What are Plasmids?

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What are Plasmids?..

Answer / g.ravi sankar

plasmids are the closed circle self replicating
extrachromosomal gentic material

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What are Plasmids?..

Answer / pradeep

Many types of bacteria contain plasmid DNA. Plasmids are
extra chromosomal, double-stranded circular DNA molecules
generally containing 1,000 to 100,000 base pairs. Even the
largest plasmids are considerably smaller than the
chromosomal DNA of the bacterium, which can contain several
million base pairs. Certain plasmids replicate
independently of the chromosomal DNA and can be present in
hundreds of copies per cell. A wide variety of genes have
been discovered in plasmids. Some of them code for
antibiotic resistance and restriction enzymes. Plasmids are
extremely important tools in molecular cloning because they
are useful in propagating foreign genes. When plasmids are
used for these purposes, they are referred to as vectors.
Through the use of recombinant DNA technology, hundreds of
artificial vectors have been constructed from elements of
naturally occurring plasmids. These vectors have
specifically designed properties that make them useful in
solving particular experimental problems. For example,
synthetic oligodeoxynucleotide linkers have been
incorporated into many plasmid vectors. These linkers
contain many different restriction enzyme recognition sites
to facilitate the insertion of foreign DNA. The linkers are
often placed near characteristic marker genes or high
efficiency transcriptional promoters, both of which aid in
the isolation and expression of the cloned DNA.
Plasmid DNA naturally exists as a supercoiled molecule.
Supercoiling arises from alterations in the winding of the
two DNA strands around each other. In certain areas of the
molecule, the DNA strands are wound around each other less
frequently than in non-supercoiled DNA. The strain caused
by these alterations creates deformations in the DNA. These
deformations partially relieve the strain and ultimately
lead to supercoiling. Supercoiled DNA is folded onto itself
and has a more condensed and entangled structure than the
same DNA which is relaxed. As an analogy to supercoiling,
consider a rubber band. When the rubber band is twisted, it
eventually becomes knotted and collapses onto itself as an
entangled ball.
Purified DNA must be a covalently closed circle to exist as
a supercoiled molecule. Supercoiled plasmid DNA is often
called Form I DNA. Supercoiling in the cell is caused by
the action of enzymes called DNA gyrases. These enzymes use
the chemical energy in ATP to introduce supercoiling into a
relaxed molecule. In addition, there are enzymes that relax
supercoiled DNA and are called unwinding or relaxing
enzymes. Supercoiling has important biological
consequences. Very large DNA molecules would simply not fit
in the cell if they were not supercoiled. Gene expression
can also be influenced by supercoiling. If one or more
phosphate bonds anywhere in the backbone of supercoiled DNA
are broken, the molecule unravels to a relaxed form called
open circular DNA or Form II DNA. These breaks in the
phosphate backbone are called nicks. Nicked double-stranded
DNA is not covalently closed. The two strands of nicked DNA
are still held together by hydrogen bonds between the
bases. With time, purified supercoiled DNA slowly develops
nicks and converts to Form II. This is because supercoiled
DNA is not as stable as its relaxed or open circular forms.
Endonucleases, such as DNAse I, will randomly nick
supercoiled DNA when used in low amounts. Nicking can also
be introduced by mechanical manipulations during plasmid
purification. During replication, several of the same
plasmid molecules can form interlocked rings. These
multimers of plasmid are called catenanes. A catenane
containing two of the same plasmid molecules is called a
dimer. Similarly, those containing three or four molecules
are called trimers and tetramers, respectively. Each
plasmid molecule in a catenane can be supercoiled, however,
for clarity; they are represented as relaxed circles.
Agarose gel electrophoresis is a powerful separation method
frequently used to analyze plasmid DNA. The agarose gel
consists of microscopic pores that act as a molecular
sieve. Samples of DNA can be loaded into wells made in the
gel during molding. When an electric field is applied, the
DNA molecules are separated by the pores in the gel
according to their size and shape. Generally, smaller
molecules pass through the pores more easily than larger
ones. Since DNA has a strong negative charge at neutral pH,
it will migrate towards the positive electrode in the
electrophoresis apparatus. The rate at which a given DNA
molecule migrates through the gel depends not only on its
size and shape, but also on the type of electrophoresis
buffer, the gel concentration and the applied voltage.
Under the conditions that will be used for this experiment,
the different forms of the same plasmid DNA molecule have
the following rates of migration (in decreasing order):

Supercoiled > linear > Nicked Circles >dimer > trimer > etc.

Supercoiled DNA has the fastest migration rate of the
different forms of plasmid. In the plasmid extraction
experiment you will be doing, there will be some residual,
degraded RNA which consists of transfer RNA and digested
ribosomal and messenger RNA. Degraded RNA has a faster
migration rate than supercoiled plasmid DNA because it is
much smaller in size.
In the first step of the experiment a cell lysis solution
is added to the cells. This solution contains the detergent
sodium dodecyl sulfate (SDS) which dissolves the cell
membrane and denatures proteins. The solution is very
alkaline (pH > 12) due to the presence of sodium hydroxide.
The high pH aids in denaturing proteins and causes the
cleavage of the phosphate bonds in RNA. This eliminates
interference from high
molecular weight RNA during the plasmid purification. Under
highly alkaline conditions, the two strands in non-
supercoiled DNA (linear fragments of chromosomal DNA,
relaxed and nicked circular DNA) separate and are partially
removed from solution. However, this does not occur with
supercoiled forms of plasmid DNA because the two strands
are intertwined and entangled in a way that prevents them
from coming apart. Therefore, supercoiled plasmid remains
free in solution.
The potassium acetate neutralization buffer contains acetic
acid and potassium salts. The acidic buffer neutralizes the
alkaline conditions created by the sodium hydroxide. The
potassium causes the SDS, with its associated membrane
fragments and proteins, to precipitate. The chromosomal DNA
of E. coli is attached at several points to the cell
membrane. Centrifugation of the potassium-SDS-membrane
complexes also removes large amounts of entrapped
chromosomal DNA. The addition of isopropanol precipitates
the plasmid and remaining RNA. Tris buffer (diluted buffer
concentrate for RNase) is used to resuspend the DNA
precipitate in a higher concentration. The buffer contains
the enzyme RNase, which further degrades RNA. The
concentrated gel loading solution prepares the sample for
electrophoresis by making it denser than the
electrophoresis buffer. This enables the sample to sink
into the wells of the submerged gel. A negatively charged,
blue tracking dye is also present to monitor the
electrophoresis and to make sample loading easier.
In this experiment, a 3000 base pair plasmid will be
extracted from E. coli cells. The restriction map for this
plasmid has a single site for Eco RI. Digestion with the
enzyme will yield a single band measuring 3,000 ± 300
nucleotides. The multiple forms of the plasmid will be
converted to the linear form

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6 Yes

1 No

What are Plasmids?..

Answer / lakshmi tulasi

plasmids are extrachromosomal, circular, dsDNA molecules
which are self replecating molecules. plasmids are used as
vectors for the TRANSFER of desired DNA in r-dna technology.

Is This Answer Correct ?

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