Joining together of DNA molecules from two different species that are inserted into a host organism to produce new genetic combinations (i.e recombinant DNA) that are of value to science, medicine, agriculture and industry RESTRICTION ENDONUCLEASES AND OTHER ENZYMES USED IN GENETIC ENGINEERING • Also called restriction enzymes or molecular scissors • They are enzymes that cut DNA at or near specific recognition nucleotide sequences known as restriction sites • They are found in bacteria and archaea • A bacterium uses a restriction enzyme to defend against bacterial viruses called bacteriophages or phages. • When a phage infects a bacterium, it inserts its DNA into the bacterial cell so that it might be replicated. Restriction enzyme prevents replication of the phage DNA by cutting it into many pieces • The bacterial DNA is prevented from the action of the restriction enzyme by another set of enzymes known as DNA methyltransferases or methylases • DNA methylase is synthesized by the bacteria. It adds methyl to the DNA sequence of the bacteria for protection against restriction enzyme • The combination of restriction endonuclease and methylase is called RESTRICTION-MODIFICATION SYSTEM

2. • Joining together of DNA molecules from two
different species that are inserted into a host
organism to produce new genetic
combinations (i.e recombinant DNA) that are
of value to science, medicine, agriculture and
industry

3. RESTRICTION
ENDONUCLEASES AND
OTHER ENZYMES USED
IN GENETIC
ENGINEERING

4. • Also called restriction enzymes or molecular
scissors
• They are enzymes that cut DNA at or near specific
recognition nucleotide sequences known as
restriction sites
• They are found in bacteria and archaea
• A bacterium uses a restriction enzyme to defend
against bacterial viruses called bacteriophages or
phages.

5. • When a phage infects a bacterium, it inserts its DNA
into the bacterial cell so that it might be replicated.
Restriction enzyme prevents replication of the phage
DNA by cutting it into many pieces
• The bacterial DNA is prevented from the action of the
restriction enzyme by another set of enzymes known
as DNA methyltransferases or methylases
• DNA methylase is synthesized by the bacteria. It adds
methyl to the DNA sequence of the bacteria for
protection against restriction enzyme
• The combination of restriction endonuclease and
methylase is called RESTRICTION-MODIFICATION
SYSTEM

6. Restriction Enzymes
Enzymes
AluI
BamHI
EcoRI
HindIII
KpnI
NotI
Sau3A
Sources
Arthrobacter luteus
Bacillus amyloliquefaciens
Escherichia coli
Haemophilus influenzae
Klebsiella pneumoniae
Nocardia otitidis
Staphylococcus aureus

7. Nomenclature
• Each restriction endonuclease is named after the
bacterium from which it was isolated using a
naming system based on bacterial genus, species
and strain
• The first letter of the generic name and the first
two letters of the specific name of the organism is
used to form a three letters abbreviation. For
example, Escherichia coli- Eco and Haemophilus
influenza- Hin
• The strain is identified by the next letter. For
example, EcoR i.e from strain Ry13

8. • The order of identification of the enzyme in a
bacterium is designated with a Roman letter. For
example, EcoRI ie first endonuclease to be
discovered in Escherichia coli
• EcoRI is from Escherichia (E) coli (co), strain Ry13
and first endonuclease (I) to be discovered.
• HindIII is from Haemophilus (H) influenzae (in),
strain Rd (d) and the third endonucleases (III) to
be discovered.

9. Recognition Sequences
• All restriction endonucleases require specific
nucleotide sequences to carry out cleveage. These
sequences are known as recognition sequences
• Characteristics of recognition sequences:
1. Length of recognition sequences varies. For
example, EcoRI recognizes sequence of six base pair
in length while NotI recognizes a sequence of eight
base pair in length

11. 2. Different restriction enzymes have the same
recognition sequence. These are better known
as isoschizomers. For example, SphI and BbuI
restriction enzymes has the same recognition
sequence.

13. 3. Recognition sequence of one enzyme contains
recognition sequence for the other. For example,
the restriction enzyme BamHI also contains the
recognition sequence for another enzyme
Sau3AI.

15. 4. Recognition sequences are palindromic,
meaning the base sequence reads the same
backwards and forwards. There are two types of
palindromic sequences that can be possible in
DNA:

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

17. TYPES
• Naturally occurring restriction endonucleases
are categorized into four groups (Types I, II III,
and IV) based on their composition and
enzyme cofactor requirements, the nature of
their target sequence, and the position of
their DNA cleavage site relative to the target
sequence.

18. Type I
• Type I restriction enzymes were the first to be
identified and were first identified in two
different strains (K-12 and B) of E. coli.
• These enzymes cut at a site that differs, and is
a random distance (at least 1000 bp) away,
from their recognition site.

19. Type II
• They recognize and cleave DNA at the same
site
• They are the most commonly available and
used restriction enzymes
• Some examples of the type II restriction
endonucleases include BamHI, EcoRI, EcoRV,
and Haelll

20. Type III
• They cut DNA about 20-30 base pairs after the
recognition site.

21. Type IV
• Type IV enzymes recognize modified, typically
methylated DNA

22. Cleavage Patterns
The restriction enzymes generate three different types
of ends after cleavage within the recognition
sequences.
• 1. Sticky ends: The restriction enzymes cut
asymmetrically within the recognition sequences
leaving single -stranded overhangs and are also
called as cohesive ends. There are two types of sticky
ends.
• a) 5’ overhangs: The restriction enzymes cut
asymmetrically within the recognition sequence in
such a way that it leaves an overhanging short single
stranded 5’ end. For example,

24. b) 3’ overhangs: The restriction enzymes cut
asymmetrically within the recognition sequence
in such a way that it leaves an overhanging
single-stranded 3’ ends. For example,

26. • But, the limitation of the sticky end is that they
only stick to fragments which are compatible.
For example, two EcoRI fragments can join
together but EcoRI cannot join with any other
fragments produced by other restriction enzyme.
• 2. Blunt ends: The restriction enzymes cleaves
exactly on the same site on both the strands
without leaving any overhangs. For example,

28. • The DNA fragments with sticky ends are
particularly useful for recombinant DNA
experiments as the single-stranded sticky ends
can easily pair with any other DNA fragment
having complementary sticky ends.
• A selected list of enzymes, recognition
sequences and their products having either
sticky or blunt ends formed are given below

29. List of Restriction Enzymes, Recognition
Sequences and its Products.
Enzymes Recognition
sequence
1. Alu1 5' AGCT
3' TCGA
2. BamH1 5' GGATTC
3' CCTAAG
3. EcoRI 5' GAATTC
3' CTTAAG
4. HindIII 5' AAGCTT
3' TTCGAA
Cuts
5'---AG CT--- 3'
3'---TC GA---5'
5'---G GATTC---3'
3'---CCTAA G
5'---G AATTC---3'
3'---CTTAA G---5'
5'---A AGCTT---3'
3'---TTCGA A---5'

30. 5. KpnI 5' GGTACC
3' CCATGG
6. NotI 5'CGGCCGC
3'GCCGGCG
7. PstI 5' CTGCAG
3' GACGTC
8. Sau3A 5' GATC
3' CTAG
5'---GGTAC C---3'
3'---C CATGG---5'
5'---GC GGCCGC---3'
3'---CGGGGG CG---5'
5'---CTGCA G---3'
3'---G ACGTC---5'
5'------- GATC---3'
3'----CTAG -----5'

31. DNA LIGASE

32. • This enzyme repairs broken DNA by joining two
nucleotides in a DNA strand. It is commonly
used in genetic engineering to do the reverse of
a restriction enzyme, i.e. to join together
complementary restriction fragments.
• The sticky ends allow two complementary
restriction fragments to anneal, but only by
weak hydrogen bonds, which can quite easily be
broken by gentle heating. The backbone is still
incomplete.

33. • DNA ligase completes the DNA backbone by
forming covalent bonds. Restriction enzymes
and DNA ligase can therefore be used
together to join lengths of DNA from
different sources.

34. CLONING VEHICLES
(TYPES AND
CONSTRUCTION)

35. CLONING
• Production of multiple copies of a specific
DNA molecule
• Also used to describe the production of
genetically identical cells or even organisms

36. CLONING VEHICLE
• Also known as cloning vector
• A length of DNA that carries the gene to be
cloned into a host cell
• A cloning vehicle is needed because a length of
DNA containing a gene on its own can not
actually do anything inside a host cell. Since it is
not part of the cell’s normal genome it won’t be
replicated when the cell divides, it won’t be
expressed, and in fact it will probably be broken
down

37. FEATURES
• It must be big enough to hold the gene to be
cloned
• It must be circular (or more accurately a
closed loop), so that it is less likely to be
broken down
• It must contain control sequences, so that the
gene will be replicated or expressed
• It must contain marker genes, so that cells
containing the vector can be identified

38. TYPES

39. PLASMID VECTORS
• A plasmid is a DNA molecule that can
replicate independently of the chromosomal
DNA
• Plasmids are circular molecules of DNA
• Found in all three major domains: Archaea,
Bacteria, and Eukarya
• Plasmid vectors are used to clone DNA ranging
in size from several base pairs to several
thousands of base pairs (100bp -10kb).

40. BACTERIOPHAGE LAMBDA
• Phage lambda is a bacteriophage or phage, i.e. bacterial
virus, that uses E. coli as host.
• Its structure is that of a typical phage: head, tail, tail fibres.
• Lambda viral genome: 48.5 kb linear DNA with a 12 base
ssDNA "sticky end" at both ends; these ends are
complementary in sequence and can hybridize to each
other (this is the cos site: cohesive ends).
• Infection: lambda tail fibres adsorb to a cell surface
receptor, the tail contracts, and the DNA is injected.
• The DNA circularizes at the cos site, and lambda begins its
life cycle in the E. coli host

41. TYPICAL CLONING EXPERIMENT
THE CLONING OF DOLLY

42. • Dolly is the first sheep created by a cloning
expriment carried out by Wilmut and his
colleagues at Roslin Institute, Scotland.
• Cells from an adult Finn Dorset breed sheep’s
mammary gland was removed. Finn Dorset is a
pure white breed of sheep.
• The cells were grown in a tissue culture.
• The cells were starved of important nutrients.
They stopped growing, dividing and became
quiescent.

43. • An oocyte was collected from a Scottish
Blackface ewe (Ewes are female sheep)
• The Scottish Blackface breed is a common
breed of sheep in Scotland easily identified by
its black face
• The nucleus of the oocyte was removed while
nucleus ‘donor’ collected from the quiescent
mammary cell was injected in the enucleated
oocyte

44. • The nucleus was allowed to fuse with the
cytoplasm of the enucleated oocyte and
transferred into the reproductive chamber of a
Blackface ewe
• After 148 days, a normal length of time for the
Finn Dorset breed of sheep, Dolly was born as a
healthy, normal looking Finn Dorset
• This proves that Dolly is not a product of a sneaky
mating. This is because from the genetics of
sheep breed, a Blackface breed can not produce a
Finn Dorset breed

45. • A DNA fingerprint was conducted which
showed that Dolly’s DNA matched the cells
from the tissue culture, not the cells from the
ewe that gave birth to her.