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