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
Nov 16, 2014
Restriction Digestion of Plasmid DNA
Group # 5Sim, 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 Vectorsplasmids 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 MapGenerated 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 Plasmid pCH (0.2ug/uL) Restriction enzymes
BamHI EcoRI HindIII PstI
Restriction enzyme buffers Micropipettors 0.5mL microcentrifuge tubes Pipette tips Crushed ice Styrofoam cup
Special Equipment Microcentrifuge
ProcedureLabel 5 0.5mL microtubes (C B E H P)
Place them in the microtube rack
To each tube, add 7.5uL of distilled water, 1uL of plasmid pCH and 1uL of appropriate 10X restriction buffer
To the appropriate labeled tubes, add 0.5uL of C, B, E, H, and P
Securely fasten the cap on each tube
Spin for several seconds in a microcentrifuge to collect the sample at the bottom of the tube
Incubate the tubes in a 370C water bath for approximately 1 hour
After the incubation, remove the tubes from the water bath and store them in the refrigerator until the next laboratory period
C = no restriction enzyme just uncut plasmid pCHB = BamHI restriction digest of plasmid pCHE = EcoRI restriction digest of plasmid pCHH = HindIII restriction digest of plasmid pCHP = PstI restriction digest of plasmid pCH
C = distilled waterB = BamHIE = EcoRIH = HindIIIP = PstI
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
Fragments
Expected Sizes of the Fragments
(bp)
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 Fragments
Expected Sizes of the Fragments (bp)
BamHI + EcoRI 24359 – 375 = 3984 bp
4361 – 3984 = 377 bp
HindIII + EcoRI 24359 – 29 = 4330 bp
4361 – 4330 = 31 bp
PstI + EcoRI 24359 – 3607 = 752
4361 – 752 = 3609