Restriction Digestion of

Plasmid DNA
Group # 5
Sim, Michelle D.
Suderio, Gellina Ann R.
Teope, Jonnah Kristina C.
Timbol, Danica Kaye P.
Uy, regina Celine DG
 Plasmids
• First introduced by Joshua
Lederberg in 1952
• mostly circular double-stranded
DNA , few are linear, varies in
size
• extra-chromosomal DNA
molecule, capable of self
replicating
– replication is dependent on
host-cell proteins
• commonly used as cloning or
expression vectors
 Types of Plasmids
(according to their ability to transfer to another bacteria)

• Conjugative Plasmids
– contain so-called tra-genes, which perform
conjugation
• Non-conjugative Plasmids
– incapable of initiating conjugation
– can only be transferred with the assistance of
conjugative plasmids
• Mobilizable Plasmids
– carry only a subset of the genes required for transfer
– can “parasitize” a conjugative plasmid
 Types of Plasmids
(according to their function)

• Fertility (F) plasmids

– contain tra-genes.
– capable of conjugation
• Resistance (R) plasmids
– contain genes that can build a resistance against antibiotics or poisons
and help bacteria produce pili
– historically known as R-factors, before the nature of plasmids was
understood.
• Col-plasmids
– contain genes that code for bacteriocins (protein that can kill other
bacteria)
• Degradative plasmids
– enable the digestion of unusual substances such as toluene or salicylic
acid
• Virulence plasmids
– turn the bacterium into a pathogen
 Plasmid Conformations
• Nicked / Open-Circular DNA
– has one strand cut
• Relaxed Circular DNA
– fully intact with both strands uncut, but has been
enzymatically "relaxed" (supercoils removed)
• Linear DNA
– has free ends, either because both strands have
been cut, or because the DNA was linear in vivo
• Supercoiled or Covalently Closed-
Circular DNA
– fully intact with both strands uncut, and with a twist
built in, resulting in a compact form
• Supercoiled Denatured DNA
– like supercoiled DNA, but has unpaired regions that
make it slightly less compact
– can result from excessive alkalinity during plasmid
preparation.
 Functions of Plasmids
• Resistance to:
– Heavy metals
– Antibiotics
– Bacteriophages
– Viral infection
• Production of:
– Restriction enzymes
– Rare amino acids
– Toxins
• Ability to:
– Form symbiotic relationships
– Catabolyze complex organic molecules
 Plasmid Vectors
plasmids used in genetic engineering
• Replicator Site
– Origin of DNA replication
– Contains genes encoding for
RNAs and proteins needed for
replication
• Selectable Marker Site
– For selection of successful
plasmid transformants
– Genes encoding resistance to
ampicillin, tetracycline,
kanamycin and
chloramphenicol
• Multiple Cloning Site
– Polylinker regions
– Contains restriction enzyme
sites used in cloning DNA
fragments
 Restriction Endonucleases
• Discovered 40 years ago during investigations into the
phenomenon of host-specific restriction and modification of
bacterial viruses
• Recognize specific palindromic sequences and cleave a
phosphodiester bond on each strand at that sequence
• Function as microbial immune systems
• After digestion, the resulting DNA fragments can be separated by
agarose gel electrophoresis and their size can be estimated
 Restriction Map
Generated by using the fragment size data to determine the location of the
specific endonuclease recognition sequences on the plasmid
 Restriction Endonuclease
Nomenclature
 First letter (capital letter) – first letter of the genus where RE was
isolated
 Second and third letter (small letters) – first two letters of the
species name (specific epithet) where RE was isolated
 Fourth letter – first/second letter of strain name of organism where
RE was isolated
 Roman numeral – number (according to order of discovery) of RE
isolated from the species
 Examples:
 EcoR I – Escherichia coli
 Hind III – Haemophilus influenzae
 Sma I – Serratia marcescens
 Taq I – Thermophilus aquaticus
 Kpn I – Klebsiella pneumoniae
 Restriction Enzymes
• enzymes that cut double-stranded or single stranded
DNA at specific recognition nucleotide sequences
• Type 1
– the first to be identified
– cut at a site that differs and at least 1000 bp away from their recognition site
– recognition site is asymmetrical and is composed of two portions
• one containing 3-4 nucleotides, and another containing 4-5 nucleotides
• Type 2
– composed of only one subunit
– recognition sites are usually undivided and palindromic and 4-8 nucleotides
in length
– recognize and cleave DNA at the same site
• Type 3
– recognize two separate non-palindromic sequences that are inversely
oriented
– cut DNA about 20-30 base pairs after the recognition site
Restriction
Enzymes
 Recognition Sites
• Sequence specific
• Variable length
• Recognize mostly palindromic sequences (4-8 bp)

Enzyme Restriction Site

BamHI G•GATCC
EcoRI G•AATTC
HindIII A•AGCTT
PstI CTGCA•G
Types of Restriction Fragments
• Blunt-End Restriction Fragments
– are the result of restriction digestion which yields "blunt" or
"non-sticky" end DNA fragments
– the fragments do not have overhangs
– allows the cloning of incompatible DNA fragments
• Sticky-End Restriction Fragments
– are quite useful as they can be ligated with other compatible
restriction fragment ends (similar to the way lego pieces are
stuck together)
– allowed the cloning of DNA fragments into other DNA pieces
 Restriction Enzyme Digestion
• employs the function of one or more restriction enzyme
to selectively cut DNA strands into shorter restriction
fragments
• Some restriction enzymes cut in the middle of their
recognition site, creating blunt-ended DNA fragments.
• However,the majority of enzymes make cuts staggered
on each strand, resulting in a few base pairs of single-
stranded DNA at each end of the fragment, known as
“sticky” ends.
• Some enzymes create 5' overhangs and others create
3‘ overhangs.
Restriction Enzyme Digestion
Experiment # 3
Materials Special Equipment
 Plasmid pCH (0.2ug/uL)  Microcentrifuge
 Restriction enzymes
 BamHI
 EcoRI
 HindIII
 PstI
 Restriction enzyme buffers
 Micropipettors
 0.5mL microcentrifuge tubes
 Pipette tips
 Crushed ice
 Styrofoam cup
 Procedure
Label 5 0.5mL microtubes (C B E H P)
C = no restriction enzyme
just uncut plasmid pCH
Place them in the microtube rack B = BamHI restriction digest
of plasmid pCH
E = EcoRI restriction digest
To each tube, add 7.5uL of distilled water, 1uL of
of plasmid pCH
plasmid pCH and 1uL of appropriate 10X restriction buffer
H = HindIII restriction digest
of plasmid pCH
To the appropriate labeled tubes, add 0.5uL of
C, B, E, H, and P P = PstI restriction digest
of plasmid pCH

Securely fasten the cap on each tube

Spin for several seconds in a microcentrifuge to collect C = distilled water

the sample at the bottom of the tube B = BamHI
Incubate the tubes in a 370C water bath E = EcoRI
for approximately 1 hour H = HindIII
P = PstI
After the incubation, remove the tubes from the water bath
and store them in the refrigerator until the next laboratory period
Post Laboratory Questions
 Given below is the restriction map of plasmid pCH. Predict the number of
fragments and their sizes that will be obtained when the plasmid is cut with the
restriction enzymes used in the experiment.

Restriction Enzyme Number of Expected Sizes of

Expected the Fragments (bp)
Fragments

BamHI 1 4361 bp

EcoRI 1 4361 bp

HindIII 1 4361 bp

PstI 1 4361 bp
 Predict the number of fragments and their sizes that will be obtained when the
plasmid is cut by a combination of enzymes

Restriction Enzyme Number of Expected Expected Sizes of the

Fragments Fragments (bp)
BamHI + EcoRI 2 4359 – 375 = 3984 bp
4361 – 3984 = 377 bp

HindIII + EcoRI 2
4359 – 29 = 4330 bp
4361 – 4330 = 31 bp

PstI + EcoRI 2
4359 – 3607 = 752
4361 – 752 = 3609