Isolation of Plasmid DNA

Introduction

Many variations on a theme exist for the isolation of plasmid DNA from bacterial cells. They usually involve the following steps (not necessarily in this order):

  1. growing cells under conditions allowing intracellular accumulation of large amounts of plasmid; harvesting the cells by centrifugation;
  2. cell lysis (often using lysozyme to cleave cell wall peptidoglycans and surfactants to dissolve cell walls/membranes);
  3. separation of chromosomal DNA; removal of cellular RNA with ribonuclease;
  4. deproteinization;
  5. precipitation of the plasmid DNA with alcohol.

EDTA is included in most solutions to chelate Mg2+ ions and thus inhibit deoxyribonucleases that would otherwise degrade the plasmid DNA (RNases do not require Mg2+ as a rule).

We will use a recently-reported method designed for medium scale isolation that is quick and that yields good quality plasmid DNA, free of RNA, chromosomal DNA and impurities that interfere with restriction enzymes and other subcloning operations.

Ausebel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K. (eds.) 1989. In: Short Protocols in Molecular Biology, pp. 22-23, John Wiley & Sons, New York.

We will prepare plasmid pO.380, a vector which contains 3.8 kb of Drosophila DNA subcloned into the EcoRI restriction site within the polylinker region of pBluescribe (Stratagene) (McLean, J.R., Boswell, R., and O’Donnell, J., 1990. Cloning and molecular characterization of a metabolic gene with developmental functions in Drosophila: Analysis of the head function of Punch. Genetics 126:1007 - 1019). The strain that harbors this plasmid is ampicillin-resistant and therefore may be selectively grown in medium containing this antibiotic. It is in general advisable to grow plasmid-containing strains under selective conditions (e.g., in the presence of an appropriate antibiotic) to insure that the plasmids are not eliminated from the cells.

Background Notes

Bacterial plasmids are double-stranded closed circular (supercoiled) DNA molecules that range in size from 1 kb to 200 kb.

Plasmids are found in a variety of bacterial species.

Plasmids behave as accessory genetic units that replicate and are inherited independently of the bacterial chromosome (extrachromosomal genetic elements).

Plasmids rely on cellular enzymes and structural proteins for their replication and transcription.

Plasmids can confer a selective advantage to the host cell (resistance to antibiotics).

History: Dr. Lederberg in 1952 first discovered plasmids (F factor) (sex factor) in bacteria.

F factor is a special type of plasmid = episome

Episome is a segment of DNA capable of existing in 2 alternate forms, one replicating autonomously in the cytoplasm (plasmid), the other replicating as part of the bacterial chromosome (DNA can integrate into the host genome).

Current plasmid vectors are derived from a plasmid isolated from a clinical specimen in the 1970's (pMB1).

pBluescribe (also pUC) is a derivative of pMB1.

Mobilization and Selectable Markers of Plasmids:

Under natural conditions, plasmids are transmitted to a new host by a process called bacterial conjugation.

In the laboratory, plasmid DNA can be introduced into bacteria by the artificial process of transformation.

In this process, bacteria are treated with divalent cations (Ca2+) to make them temporarily permeable to small DNA molecules.

Efficiency of transformation is very low, so in order to identify transformed cells (cells which incorporated plasmids via transformation), one monitors a selectable marker which is encoded by the plasmid.

A selectable marker confers a new phenotype to the host cell that allows bacteria that have been successfully transformed to be selected (with ease).

Commonly used selectable markers are genes that confer resistance to antibiotics.

Ampicillin: inhibits enzymes that are involved in the synthesis of the cell wall.

Tetracycline: binds to a protein on the 30S subunit of the ribosome and inhibits ribosomal translocation.

Chloramphenical: binds to the 50S subunit of the ribosome and inhibits protein synthesis.

Kanamycin and neomycin: are deoxystreptamine aminoglycosides that bind to ribosomal components and inhibit protein synthesis.

pBluescribe (pUC) carries an ampicillin resistance gene. (bla) = encodes an enzyme that is secreted into the cell wall and breaks down ampicillin

Today's experiment (and next week's) will follow a procedure to isolate a pBluescribe plasmid.

pBluescribe has a polycloning site (a DNA sequence that was genetically constructed in vitro and contains many sites which are recognized by restriction endonucleases (enzymes): one of a large number of nucleases (enzymes that degrade nucleic acids) that can cleave a DNA molecule at any site where a specific short sequence of nucleotides occurs. Restriction enzymes are extensively used in recombinant DNA technology.

pBluescribe also contains a selectable marker (bla) so it confers an ampicillin resistant phenotype.

General Procedure:

Cells containing plasmids are concentrated and resuspended in an isotonic solution of sucrose.

Cells containing plasmids are treated with lysozyme to break down the cell wall and outer membrane.

Plasmids are released from cells by gentle lysis (addition of a nonionic detergent).

Cell lysate is heated to release carbohydrates and nick chromosomal DNA.

Plasmid DNA, RNA and proteins are concentrated by alcohol precipitation.

Proteins are separated from plasmid DNA and RNA with saturated phenol.

Phenol removed with chloroform.

Plasmid DNA and RNA are concentrated by alcohol precipitation.

RNA removed with RNase.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Biology 3403 Dr. Mackay

Handout

 

Polymerase Chain Reaction (PCR) ñ used to amplify a segment of DNA (ìtarget DNAî) that lies between 2 regions of known sequence

Relatively new technique developed by K.B. Mullis and F. Fulcomer in 1983 (Mullis Nobel Prize 1993). Used in:

diagnosis of genetic disorders

detection of nucleic acid sequences of pathogenic organisms in clinical samples

genetic identification of forensic samples (includes DNA extracted individual hairs or a single sperm)

Analysis of alterations (mutations) in cancer genes (oncogenes, tumor suppressor genes)

In recombinant DNA technology for molecular cloning and analysis of DNA

PCR Procedure

two oligonucleotides are used as primers for a series of synthetic reactions that are catalyzed by a DNA polymerase.

Oligonucleotides have different sequences and are complementary to sequences that:

lie on opposite strands of the template DNA

flank the segment of DNA that is to be amplified

template DNA is dentatured by heating in the presence of a large molar excess of each of the 2 oligonucleotides and the four deoxynucleoside triphosphates.

Reaction mixture is then cooled to a temperature that allows the oligonucleotide primers to anneal to their DNA template (target sequence).

The annealed primers are lengthened (extended) using DNA polymerase.

Cycle of denaturation, annealing and DNA replication (synthesis) is then repeated many times (usually 30 cycles).

Features of PCR

PCR referred to as a ìchain reactionî since the formation of product increases exponentially with time.

Each successive cycle essentially doubles the amount of the desired DNA product because the products of one round of amplification reverse as templates for the next round.

The major product of the exponential reaction is a segment of double-stranded DNA where the ends are defined by the 5í ends (terminus) of the oligonucleotide primers and the length of the product is defined by the distance between the primers.

In addition to the major product, there are also minor products formed that are longer in size.

For example, the products formed after the round of amplification are different sized DNA molecules whose lengths exceed the distance between the binding sites of the two primers.

These longer molecules are continually produced but accumulate at a linear rate where the major product accumulates at an exponential rate.

Original PCR protocols used DNA polymerase I from E.coli ( fragment) to catalyze the extension of the annealed oligonucleotide primers.

Since this enzyme is inactivated at high temperatures that are needed to denature DNA template, each round of DNA synthesis (replication) required the addition of a fresh of enzyme.

Only worked with small templates ( bp)

Yield was low

Products were heterogeneous in size caused by ìmisprimingî which occurred during the extension reactions.

These problems were solved by using a thermostable DNA polymerase which was purified from the thermophilic bacterium Thermus aquaticus (Tag DNA polymerase).

Enzyme can survive extended incubation at 95∫C.

Is not inactivated by the heat denaturation step and does not need to be replaced at every round of the amplification cycle.

In addition, because annealing and extension of oligonucleotides can be carried out at higher temperature, mispriming is greatly reduced.

Use of Tag I results in substantial improvements in the:

specificity of amplification reaction

yield of amplification reaction

size of amplified product

For example, 0.5mg-1 mg of target DNA up to 2kb (kilobases) (2000 bases) in length can be obtained in 30 cycles of amplification with only 10 mg (1pg) of starting DNA.

Amplification of DNA by PCR is not unlimited

30 cycles gives a 10 fold amplification

can then dilute 1/10 and gives 10 fold additional amplification

10 amplification can be achieved with 60 cycles

Todayís Experiment:

Amplify a part of the Maedi Visna Viral Genome.

lenti virus (close relative of HIV) infects sheep

viral genome 9.2kb injected into a plamid vector

p Bluescript

 

 

 

 

 

 

Reaction (in order)

39ml dH O

5ml 10x PCR buffer (with Mg)

1ml dNTP

2ml primers

2ml Tag. Vortex.

1ml template (spin)

20ml mineral oil

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Biology 3403 Dr. Mackay

Transformation of E.coli with pUC19

 

Genes can be transferred between bacteria in several ways: conjugation, transduction, and transformation.

Conjugation ñ mating process during which genetic material (F factor) is transferred from one cell to a second cell.

Transduction ñ a virus (bacteriophage) transfers genes from one cell to another.

Bacterial Transformation ñ involves the transfer of genetic information into a cell by direct absorption of the DNA.

Transformation can confer a new phenotype to the recipient cell (today will be ampicillin resistance due to the uptake of pUC19 DNA).

Bacterial Transformation using plasmid DNA was first demonstrated by Dr. Cohen in the 1970ís.

The efficiency of transformation is defined as: the number of transferred colonies (mg of DNA).

To increase the efficiency of transformation, the cell membrane is made temporarily permeable to DNA uptake through the treatment of Ca in the form of CaCl (these cells are called ìcompetentî cells). Efficiency of CaCl transformation is = 10 colonies/mg DNA.

In the late 1980ís, there was the development of a high efficiency transformation technique using high electrical voltage.

Process is called ìelectroporationî

High voltage temporarily disrupts the structural integrity of the bacterial cell membrane and allows for the uptake of foreign DNA (for example: plasmid DNA).

Efficiency of electroporation is = 10 cells/mg DNA.

Today, we are going to use a variation of the CaCl transformation procedure. The advantage of this procedure is that one does not have to make ìcompetentî bacterial cells.

In the procedure, bacterial cells are exposed to a solution containing polyethylene glycol (PEG), dimethylsulfoxide (DMSO), MgCl (Mg ). This solution temporarily make permeable the cell membrane to DNA template and it is not mutagenic.

Procedure (3 tubes/pair)

Transfer 1.0 ml of O/N to 3 Eppendorf Tubes.

Centrifuge at high speed for 15 seconds.

Decant supernatant.

Resuspend end tube in 100ml 2xTSS (ice-cold)

10% PEG

5% DMSO

50mM MgCl

Add 2ml/5ml DNA to tubes 1 and 2, respectively. (Tube 3 does not receive DNA-negative control).

Add 900ml of (TSS/LB) with 20mM glucose).

Incubate 1 hour at 37∫C.

Plate on LB Amp. Incubate O/N at 37∫C.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Biology 3403 Dr. Mackay

Aseptic Transfer Techniques

 

Many microbes are present on your workbench, hands, and transfer instruments. How is it possible to avoid contamination, the introduction of unwanted organisms into your pure culture?

Pure Culture ñ contains only one kind of bacteria; it consists of the descendents from on bacterial cell (clonal population).

Pure cultures are essential if a biologist is to:

identify bacteria with biochemical tests,

perform antibiotic sensitivity testing,

maintain stock cultures, or

use microorganisms in recombinant DNA technology.

Today, you will practice aseptic techniques (ìsterileî techniques) procedures that:

protect the culture, and

protect you and the environment.

Colony ñ several million bacterial cells derived from one parental cell that grow on a solid surface.

Rules for Aseptic Techniques

Disinfect your area

Sterilize instruments

Put on gloves

Work near a flame

Keep tubes covered

Work quickly, but safely

In general:

Avoid producing bacterial aerosols, microbes floating in the air.

Keep cultures closed.

Keep all items away from your mouth.

Quantifying Bacteria

Objective: to determine the number of microorganisms per milliliter of a solution.

Evaluation employed frequently by biologists in public health, industry, and medicine.

Employ Dilution Plate Counts

Useful quantification method for examining highly contaminated liquids, and fluids that clog filters.

Spread-plate technique: 100ml of solution added to a plate and liquid spread evenly over the surface of the plate.

Perform serial dilutions: 1:10 dilution 100ml + 900ml liquid

1:100 dilution 10ml + 900ml liquid

Today we will:

start with 10 cells/ml

dilute 10 , 10 , 10 , 10 ; plate 100ml of each on LB Amp plate.

Next lab session, we will count bacterial colonies.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Biology 3403 Dr. Mackay

Gel Electrophoresis/Restriction Digests

 

Last 2 labs you isolated plasmid pUC19.

Resuspended in TE buffer containing RNase (degrades ribonucleotides ñ rRNA, tRNA, mRNA).

Detect DNA using agarose gel electrophoresis (separation via an electric field) with ethidium bromide, a compound which binds to nucleic acids, fluoresces strongly when activated by UV light - can detect 20 nanograms of DNA.

Agarose, which is extracted from seaweed, is a linear polymer which is composed of galactose subunits.

Agarose gels can separate DNA fragments from 200 bp to 50 kb.

molecules of linear double-stranded DNA migrate through agarose gels at rates that are inversely proportional to the number of base pair pairs.

Migration is also affected by the concentration of agarose.

% of agarose Efficient rate of separation (kb)

5-60

0.7-8

0.1-2

DNA can exist in different conformational isomers:

superhelical circular (form I) ñ supercoiled or closed circular

nicked circular (form II) ñ open circular

linear (form III).

These DNAs migrate through agarose gels at different rates.

Under our conditions, closed circular will migrate the fastest, then linear, then open circular.

Preparation of gel.

Direction of migration ñ since DNA is a negative molecule due to the fact that PO groups in DNA are ionized ìpolyanionsî, it will migrate toward the positive electrode (anode).

Restriction Endonucleases

Enzymes found in microorganisms and catalyze double-strand break reactions in DNA yield restriction fragments.

Because a restriction enzyme reaction is very specific for a given DNA sequence, whole genomes can be divided into smaller, discrete pieces of DNA ñ extensively used in recombinant DNA technology.

1st discovered in 1960ís by Dr. Arber.

Use two today: Pst I and Sca I (both are high salt buffers).

Pst I

 

 

 

1) Incubate 90 minutes at 37∫C.

2) Add 1.0 ml EDTA to stop reaction.

3) Uncut ñ 3 forms of DNA.

4) Single cut ñ 1 2.7 kb

5) Double cut ñ 2 1.7 kb (approx.)

1.0 kb (approx.)

Generation of a restriction map

pUC19 2.7 kb plasmid

double digest 1.7 kb

kb fragments

Generate a Restriction Map of pUC19

restriction map ñ shows the distribution of short nucleotide sequence along a chromosome.

Can be constructed by comparing the sizes of the DNA fragments (restriction fragments) produced from a particular genetic region after treatment with a combination of different restriction nucleases.

Map reflects the arrangement of restriction sites on the region.

Fragments are separated by agarose gel electrophoresis.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Biology 3403 Dr. Mackay

Isolation of Plasmid DNA

 

Organic Extractions

Phenol Extraction ñ is used to remove proteins from solutions containing nucleic acids.

Add 1 volume of phenol to 1 volume of DNA

Vortex at high speed

Centrifuge for 2 minutes at 13K to separate the phases (phenol is more dense and will be at the bottom). DNA is the upper (aqueous phase).

Transfer aqueous phase to new microfage tube.

Chloroform Extraction ñ used to remove phenol from the aqueous phsase.

Add 1 volume of chloroform to 1 volume of DNA sample (aqueous phase)

Vortex at high speed

Centrifuge for 2 minutes at 13 K to separate phases (chloroform will be the bottom phase)

Transfer aqueous phase to new microfage tube

Alcohol Precipitation ñ NaO Acetate / Ethanol precipitate

Add 0.1 volume 3M NaOAc ñ pH 5.2

Add 2 volume ethanol

Inver tube 4x

Ice 10 minutes

Centrifuge 20 minutes at 13K

Transfer supernatant to new microfuge tube

Add 150 ml 70% ethanol to first tube

Inver tube 3x

Spin 5 minutes at 13K

Remove 70% EtOH and add to 2nd tube

Air dry for 90 minutes