Download enzymes and vectors

RESTRICTION ENZYMES
• Restriction Enzymes scan the DNA code
• Find a very specific set of nucleotides
• Make a specific cut
RESTRICTION ENZYMES
• Restriction enzymes, also called restriction endonucleases, recognize,
bind to specific sequences in double-stranded DNA, and cleave the
DNA.
• They are usually isolated from bacteria.
• The role of these enzymes in bacteria is to "restrict" the invasion of
foreign DNA by cutting it into pieces.
• Hence, these enzymes are known as restriction enzymes.
• The cell's own DNA is not degraded, because the sites recognized by
its own restriction enzymes are methylated.
• Many restriction enzymes have been purified and characterized.
• The names of restriction enzymes consist of a three-italic-letter
abbreviation for the host organism.
• For example, restriction enzyme EcoRⅠis from Escherichia coli.
• The first three letters in the name of the enzyme consist of the first
letter of the genus (E) and the first two letters of the species (co), which
are followed by a strain designation (R) and a roman numeral (Ⅰ)to
indicate the order of discovery.
NOMENCLATURE OF RESTRICTION ENZYME
• Each enzyme is named after the bacterium from
which it was isolated using a naming system based
on bacterial genus, species and strain.
For e.g EcoRI
Derivation of the EcoRI name
Abbreviation
Meaning
Description
E
Escherichia
genus
co
coli
species
R
RY13
strain
I
First identified
order of identification
in the bacterium
Restriction enzyme nomenclature
Why the funny names?
• EcoRI –
• BamHI –
• DpnI –
• HindIII –
• BglII –
• PstI –
• Sau3AI –
• KpnI –
Escherichia coli strain R, 1st enzyme
Bacillus amyloliquefaciens strain H, 1st enzyme
Diplococcus pneumoniae, 1st enzyme
Haemophilus influenzae, strain D, 3rd enzyme
Bacillus globigii, 2nd enzyme
Providencia stuartii 164, 1st enzyme
Staphylococcus aureus strain 3A, 1st enzyme
Klebsiella pneumoniae, 1st enzyme
• There are three types of restriction enzymes, designatedⅠ,Ⅱ,andIII.
• TypesⅠand Ⅲ contain the activities of both the endonuclease and
methylase.
• TypeⅠ restriction enzymes cleave DNA at random sites.
• Type Ⅲ restriction enzymes cleave the DNA about 25 bp from the recognition
sequence.
• Both types of enzymes require ATP for energy supply.
• TypeⅡ restriction enzymes, require no ATP, and usually cleave the
DNA within the recognition sequence itself.
• So typeⅡ restriction enzymes have extraordinary utility in DNA
recombination.
• Many type Ⅱ restriction enzymes recognize specific sequences of 4
to 6 base pairs and cleave a phosphodiester bond in each strand in
this region.
• One unique feature of restriction enzymes is that the nucleotide
sequences they recognize are palindromic, or inverted repeats.
• It cuts one strand of the DNA double helix at one point and the
second strand at a different, complementary point.
• For example, the sequence recognized by a restriction enzyme EcoRⅠ is
GAATTC.
• In each strand, the enzyme cleaves the GA phosphodiester bond on the 5'
side of the symmetric axis.
• The arrow indicates the cleavage site.
•If the cleavage site is not at the center, the
restriction enzyme ( e.g., EcoRⅠ) will generate
cohesive ends(sticky ends), which can base-pair
with other DNA fragments cleaved by the same
restriction enzyme.
• If the cleavage site is at the center, the restriction
enzyme (e.g., HpaⅠ) will generate blunt ends
blunt end
sticky end
The specificities of several of these enzymes are
shown in Table 6-2.
Table 6-2 Commonly used restriction enzymes
Some more examples of restriction sites of
restriction enzymes with their cut sites:
HindIII: 5’ AAGCTT 3’
3’ TTCGAA 5’
BamHI: 5’ GGATCC 3’
3’ CCTAGG 5’
AluI: 5’ AGCT 3’
3’ TCGA 5’
HaeIII
HaeIII is a restriction enzyme that searches
the DNA molecule until it finds this
sequence of four nitrogen bases.
5’ TGACGGGTTCGAGGCCAG 3’
3’ ACTGCCCAAGGTCCGGTC 5’
5’ TGACGGGTTCGAGGCCAG 3’
3’ ACTGCCCAAGGTCCGGTC 5’
Once the recognition site was found HaeIII
could go to work cutting (cleaving) the DNA
5’ TGACGGGTTCGAGGCCAG 3’
3’ ACTGCCCAAGGTCCGGTC 5’
• This enzyme is used to covalently link or ligate fragments
of DNA together
• Isolated from viruses
• Also occurs in E.coli and eukaryotic cells
• It also participates in DNA repair process
• DNA ligase catalyses the formation of phosphodiester bond between
the 5’-phosphate of one strand of DNA or RNA and the 3’-hydroxyl of
another.
• The DNA ligase used in molecular cloning differ in their abilities to
ligate substrate,such as blunt ended duplex DNA:RNA hybrid or
ssDNAs.
MECHANISM OF DNA LIGASE
• The mechanism of DNA ligase is to form two covalent phosphodiester
bonds between 3' hydroxyl ends of one nucleotide, ("acceptor") with the
5' phosphate end of another ("donor").
ATP is required for the ligase reaction, which proceeds in three steps:
(1) Adenylation (addition of AMP) of a residue in the active center of the
enzyme, pyrophosphate is released.
(2) Transfer of the AMP to the 5' phosphate so-called donor, formation of a
pyrophosphate bond;
(3) Formation of a phosphodiester bond between the 5' phosphate of the
donor and the 3' hydroxyl of the acceptor.
• Depending up on the source,the enzyme requires
either ATP or NAD+ as cofactors
Bacteriophage T4 DNA Ligase (ATP)
• The most widely used DNA ligase is derived from the
T4 bacteriophage.
• It is a monomeric polypeptide
• MW 68KDa is encoded by bacteriophage gene30.
• It has broder specificity and repairs single stranded
Nicks in duplex DNA, RNA or DNA:RNA hybrids.
APPLICATION
1. Ligation of cohesive ends
2. Ligation of blunt ended termini
3. Ligation of synthetic linkers or adapter
E.Coli DNA ligase
• It is derived from E.coli cell and requires NAD+ as
cofacter.
• It is a monomeric enzyme of MW 74KDa which
catalyzes the formation of the phosphodiester
bond in duplex DNA containing cohesive ends.
• This enzyme has narrower substrate specificity,
making it a useful tool in specific application.
APPLICATION
1) Ligation of cohesive ends
2) Cloning of full length cDNA
E.coli DNA ligase has been employed in a procedure
for high efficiency cloning of full length cDNA
Taq DNA ligase [NAD+ ]
• The gene encoding thermostable ligases have been
identified from several thermophilic bacteria.
• Several of this ligase have been cloned and expressed
to high levels in E.coli
• Retain their activities after exposure to higher temp for
multiple rounds.
• It is uses in DNA amplificaton reaction to detect
mutation in mammalian DNA.
T4 RNA Ligase
• T4 RNA ligase is the only phage RNA ligase that used
in genetic engineering.
• This enzyme catalyzed the phosphodiester bond
formation of RNA molecule with hydrolysis of ATP
to PPI
• It is monomeric enzyme a product of the T4 gene
63
APPLICATION
1) Production of elongated molecules
2) Modification of internal nucleotide
3) Stimulation of T4 DNA ligase activity
ALKALINE PHOSPHATASE
• Alkaline phosphatase(ALP), is a hydrolase enzyme responsible for
removing phosphate groups from many types of molecules, including
nucleotides, proteins and alkaloids.
• The process of removing the phosphate group is called
Dephosphorylation.
ENZYMES USED IN MOLECULAR
BIOLOGY
ALKALINE PHOSPHATASE
• Alkaline
phosphatase
removes 5' phosphate
groups from DNA and RNA.
• It
will
also
remove
phosphates
from
nucleotides and proteins.
• These enzymes are most
active at alkaline pH
ALKALINE PHOSPHATASE
• There are two primary uses for alkaline phosphatase in DNA
manipulations:
• Removing 5' phosphates from plasmid and bacteriophage
vectors that have been cut with a restriction enzyme. In
subsequent ligation reactions, this treatment prevents self-ligation
of the vector and thereby greatly facilitates ligation of other DNA
fragments
into
the
vector
(e.g.
subcloning).
• Removing 5' phosphates from fragments of DNA prior to labeling
with radioactive phosphate. Polynucleotide kinase is much more
effective in phosphorylating DNA if the 5' phosphate has
previously been removed
DEPHOSPORYLATED VECTOR
R.E.S WITH COMPATIBLE ENDS
POLYMERASES
• Group of enzymes that catalyses the synthesis of nucleic acid
molecules are collectively referred to as polymerases.
• Three important polymerases are given below:• DNA –dependant DNA polymerase :- that copies DNA from DNA.
• RNA dependant DNA polymerase (Reverse Transcriptase): that
synthesizes DNA from RNA.
• DNA dependant RNA polymerases: that produces RNA from DNA
DNA POLYMERASE
• A DNA polymerase is an enzyme that catalyzes the
polymerization of deoxyribonucleotides into a DNA
strand.
• DNA polymerases are best known for their role in
DNA replication in which the polymerase" reads” an
intact DNA strand as a template and uses it to
synthesize the new strand.
• The process copies a pieces of DNA.
• DNA polymerases use a mg++ for catalytic activitiy.
• Type . Pol I, pol II, pol III
EXONUCLEASE
• Exonucleases are enzymes that work by cleaving
nucleotides one at a time from the end of a
polynucleotide chain.
• A hydrolyzing reaction that breaks phosphodiester
bonds at either the 3’ or the 5’ends occurs.
TERMINAL DEOXYNUCLEOTIDYL
TRANSFERASE
• Terminal Deoxynucleotidyl Transferase, also known
as TdT and terminal transferase.
• TdT catalyses the addition of nucleotides to the 3’
terminus of a DNA molecule.
• Cobalt is a necessary cofactor
POLYNUCLEOTIDE KINASE
• Polynucleotide kinase (PNK) is an enzyme that catalyzes the transfer
of a phosphate from ATP to the 5’ end of either DNA or RNA.
REVERSE TRANSCRIPTASE
• This enzyme by using the template of RNA ,synthesize the new strand
of DNA .
RNA
cDNA
dsDNA
NUCLEASES
•Nucleases are a class of enzymes called
hydrolases that catalyzes the hydrolysis of
nucleic acids(DNA,RNA) in all organisms
including plants and humans.
•Nucleases are usually specific in action,
ribonucleases acting only upon ribonucleic
acids (RNA) and deoxyribonucleases acting only
upon deoxyribonucleic acids (DNA).
•There are two types of nucleases: Endonucleases and
exonucleases
Exonucleases degrade nucleic acids from one
end of the molecule. They opearate either in 5’
 3’ or 3’  5’ direction.
Endonucleases degrade nucleic acids at specific
internal sites, reducing it to smaller and
smaller fragments.
Restriction enzyme,a endonuclease due to it’s
cleavage at specific nucleotide sequence find so
much importance in recombinant DNA
technlology.
•Nuclease cleavage sites
•(phosphodiester linkage)
•Cleavage at bond A
generates a 5’phosphate and a 3’ OH
terminus
•Cleavage at bond B
generates a 3’phosphate and a 5’hydroxyl terminus
ROLE OF NUCLEASES
•Processes under control of nucleases are
protective mechanisms against "foreign”
(invading) DNA
•degradation of host cell DNA after virus
infections
•DNA repair
•DNA recombination
• DNA synthesis
III. Vectors for Gene Cloning
INTRODUCTION
• A cloning vector is a DNA molecule in which foreign DNA can be inserted
or integrated and which is further capable of replicating within host cell
to produce multiple clones of recombinant DNA.
• Examples: Plasmids,phage or virus
Characteristics
It should be able to replicate autonomously.
Origin of replication.
Selectable markers.
Restriction sites.
A. Requirements of a vector to serve as a
carrier molecule
• The choice of a vector depends on the design of the
experimental system and how the cloned gene will be
screened or utilized subsequently
• Most vectors contain a prokaryotic origin of
replication allowing maintenance in bacterial cells.
• Some vectors contain an additional eukaryotic origin
of replication allowing autonomous replication in
eukaryotic cells.
 All
cloning vectors have in common at least one unique cloning
site, a sequence that can be cut by a restriction endonuclease to
allow site-specific insertion of foreign DNA.
 The most useful vectors have several restriction sites grouped
together in a multiple cloning site (MCS) called a polylinker
B. Main types of vectors
• Plasmid,
• bacteriophage,
• cosmid,
• bacterial artificial chromosome (BAC),
• yeast artificial chromosome (YAC),
• retrovirus,
• baculovirus vector……
C. Choice of vector
• Depends on nature of protocol or experiment
• Type of host cell to accommodate rDNA
• Prokaryotic
• Eukaryotic
PLASMID VECTORS
 Plasmids
are circular, double-stranded DNA (dsDNA) molecules
that are separate from a cell’s chromosomal DNA.
 These extra chromosomal DNAs, which occur naturally in bacteria
and in lower eukaryotic cells (e.g., yeast), exist in a parasitic or
symbiotic relationship with their host cell.
 Naturally occurring bacterial plasmids size range is 5000 to
400,0000 bp.
Plasmid
PLASMID VECTORS
 Advantages:
 Small, easy to handle
 Straightforward selection strategies
 Useful for cloning small DNA fragments
(< 10kbp)
 Disadvantages:
 Less useful for cloning large DNA fragments
(> 10kbp)
A plasmid vector for cloning
1. Contains an origin of replication, allowing for
replication independent of host’s genome.
2. Contains Selective markers: Selection of cells
containing a plasmid
twin antibiotic resistance
blue-white screening
3. Contains a multiple cloning site (MCS)
4. Easy to be isolated from the host cell.
4362 bp
SELECTIVE MARKER
• Selective marker is required for
maintenance of plasmid in the cell.
• Because of the presence of the selective
marker the plasmid becomes useful for
the cell.
• Under the selective conditions, only cells
that contain plasmids with selectable
marker can survive
• Genes that confer resistance to various
antibiotics are used.
• Genes that make cells resistant to
ampicillin, neomycin, or chloramphenicol
are used
ORIGIN OF REPLICATION
• Origin of replication is a DNA
segment recognized by the
cellular
DNA-replication
enzymes.
• Without replication origin,
DNA cannot be replicated in
the cell.
MULTIPLE CLONING SITE
• Many cloning vectors contain a
multiple cloning site or polylinker: a
DNA segment with several unique
sites for restriction endo- nucleases
located next to each other
• Restriction sites of the polylinker are
not present anywhere else in the
plasmid.
• Cutting plasmids with one of the
restriction enzymes that recognize a
site in the polylinker does not disrupt
any of the essential features of the
vector
TYPES OF PLASMIDS
Conjugative:- (stringent plasmid)
• Carry a set of transfer genes that facillitates bacterial conjugation
• Are large, show stringent control of DNA replication and present in low
numbers
• Low copy number = 1-4 copies / cell
Non conjugative:- (relaxed plasmid)
• If they do not posses such genes.
• Are small,show relaxed control of DNA replication and present in high
number
• High copy number = 10-100 copies / cell
F plasmid :
• posses genes for their own transfer from one cell to another
R plasmid:
• carry genes resistance to antibiotics.
pBR322
 pBR322
was one of the first versatile plasmid vectors developed;
it is the ancestor of many of the common plasmid vectors used in
laboratories.
• Derived from E. coli plasmid ColE1), which is 4,362 bp DNA.
• pBR322 is named after Bolivar and Rodriguez, who prepared this
vector.
 pBR322 contains an origin of replication (ori) and a gene (rop)
that helps regulate the number of copies of plasmid DNA in the
cell.
 There are two marker genes: confers resistance to ampicillin, and
confers resistance to tetracycline.
 pBR322 contains a number of unique restriction sites that are
useful for constructing recombinant DNA.
 It has unique restriction sites for 20 restriction endonucleases.
pBR322
1. Origin of
replication
2. Selectable
marker
3. unique
restriction
sites
• Another series of plasmids that are used as cloning vectors belong to pUC
series (after the place of their initial preparation I.e. University of
California).
• These plasmids are 2,700 bp long and possess
• Ampicillin resistance gene
• The origin of replication derived from pBR322 and
• The lacz gene derived from E.coli.
• Within the lac region also having unique restriction sites.
• When DNA fragments are cloned in this region of pUC, the lac gene is
inactivated.
• On the other hand, pUC having no inserts are transformed into
bacteria, it will have active lac Z gene and therefore will produce blue
colonies, thus permitting identification of colonies having pUC vector
with cloned DNA segments.
pUC19
2.68kbp
Bacteriophage lambda (λ)
A virus that infects
bacteria
o
BACTERIOPHAGE VECTORS
• BACTERIAL VIRUS
• Infects bacterial cells by injecting their genetic material (DNA or RNA)
• Follow either Lytic cycle and Lysogenic cycle
• Commonly used E.Coli phages are l (lambda) , M 13 ,Fd phages.
• Most efficient than plasmid for cloning of large fragments of over 25
kb.
• Easy to screen
• MAINLY l - widely used
• Larger capacity of insert than PLASMIDS
Bacteriophage vectors
• Advantages:
• Useful for cloning large DNA fragments
(10 - 23 kbp)
• Inherent size selection for large inserts
• Disadvantages:
• Less easy to handle
Bacteriophage
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.
Bacteriophage lambda
COS site: Cohesive
“sticky” ends
Lysis
Replication
ori
Lysogeny
Head
Tail
Circularized
lambda
• Lambda genome is approximately 49 kb in length.
• Only 30 kb is required for lytic growth.
• Thus, one could clone 19 kb of “foreign” DNA.
• Packaging efficiency 78%-100% of the lambda genome.
BACTERIOPHAGE LAMBDA
.
DNA cloning using phages as vectors
PHAGE M13 VECTOR
COSMID VECTORS
• Hybrid molecules containing components of both lambda and plasmid DNA
• Lambda components: COS sequences (required for in vitro packaging into phage
coats)
• Plasmid DNA components: ORI + Antibiotic resistance gene
• Cosmids can carry up to 50 kb of inserted DNA.
• Cloning sites will be part of vector
COSMID VECTORS
• RDNA is packaged using extracts of coat and tail proteins derived from
normal lambda components  BUT cannot be packaged after introduced
into host cell because rDNA does not encode the genes required for coat
proteins
• The cos sequences occurs at one end of lambda DNA molecules and it is
responsible for its insertion into the phage capsid.
• Cos sites allows them to be packaged into capsids.
• After packaged it used to infect E. coli.
• After infection of E. coli, rDNA molecules replicate as plasmids
CLONING BY USING COSMID VECTORS
SHUTTLE VECTORS
• Hybrid molecules designed for use in multiple cell types
• Multiple ORIs allow replication in both prokaryotic and eukaryotic
host cells allowing transfer between different cell types
• Examples:
• E. coli  yeast cells
• E. coli  human cell lines
• Selectable markers and cloning sites
SHUTTLE VECTORS
• Possess two origin for replication (ori E & ori Euk).
• Can be expressed in either host
• Can be grown in one host and shifted to another host
• ori E functions in E. coli & ori Euk functions in eukaryotic cells like
yeast.
YEAST VECTORS
• Yeast is a unicellular eukaryotic micro organism.
• Reproduce sexually as well as asexually
• Contains their own plasmid (6318 bp long)
• Present in high number copy
Yeast plasmid contains:
• Origin of replication (ori)
• Cis action region (REP 3)
• Two genes ( REP 1 & REP 2)
Yeast artificial chromosomes (YACs)
• Hybrid molecule containing components of yeast,
protozoa and bacterial plasmids
• Yeast:
• ORI = ARS (autonomously replicating sequence)
• Selectable markers on each arm (TRP1 and URA3)
• Yeast centromere
• Protozoa= Tetrahymena
• Telomere sequences (yeast telomeres may also be used)
• Bacterial plasmid
• Polylinker
• Can accommodate >1Mb (1000kbp = 106 bp)
YAC vector
large
inserts
URA3
HIS3
ARS
telomere
telomere
centromere markers
replication
origin
 Capable of carrying inserts of 100 kbp in yeast
Bacterial artificial chromosomes (BACs)
• Based on F factor of bacteria (imp. In conjugation)
• Can accommodate 300 kb of inserts
• Advantage is the instability problems of YACs can be
avoided
• F factor components for replication and copy #
control are present
• Selectable markers and cloning sites available
BAC vector
 oriS and oriE mediate
replication
 parA and parB
maintain single copy
number
 ChloramphenicolR
marker
Human artificial chromosomes
• Developed in 1997 – synthetic, self-replicating
• ~1/10 size of normal chromosome
• Microchromosome that passes to cells during
mitosis
• Contains:
• ORI
• Centromere
• Telomere
• Protective cap of repeating DNA sequences at ends of
chromosome (protects from shortening during mitosis)
• Histones provided by host cell
What determines the choice vector?
 insert size

vector size

restriction sites

copy number

cloning efficiency

ability to screen for inserts
APPLICATIONS OF RDNA TECHNOLOGY IN
PHARMACEUTICAL
APPLICATIONS
• Several proteins are created from recombinant DNA (recombinant
proteins) and are used in medical applications.
• Hematopoietic growth factor.
• Interferon’s
• Hormones
• Recombinant protein vaccines
• Tissue/bone growth factors and clotting factors
• Biological response modifiers
• Monoclonal/Diagnostic/Therapeutic antibodies
• Recombinant proteins is extensively used in biotechnology,
medicine and research.
100
PRODUCTS EXTRACTED FROM TISSUE/
PRIMARY CELLS
Product
 Insulin
 Growth hormone
 Interferon
 Urokinase
 Factor VIII
Extracted from....
Pancreas; bovine or porcine
Human pituitary glands
Viral activation of cells
Human urine
Pooled human blood
PROBLEMS OF EXTRACTION FROM
ANIMAL/ HUMAN SOURCES
• Small quantities available
• Non-human proteins cause immunogenicity
• contamination with viruses or prions
INSULIN
Insulin
•
Hormone produced by beta cells in the pancreas
→ allows glucose to pass into cells
→ suppresses excess production of sugar in the liver
and muscles
→ suppresses breakdown of fat for energy
ANIMAL CELL PRODUCTS – RECOMBINANT PROTEINS
beta cells in pancreas
Preproinsulin (109 A.A)
Proinsulin (86 A.A)
Insulin(51 A.A) + C-peptide
Computer-generated image of insulin hexamers highlighting the threefold
symmetry, the zinc ion holdin it together and the histidine residues invlolved in zincbinding
INSULIN
51 amino acids
5,8808 molecular weight
ANIMAL CELL PRODUCTS – RECOMBINANT PROTEINS
• Insulin produced from pig pancreas cells
→ structure of insulin differs slightly between species
→ the C-terminal amino acid of the B chain = alanine
(threonine in humans)
• two problems associated with porcine insulin
→ causes immunogenic response in some diabetic
patients
→ supply of pancreas fluctuates with meat trade
INSULIN SYNTHESIS
• Prepared by synthetic gene or from mRNA separated from rat pancrease.
• Chemically synthesized DNA sequence for A & B chains of insulin.
• The synthetic A & B genes are separately inserted into two pBR 322 plasmid
by the side of galactosidase gene.
• The recombinant plasmids are separately transferred into E.Coli cells.
INSULIN SYNTHESIS
• The bacterial cells are grown in large fermenter by using proper nutrients
and optimized physical conditions.
• The product contains large chimeric protein consisting of the A and B
chain attached to naturally occurring E.Coli protein.
• These chains are detached from protein through cyanogen bromide.
• Chain A and B are joined in vitro to form insulin by sulphonating the two
peptides with sodium disulphonate and sodium sulphite.
Producing A and B chains separately
INSULIN PRODUCTION
112
THANK YOU
-PHARMA STREET