion

4. Restriction enzyme digestion

5. STICKY ENDS ARE MORE USEFUL
1. DNA fragments with complementary sticky ends can combine to create
new recombinant DNA.
2. Hence, it allows the creation and manipulation of DNA sequences from
different sources.
STICKY ENDS
• Also known as COHESIVE ENDS
• Sticky ends are DNA fragments with overhanging single-stranded ends
resulting from staggered cuts by restriction enzymes.

7. Linkers
• Linkers are the chemically synthesized double stranded DNA oligonucleotides
containing one or more restriction sites for cleavage by restriction enzymes.
• Linkers are ligated to blunt end DNA by using DNA ligase.
• Both the vector and DNA are treated with a restriction enzyme to develop sticky ends

8. Adaptors
• Short double-stranded oligonucleotides that carry an internal RE site with single-
stranded tails at one or both ends.
• The ends are treated with Polynucleotide kinases to restore it and ligate to vectors.
Vector

9. Nomenclature of Restriction enzymes
• Each enzyme is named after the bacterium from which it is
isolated.
• Based on Bacterial genus, species and strain
Derievation of EcoRI name
Abbreviation Meaning Description
E Escherichia Genus
co coli species
R RY13 strain
I First isolated Order of identification in this
bacteria

10. Examples of restriction endonucleases nomenclature

11. Types of Restriction Enzymes
RE is categorised into sub-types based on
1. Composition- The structural and functional complexity
of the enzyme.
2. Enzyme cofactor requirement- specific cofactors such
as ATP, Mg² , or S-adenosylmethionine for enzymatic
⁺
activity
3. The nature of their target sequence - Specific
recognition sequences on the DNA, typically palindromic,
vary between types.
4. Position of their DNA cleavage site relative to the
target sequence

12. Type I RE
• Capable of both restriction and modification activities.
• Recognize specific DNA sequences but cut at a random site far (up to 1000 bp) from the
recognition sequence.
• The co-factors S-adenosyl Methionine (SAM), ATP, and Mg2+
are required for their full
activity.
• Contains:
Two R (restriction) subunits
Two M (methylation) subunits
one S (specificity) subunit
• Example: EcoKI

13. Type II RE
• These are most commonly available and used as restriction enzymes
• They are composed of only one subunit.
• Their recognition sites are usually undivided, palindromic and 4-8 nucleotides in
length.
• They recognize and cleave DNA at the same site.
• They do not use ATP for their activity.
• They usually require only Mg2+
as a cofactor
• Example: EcoRI,
HindIII,
BamHI

14. Type IIs RE
• It is the subtype of TYPE II restriction enzyme
• Recognize specific asymmetric (non-palindromic) sequences.
• Cut DNA at a defined distance from their recognition site.
• Require Mg²⁺ as a cofactor.
• Example :
FokI RE: Recognizes 5'-GGATG-3' but cuts several
bases away from the recognition sequence.

15. Type III RE
• It recognizes two separate non – palindromic sequences that are inversely
oriented.
• They cut DNA about 20-30 nucleotide bp after the recognition site.
• These enzymes contain more than one subunit.
• Require SAM and ATP as cofactors for their activity of DNA methylation and
restriction digestion.
• Example: EcoP15I recognizes the DNA sequence 5' - CAGCAG - 3’, and
cleaves approximately 25–27 base pairs downstream from the recognition sequence.
 It requires two inversely oriented recognition sites on the DNA to cleave
effectively.
 5’------ CAGCAG ------------------------- CAGCAG ----- 3’

16. Type IV RE
 Recognize and cut modified DNA, such as methylated, hydroxy-
methylated, or glucosylated bases.
 They don't work on unmodified DNA.
 Cleavage sites can be variable and depend on the specific enzyme and
modification.
 Cleavage takes place ~30 bp away from one of the restriction site.
 Recognition sequences are not well defined.
 Cofactors such as Mg²⁺ and sometimes additional substrates like GTP
are required
 Example: McrBC Recognizes methylated cytosine residues and cuts at
random sites near the modification

17. Type V RE
•Use RNA molecules as guides to target specific DNA sequences, similar to
CRISPR-Cas systems.
•CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats
Cas9 (CRISPR-associated protein 9):
An enzyme that uses CRISPR sequences as a guide to recognize and cut at
specific strands of DNA.