Restriction Digestion Of Plasmid Dna

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

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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

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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.

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Col-plasmids – contain genes that code for bacteriocins (protein that can kill other bacteria)

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Degradative plasmids – enable the digestion of unusual substances such as toluene or salicylic acid

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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 ClosedCircular 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

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Selectable Marker Site – For selection of successful plasmid transformants – Genes encoding resistance to ampicillin, tetracycline, kanamycin and chloramphenicol

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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 EcoRI HindIII PstI

G•GATCC G•AATTC A•AGCTT 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 singlestranded 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

Procedure Label 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

C = no restriction enzyme just uncut plasmid pCH B = BamHI restriction digest of plasmid pCH E = EcoRI restriction digest of plasmid pCH H = HindIII restriction digest of plasmid pCH P = PstI restriction digest of plasmid pCH

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 = distilled water B = BamHI E = EcoRI H = HindIII P = 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

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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

HindIII + EcoRI

2

PstI + EcoRI

2

4359 – 375 = 3984 bp 4361 – 3984 = 377 bp 4359 – 29 = 4330 bp 4361 – 4330 = 31 bp 4359 – 3607 = 752 4361 – 752 = 3609