Transcript No Slide Title

DNA Recombinant Technology
What and Why?
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What?: A gene of interest is inserted
into another organism, enabling it to
be cloned, and thus studied more
effectively
Why?: Detailed studies of the
structure and function of a gene at
the molecular level require large
quantities of the individual gene in
pure form
Cloning
A collection of molecules or cells, all identical
to an original molecule or cell
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To "clone a gene" is to make many copies of it - for
example, in a population of bacteria
Gene can be an exact copy of a natural gene
Gene can be an altered version of a natural gene
Recombinant DNA technology makes it possible
Terms to Know
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Vector: an autonomously replicating genetic
element used to carry DNA fragments into a
host, typically E. coli, for the purpose of gene
cloning
• Plasmid vector
• Bacteriophage gamma vector
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Recombinant DNA: any DNA molecule composed
of sequences derived from different sources
Cleavage
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It is done by Restriction enzymes
Restriction enzymes :
Enzyme produced by bacteria that
typically recognize specific 4-8 base pair
sequences called restriction sites, and
then cleave both DNA strands at this site
Inserting DNA Fragments
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DNA fragments are inserted into
vector DNA with the aid of DNA
ligases
Ligases catalyze the end-to-end
joining of DNA fragments
Plasmids
Naturally occurring extrachromosomal
DNA
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Plasmids are circular dsDNA
Plasmids can be cleaved by restriction
enzymes, leaving sticky ends
Artificial plasmids can be constructed by
linking new DNA fragments to the sticky ends
of plasmid
Restriction Enzyme
 Molecular scissors; isolated from bacteria
where they are used as Bacterial defense
against viruses.
 Molecular scalpels to cut DNA in a precise and
predictable manner
 Members of the class of nucleases
Nuclease
Breaking the phosphodiester bonds that link
adjacent nucleotides in DNA and RNA
molecules
 Endonuclease
Cleave nucleic acids at internal position
 Exonuclease
Progressively digest from the ends of the
nucleic acid molecules
Endonuclease
Type
Characteristics
I
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II
III
Have both restriction and modification activity
 Cut at sites 1000 nucleotides or more away from
recognition site
 ATP is required
 It has only restriction site activity
 Its cut is predictable and consistent manner at a site
within or adjacent to restriction site
 It require only magnesium ion as cofactor
Have both restriction and modification activity
Cut at sites closed to recognition site
ATP is required
Restriction Enzyme
 There are already more than 1200 type II enzymes isolated
from prokaryotic organism
 They recognize more than 130 different nucleotide sequence
 They scan a DNA molecule, stopping only when it recognizes
a specific sequence of nucleotides that are composed of
symetrical, palindromic sequence
Palindromic sequence:
The sequence read forward on one DNA strand is identical to
the sequence read in the opposite direction on the
complementary strand
 To Avoid confusion, restriction endonucleases are named
according to the following nomenclature
Nomenclature
 The first letter is the initial letter of the genus name of the
organism from which the enzyme is isolated
 The second and third letters are usually the initial letters of
the organisms species name. It is written in italic
 A fourth letter, if any, indicates a particular strain organism
 Originally, roman numerals were meant to indicate the order
in which enzymes, isolated from the same organisms and
strain, are eluted from a chromatography column. More
often, the roman numerals indicate the order of discovery
Nomenclature
EcoRI
BamHI
HindIII
E : Genus Escherichia
co: Species coli
R : Strain RY13
I : First endonuclease isolated
B : Genus Bacillus
am: species amyloliquefaciens
H : Strain H
I : First endonuclease isolated
H : Genus Haemophilus
in : species influenzae
d : strain Rd
III : Third endonuclease isolated
Specificity
Enzyme
BamHI
BglII
EcoRI
EcoRII
HaeIII
HindII
HindIII
HpaII
NotI
PstI
Source
Bacillus amyloliquefaciens H
Bacillus globigii
Escherichia coli RY13
Escherichia coli R245
Haemophilus aegyptius
Haemophilus influenzae Rd
Haemophilus influenzae Rd
Haemophilus parainfluenzae
Nocardia otitidis-caviarum
Providencia stuartii 164
Sequence
GGATCC
AGATCT
GAATTC
CCTGG
GGCC
GTPyPuAC
AAGCTT
CCGG
GCGGCCGC
CTGCAG
End
Sticky
Sticky
Sticky
Sticky
Blunt
Blunt
Sticky
Sticky
Sticky
Sticky
Restriction enzymes
Restriction enzymes can be grouped by:
 number of nucleotides recognized (4, 6,8 base-cutters
most common)
 kind of ends produced (5’ or 3’ overhang (sticky), blunt)
 degenerate or specific sequences
 whether cleavage occurs within the recognition
sequence
Become familiar with the back of your molecular
biology catalog!
A restriction enzyme (EcoRI)
1. 6-base cutter
2. Specific palindromic
sequence (5’GAATTC)
3. Cuts within the
recognition
sequence (type II
enzyme)
4. produces a 5’
overhang (sticky
end)
Restriction enzymes
Cloning Vectors
Plasmids that can be modified to carry
new genes
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Plasmids useful as cloning vectors must have
• a replicator (origin of replication)
• a selectable marker (antibiotic resistance
gene)
• a cloning site (site where insertion of foreign
DNA will not disrupt replication or inactivate
essential markers
A typical plasmid vector with a
polylinker
Chimeric Plasmids
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Named for mythological beasts with body
parts from several creatures
After cleavage of a plasmid with a restriction
enzyme, a foreign DNA fragment can be
inserted
Ends of the plasmid/fragment are closed to
form a "recombinant plasmid"
Plasmid can replicate when placed in a
suitable bacterial host
Directional Cloning
Often one desires to insert foreign DNA in a
particular orientation
 This can be done by making two cleavages
with two different restriction enzymes
 Construct foreign DNA with same two
restriction enzymes
 Foreign DNA can only be inserted in one
direction