METHODS OF DNA PREPARATION

Preparation of plasmid DNA

• A plasmid is a DNA molecule that is separate from, and can replicate independently of,
the chromosomal DNA. They are double stranded and, in many cases, circular. Plasmids
usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms
(e.g., the 2- micrometre-ring in Saccharomyces cerevisiae). The term plasmid was
introduced in 1952 by the American molecular biologist Joshua Lederberg.

Basic understanding of Plasmid:

• A plasmid is a vehicle that can carry artificially inserted DNA. It will replicate in E. coli, and
with its own replication it will also replicate the inserted DNA, independent of it's origin. In a
way one can see a plasmid as a minute DNA factory. The main criteria for a 'good' plasmid is
that it takes up the insert you want to put in, and that it replicates in sufficient amounts,and that it
does not destroy your insert during the process.

Structure of Plasmids • Plasmid size varies from 1 to over 1,000 kilo base pairs (kbp). The
number of identical plasmids within a single cell can range anywhere from one to even
thousands under some circumstances.

Different forms of plasmids: Bacterial plasmid is a double stranded circular DNA exists in 3
different forms. If the both strands of circular double strands are intact then it is called as
covalently closed circles (CCC) where as if one of the strand has nick, then it acquire the
conformation of open circle DNA (OC, DNA). During the isolation of plasmid DNA from
bacteria, covalently closed circular DNA losses few number of turns and as a result it acquire
supercoiled configuration. The interchange between these different forms are possible under the
in-vitro or in-vivo conditions, such as DNA gyrase produces additional turn into the circular
DNA to adopt supercoiled conformation.

Features of different plasmids:

1. Origin of replication- Like any other replicating DNA, plasmid DNA needs its own
independent origin of replication to provide replication start site to make more copies.
2. 2. Selection marker- Selection marker in the form of either antibiotic resistance gene or
enzymatic gene is essential to give phenotypic changes in host after entry of the plasmid.
3. Promoter- Plasmid replication in host is performed by the host provided proteins such as
DNA gyrase, helicase, polymerase and DNA ligase. In addition, promoter is also needed
to express gene present on foreign DNA.

Plasmids in Genetic Engineering • Plasmids are extremely valuable tools in the fields of
molecular biology and genetics, specifically in the area of genetic engineering. They play a
critical role in such procedures as gene cloning, recombinant protein production (e.g., of human
insulin), and gene therapy research. In such procedures, a plasmid is cut at a specific site (or
sites) using enzymes called restriction endonucleases.

Plasmid DNA extraction:

Plasmids are often used to purify a specific sequence, since they can easily be purified away
from the rest of the genome. For their use as vectors, and for molecular cloning, plasmids often
need to be isolated.

There are several methods to isolate plasmid DNA from bacteria, ranging from the miniprep to
the maxiprep or bulkprep. The former can be used to quickly find out whether the plasmid is
correct in any of several bacterial clones. The yield is a small amount of impure plasmid DNA,
which is sufficient for analysis by restriction digest and for some cloning techniques.

In the latter, much larger volumes of bacterial suspension are grown from which a maxi-prep can
be performed. In essence, this is a scaled-up miniprep followed by additional purification. This
results in relatively large amounts (several hundred micrograms) of very pure plasmid DNA.

Many commercial kits have been created to perform plasmid extraction at various scales, purity,
and levels of automation.
Isolation and Purification of Plasmid DNA:

Plasmids are circular, double stranded extra cellular DNA molecules of bacterium and most
commonly used in recombinant DNA technology. The isolation of plasmid DNA involves three
major steps

1. Growth of the bacterial cell. 2. Harvesting and lysis of the bacteria.

3. Purification of the plasmid DNA

1. Growth of the bacterial cell:

It involves growth of the bacterial cells in a media containing essential nutrients.

2. Harvest and lysis of bacteria

Lysis of bacteria results in the precipitation of DNA and cellular proteins. Addition of acetate-
containing neutralization buffer results in the precipitation of large and less supercoiled
chromosomal DNA and proteins leaving the small bacterial DNA plasmids in solution.

3. Purification of Plasmid DNA

This step is same for both plasmid and genomic but former involves an additional step i.e. the
separation of plasmid DNA from the large bacterial chromosomal DNA.

Principle of the experiment “Alkaline lysis method “: using SDS in an alkaline solution:

-The SDS: will lyse the bacterial cell membrane and denature the proteins too.

-The alkaline pH : will denature the genomic DNA and denature the proteins too.

-The degraded cell wall, denatured chromosomal DNA and bacterial proteins form large
aggregated complex which and precipitated and removed by centrifugation.

-Native plasmid DNA can be collected from the supernatant. Plasmid isolation and purification

Isolation of plasmid from bacteria:

STEP 1: The bacteria containing plasmid was grown in suitable culture media in high density
(~0.8 optical density). Each Bacterial cell contains chromosomal DNA, plasmid DNA and
cellular proteins. The bacterial culture is collected by centrifugation at the bottom and
resuspended in the solution I containing 50 mM glucose, 25 mM TrisHCl pH 8.0, 10 mM EDTA
pH 8.0.

STEP 2: Alkaline Lysis: Bacterial cells are treated with lysis solution II containing 0.2N NaOH
and 1% SDS. to lyse the cells and denature DNA (both chromosomal and plasmid DNA).
STEP 3: Renaturation: In 3rd step, denatured DNA is renatured with solution III containing
potassium acetate, glacial acetic acid. In this step small DNA (plasmid) renature back quickly
whereas chromosomal DNA remained denatured.

STEP 4: Deproteination: Resulting supernatant containing plasmid DNA and protein is treated
with phenol: chloroform: isoamyl alcohol mixture to remove protein in the precipitate where as
plasmid remained in solution.

STEP 5: Precipitation : plasmid is precipitated by 100% alcohol from the solution.

Fig. steps in plasmid purification from bacterial culture

Methods for separation of plasmid DNA :

Separation of plasmid DNA is based on the several features like size and conformation of
plasmid DNA and bacterial DNA. Plasmids are much smaller than the bacterial main
chromosomes, the largest plasmids being only 8% of the size of the E. coli chromosome. The
separation of small molecules (i.e. plasmids) from larger ones (i.e. bacterial chromosome) is
based on the fact that plasmids and the bacterial chromosomes are circular but bacterial
chromosomes break into linear fragments during the preparation of the cell extract resulting in
separation of pure plasmids. The methods of separation of plasmid DNA are described as below
A. Separation based on size difference

• It involves lysis of cells with lysozyme and EDTA (function as described above in point 4-
1.2.2.) in the presence of sucrose (prevents the immediate bursting of cell). • Cells with
partially degraded cell walls are formed that retain an intact cytoplasmic membrane called as
sphaeroplasts. • Cell lysis is then induced by the addition of a non-ionic detergent (e.g. Triton
X100) or ionic detergents (e.g. SDS) causing chromosomal breakage. • Bacterial
chromosome attached to cell membrane, upon lysis gets removed with the cell debris. • A
cleared lysate consisting almost entirely of plasmid DNA is formed with very little breakage
of the bacterial DNA.

Fig. Separation of plasmid DNA on the basis of size

B. Separation based on conformation:

Plasmids are supercoiled molecules formed by partial unwinding of double helix of the
plasmid DNA during the plasmid replication process by enzymes called topoisomerases. The
supercoiled conformation can be maintained when both polynucleotide strands are intact,
hence called covalently closed-circular (ccc) DNA. If one of the polynucleotide strands is
broken, the double helix reverts to its normal relaxed state taking an alternative
conformation, called open-circular (oc). Super coiling is important in plasmid preparation
due to the easy separation of supercoiled molecules from non-supercoiled ones.

The commonly used methods of separation based on conformation are as follows:

(a). Alkaline denaturation method

This method is based on maintaining a very narrow pH range for the denaturation of non-
supercoiled DNA but not the supercoiled plasmid. • Addition of sodium hydroxide to cell
extract or cleared lysate (pH12.0-12.5) results in disruption of the hydrogen bonds of non-
supercoiled DNA molecules. • As a result, the double helix unwinds and two polynucleotide
chains separate. • Further addition of acid causes the aggregation of these denatured bacterial
DNA strands into a tangled mass which can be pelleted by centrifugation, leaving plasmid
DNA in the supernatant.

(b). Ethidium bromide-cesium chloride density gradient centrifugation

• Density gradient centrifugation can separate DNA, RNA and protein. It is a very efficient
method for obtaining pure plasmid DNA. • A density gradient is produced by centrifuging a
solution of cesium chloride at a very high speed which pulls the CsCl ions towards the
bottom. This process is referred as isopycnic centrifugation. • The DNA migrates to the point
at which it has density similar to that of CsCl i.e.1.7 g/cm3 in the gradient. • In contrast,
protein molecules having lower buoyant densities float at the top of the tube whereas RNA
gets pelleted at the bottom. Density gradient centrifugation in the presence of ethidium
bromide (EtBr) can be used to separate supercoiled DNA from non-super coiled molecules.
Ethidium bromide is an intercalating dye that binds to DNA molecules causing partial
unwinding of the double helix. Supercoiled DNA have very little freedom to unwind due to
absence of free ends and bind to a limited amount of EtBr resulting in very less decrease in
buoyant density (0.085 g/cm3 ) than that of linear DNA (0.125 g/cm3 ). As a result, they
form a distinct band separated from the linear bacterial DNA. The EtBr bound to DNA is
then extracted by n-butanol and the CsCl is removed by dialysis.