DNA Preparation and Purification

DNA Preparation and Purification

For cloning, there is a requirement of three distinct kinds of DNA i.e, total cell DNA, pure plasmid DNA and phage DNA. 

Total cell DNA preparation

Total cell DNA will often be required as a source of material from which to obtain genes to be cloned. Total cell DNA may be DNA from a culture of bacteria, from a plant, from animal cells, or from any other type of organism that is being studied. The four steps (Figure-1) in the preparation of total cell DNA are: the growth and harvesting of a bacterial culture, breaking of cells to release their contents, purification of the DNA from the cell extract and finally concentration of the DNA.

Growing and harvesting a bacterial culture

Most bacteria can be grown without much difficulty in a liquid medium otherwise known as broth culture. The culture medium must provide a balanced mixture of the essential nutrients at concentrations that will allow the bacteria to grow and divide efficiently. M9 is an example of a known defined medium in which a mixture of inorganic nutrients(nitrogen,magnesium and calcium) and glucose(source of carbon and energy) are present. LB is a complex undefined medium in which two of the ingredients, tryptone and yeast extract are complicated mixtures of unknown chemical compounds. 

Defined media must be used when the bacterial culture has to be grown under precisely controlled conditions. When the culture is being grown simply as a source of DNA, a complex medium is appropriate for it. 

In order to prepare a cell extract, the bacteria must be obtained in as small a volume as possible. Harvesting is therefore performed by spinning the culture in a centrifuge. Low centrifugation speeds will pellet the bacteria at the bottom of the centrifuge tube, allowing the culture medium to be poured off.

Cell extract preparation

Cell extract preparation is the second step of total cell DNA preparation. Breaking open of bacteral cells can be done by physical methods, and chemical methods. Chemical methods are most commonly used with bacterial cells during DNA preparation. Cell lysis by chemical method is brought about by one chemical that attacks the cell wall while the other disrupts the cell membrane (Figure-2). Cell wall weakening is brought about by lysozyme, ethylenediamine tetraacetate(EDTA) or a combination of both. Lysozyme digests the polymeric compounds that give the cell wall its rigidity. Magnesium ions are essential for preserving the overall structure of the cell envelope. EDTA removes magnesium ions and also inhibits cellular enzymes that could degrade DNA. Sometimes a detergent such as sodium dodecyl sulphate(SDS) is added along with the chemicals, because detergents aid the process of lysis by removing lipid molecules and thereby cause disruption of the cell membranes.

The insoluble cell debris can be pelleted by centrifugation, (Figure-3) leaving the cell extract 

as a reasonably clear supernatant.

 DNA purification from a cell extract

For obtaining pure DNA, Bacterial cell extract is purified from significant quantities of Protein and RNA. DNA associated proteins, as well as other cellular proteins, may be degraded with the addition of a protease. Precipitation of the protein is aided by the addition of a salt such as ammonium or sodium acetate. When the sample is vortexed with phenol-chloroform(1:1 ratio) and centrifuged, the precipitated proteins left as white coagulated mass will remain at the interface between the aqueous and organic layers and can be drawn off carefully (Figure-4). The aqueous solution of nucleic acids (DNA and RNA) can then be removed with a pipette. The only effective way to remove the RNA is with the enzyme ribonuclease, which rapidly degrades these molecules into ribonucleotide subunits.

Concentration of DNA samples and measurement of concentration

Ethanol precipitation is the most frequently used method of concentration. In the presence of a salt (only monovalent cations), absolute ethanol efficiently precipitates polymeric nucleic acids at around a temperature of -20ºC or may be less than that. With a thick solution of DNA the ethanol can be layered on top of the sample, causing molecules to precipitate at the interface. If ethanol is mixed with a dilute solution, the precipitate can be collected by centrifugation, and then redissolved in an appropriate volume of water.

DNA concentrations can be accurately measured by ultraviolet absorbance spectrophotometry.The amount of ultraviolet radiation absorbed by a solution of DNA is directly proportional to the amount of DNA in the sample. Absorbance is measured usually at 260nm, at which wavelength an absorbance(A₂₆₀) of 1.0 corresponds to 50µg of double stranded DNA/ml.A pure sample of DNA indicates the ratio of absorbances at 260 and 280nm is 1.8 i.e.,A₂₆₀/A₂₈₀ is 1.8, for a pure sample of DNA. Ratio less than 1.8 indicates contamination of protein or phenol.

DNA preparation from animal and plant cells

Preparation of DNA from plant and animal cells is different from bacterial cell. Bacterial cell wall degradating enzyme lysozyme has no effect on plant cell wall. whereas most animal cells have no cell wall at all, and can be lysed simply by treating with detergent. Plant tissues consist of large amount of carbohydrates which are not removed by phenol extraction. In this case a detergent called cetyltrimethylammonium bromide(CTAB) is used which forms an insoluble complex with nucleic acids. When CTAB is added to a plant cell extract the nucleic acid-CTAB complex precipitates, leaving carbohydrate, protein and other contaminants in the supernatant. The precipitate is then collected by centrifugation and resuspended in 1M Nacl, which causes the complex to breakdown and the RNA removed by ribonuclease treatment.

 

Plasmid DNA preparation

Plasmid DNA preparation is same as total cell DNA preparation but importantly distinct in one aspect that in plasmid DNA preparation it is always necessary to separate the plasmid DNA from the large amount of bacterial chromosomal DNA that is also present in the cells. 

       Plasmids and bacterial DNA differ in conformation(overall spatial configuration of the molecule).Plasmids and bacterial chromosome are circular, but during preparation of the cell extract the chromosome will always be broken to give linear fragments. A method of separating circular from linear molecules will therefore result in pure plasmids.

Size based separation

Bacterial cell disruption is carried out very gently to prevent wholesale breakage. Treatment with EDTA and lysozyme is carried out in the presence of sucrose, which prevents the cell from bursting. Sphaeroplasts(partially wall less cells) are formed that retain an intact cytoplasmic membrane (Figure-5). Cell lysis is induced by adding a non-ionic detergent Triton X-100 which causes minimal breakage of the bacterial DNA, therefore centrifugation will leave a cleared lysate, consisting almost entirely of plasmid DNA. A clear lysate will however, invariably retain some chromosomal DNA. Size fractionation does not sufficiently help to remove contaminants, and therefore alternative ways for it must be considered.

Conformation based separation

Most plasmids exist in the cell as supercoiled molecules. Supercoiled molecules can be easily separated from non supercoiled DNA. Two different types of conformation based separation are alkaline denaturation and EtBr-CsCl density gradient centrifugation.

Alkaline denaturation

Non-supercoiled DNA is denatured at a narrow pH range. If pH of a cell extract or cleared lysate is increased(12.0-12.5) by addition of NaOH, then the hydrogen bonding in non supercoiled DNA molecules is broken, causing the unwinding of double helix and finally separation of two polypeptide chains (Figure-6). These denatured DNA strands will re-aggregate into a tangle mass by the addition of acid. With the help of centrifugation, the insoluble network can be pelleted, leaving pure plasmid DNA in the supernatant. Under some circumstances ( cell lysis by SDS and neutralization with sodium acetate) , most of the proteins and RNA also becomes insoluble and can be removed by centrifugation.

Ethidium bromide-caesium chloride(EtBr-CsCl) density gradient centrifugation

Under high centrifugal force, a solution of cesium chloride (CsCl) molecules will dissociate, and the heavy Cs⁺ atoms will be forced towards the outer end of the tube, thus forming a shallow density gradient (Figure-7(a)). DNA molecules placed in this gradient will migrate to the point where they have the same density as the gradient (the isopycnic point). Macromolecules present in the CsCl solution when it is centrifuged will form bands at distinct points in the gradient. The gradient is sufficient to separate types of DNA with slight differences in density due to differing (G+C) content, or physical form (e.g., linear versus circular molecules). Density gradient centrifugation in the presence of ethidium bromide (EtBr)can be used to separate supercoiled DNA from non-supercoiled molecules (Figure-7(b)). EtBr binds to DNA molecules by intercalating between adjacent base pairs, causing partial unwinding of the double helix. Density gradient centrifugation can separate DNA, RNA and protein and is an alternative to phenol extraction and ribonuclease treatment for DNA purification.

       EtBr-Cscl density gradient centrifugation is a very efficient method for obtaining pure plasmid DNA. When a cleared lysate is subjected to this procedure, plasmids band at a distinct point, separated from the linear bacterial DNA, with the protein floating at the top of the gradient and RNA pelleted at the bottom. The position of the DNA bands can be seen by shining ultraviolet radiation on the tube, which causes the bound EtBr to fluoresce. The EtBr bound to the plasmid DNA is extracted with n-butanol (Figure-7(c)) and the CsCl removed by dialysis (Figure-7(d)). The resulting plasmid preparation is pure and can be used in cloning.

  

Bacteriophage DNA preparation

In bacteriophage DNA preparation, a cell extract is not the starting material, because bacteriophage particles can be obtained in large numbers from the extracellular medium of an infected bacterial culture. When such a culture is centrifuged, the bacteria are pelleted, leaving the phage particles in suspension (Figure-8). The phage particles are then collected from the suspension and their DNA extracted by a single deproteinization step to remove the phage capsid.

DNA Manipulation

After obtaining pure DNA sample preparation, the next step in gene cloning experiment is construction of the recombinant DNA molecule. Construction of a recombinant DNA molecule is done by cloning the vector as well as the DNA molecule, cutting both at specific points and then joining them together in a controlled manner respectively. Cutting and joining are two examples of DNA manipulative techniques, that underline gene cloning, are carried out by enzymes called restriction endonucleases and ligases respectively.

Restriction enzymes

To incorporate fragments of foreign DNA into a plasmid vector, methods for the cutting and rejoining of double-stranded DNA are required. The identification and manipulation of restriction endonucleases in the 1960s and early 1970s was the key discovery which allowed the cloning of DNA to become a reality. Restriction- modification systems occur in many bacterial species, and constitute a defense mechanism against the introduction of foreign DNA into the cell. They consist of two compartments; the first is a restriction endonuclease, which reconizes a short, symmetrical DNA sequence, and cuts the DNA backbone in each strand at a specific site within that sequence and the second component of the system is a methylase, which adds a methyl group to a C or A base within the same recognition sequences in the cellular DNA. This modification renders the host DNA resistant to degradation by the endonuclease. Three different classes of restriction endonuclease are recognized, each distinguished by a slightly different mode of action. Types I and III are rather complex and have only a very limited role in genetic engineering. Type II restriction endonucleases, on the other hand, are the cutting enzymes that are important in gene cloning.

Recognition sequences

Each type II restriction endonuclease has a specific recognition sequence at which it cuts a DNA molecule. A particular enzyme will cleave DNA at the recognition sequence and nowhere else. Many restriction endonucleases recognize hexanucleotide target sites, but others cut at four, five or even eight nucleotide sequences. Sau3a from (Staphylococcus aureus strain 3A) recognizes GATC, and AluI( Arthrobacter luteus ) cuts at AGCT. Some enzymes with degenerate recognition sequences, cut DNA at any one of a family of related sites. Hinf1(Haemophilus influenzae strain Rf), for instance, recognizes GANTC, so cuts at GAATC, GATTC, GAGTC and GACTC.

Blunt ends

The exact nature of the cut produced by a restriction endonuclease is of considerable importance in the design of a gene cloning experiment. The simplest DNA end of a double stranded molecule is called a blunt end. In a blunt-ended molecule both strands terminate in a base pair. Blunt ends are not always desired in biotechnology since when using a DNA ligase to join two molecules into one, the yield is significantly lower with blunt ends. When performing subcloning, it also has the disadvantage of potentially inserting the insert DNA in the opposite orientation desired. On the other hand, blunt ends are always compatible with each other. Many restriction endonucleases make a simple double stranded cut in the middle of the recognition sequence, resulting in a blunt or flush end. PuvII and AluII are blunt end cutters (Figure-9).

Sticky ends

Non-blunt ends are created by various overhangs. An overhang is a stretch of unpaired nucleotides in the end of a DNA molecule. These unpaired nucleotides can be in either strand, creating either 3' or 5' overhangs. These overhangs are in most cases palindromic. Longer overhangs are called cohesive ends or sticky ends. Quite a large number of restriction endonucleases cut DNA in a different way. Very often they cut the two DNA strands four base pairs from each other, creating a four-base 5' overhang in one molecule and a complementary 5' overhang in the other. These ends are called cohesive since they are easily joined back together by a ligase. Also, since different restriction endonucleases usually create different overhangs, it is possible to cut a piece of DNA with two different enzymes and then join it with another DNA molecule with ends created by the same enzymes. Since the overhangs have to be complementary in order for the ligase to work, the two molecules can only join in one orientation. Restriction endonucleases with different recognition sequences may produce the same sticky ends. For e.g.,BamHI(recognition sequence GGATCC) and BglII( recognition sequence AGATCT) both produce GATC sticky ends. The same sticky end is also produced by Sau3A, which recognizes only the tetranucleotide GATC (Figure-10).

Frequency of recognition sequences

The number of recognition sequences for a particular restriction endonuclease in a DNA molecule of known length can be calculated mathematically. A tetranucleotide sequence (e.g., GATC) should occur once every 4⁴ = 256 nucleotides, and a hexanucleotide sequence (e.g., GGATCC) once every 4⁶=4096 nucleotides. From these calculations it is assumed that the nucleotides are ordered in a random fashion and that the four nucleotides are present in equal proportions. In practice, neither of these assumption is entirely valid.

      Restriction sites are not evenly spaced along a DNA molecule. The fragments produced by cutting λ DNA with BglII, BamHI and SalI are not equal in size.

Restriction digests

Digestion of plasmid or genomic DNA is carried out with restriction enzymes for analytical or preparative purposes, using commercial enzymes and buffer solutions. All restriction enzymes require Mg²⁺ , at a concentration of up to 10 mM, but different enzymes require different pHs, Nacl concentrations or other solution constituents for optimum activity. The buffer solution required for a particular enzyme is supplied with it as a concentrate. The digestion of a sample plasmid with two different restriction enzymes, BamHI and EcoRI is shown in figure below (Figure-11).

       

       A restriction digest will result in a number of DNA fragments, the sizes of which depend on the exact positions of the recognition sequences for the endonuclease in the original molecule. For determining the number and sizes of the DNA fragments after restriction digestion the technique of gel electrophoresis was developed.