Biotechnology: Principles 2 Flashcards
after cutting how are dna fragments seperated?
: The cutting of DNA by
restriction endonucleases results in the fragments of DNA. These fragments
can be separated by a technique known as gel electrophoresis.
what is the principle behind gel electrophoresis
Since
DNA fragments are negatively charged molecules they can be separated
by forcing them to move towards the anode under an electric field through
a medium/matrix.
Nowadays the most commonly used matrix is agarose
which is a natural polymer extracted from sea weeds.
The DNA fragments
separate (resolve) according to their size through sieving effect provided
by the agarose gel. Hence, the smaller the fragment size, the farther it
moves.
how are the seperated dna seen?
The separated DNA fragments can be
visualised only after staining the DNA
with a compound known as ethidium
bromide followed by exposure to UV
radiation (you cannot see pure DNA
fragments in the visible light and
without staining). You can see bright
orange coloured bands of DNA in a
ethidium bromide stained gel
exposed to UV light
what is elution in gel electrophoresis
The
separated bands of DNA are cut out
from the agarose gel and extracted
from the gel piece. This step is known
as elution. The DNA fragments
purified in this way are used in
constructing recombinant DNA by
joining them with cloning vectors.
what are some things used as cloning vectors? plamids and bacteriophages are used because?
You know that plasmids and bacteriophages have the ability to replicate
within bacterial cells independent of the control of chromosomal DNA.
Bacteriophages because of their high number per cell, have very high
copy numbers of their genome within the bacterial cells. Some plasmids
may have only one or two copies per cell whereas others may have
15-100 copies per cell. Their numbers can go even higher. If we are able
to link an alien piece of DNA with bacteriophage or plasmid DNA, we can
multiply its numbers equal to the copy number of the plasmid or
bacteriophage.
what is ori point?
This is a sequence from where
replication starts and any piece of DNA when linked to this sequence
can be made to replicate within the host cells. This sequence is also
responsible for controlling the copy number of the linked DNA. So,
if one wants to recover many copies of the target DNA it should be
cloned in a vector whose origin support high copy number.
rop codes for
the proteins involved in the
replication of the plasmid.
what are selectable markers? what is its use? what are used as these markers?
In addition to ‘ori’, the vector requires a
selectable marker, which helps in identifying and eliminating nontransformants and selectively permitting the growth of the
transformants. Transformation is a procedure through which a
piece of DNA is introduced in a host bacterium (you will study the
process in subsequent section). Normally, the genes encoding
resistance to antibiotics such as ampicillin, chloramphenicol,
tetracycline or kanamycin, etc., are considered useful selectable
markers for E. coli. The normal E. coli cells do not carry resistance
against any of these antibiotics
what nature of cloning sites is preferable in cloning vectors?
In order to link the
alien DNA, the vector needs to have
very few, preferably single,
recognition sites for the commonly
used restriction enzymes. Presence of
more than one recognition sites within
the vector will generate several
fragments, which will complicate the
gene cloning.
There should be many restriction sites of different types, so that genes from diff sourc3s can be cloned into it. However it should not have more than 1 restriction site of the same enzyme/
how is antibitoic resistance gene used as a selectable marker
The ligation
of alien DNA is carried out at a
restriction site present in one of the two
antibiotic resistance genes. For
example, you can ligate a foreign DNA
at the BamH I site of tetracycline
resistance gene in the vector pBR322.
The recombinant plasmids will lose tetracycline resistance due to insertion of foreign DNA but can still be selected out from non-recombinant ones by
plating the transformants on
tetracycline containing medium.
The transformants growing on
ampicillin containing medium are then transferred on a medium
containing tetracycline. The recombinants will grow in ampicillin
containing medium but not on that containing tetracycline. But,
non- recombinants will grow on the medium containing both the
antibiotics.
In this case, one antibiotic resistance gene helps in
selecting the transformants, whereas the other antibiotic resistance gene gets ‘inactivated due to insertion’ of alien DNA, and helps in
selection of recombinants.
using beta galactosidase gene as a selectable marker? why are antibiotic resistance gene not used?
Selection of recombinants due to inactivation of antibiotics is a
cumbersome procedure because it requires simultaneous plating
on two plates having different antibiotics.
Therefore,alternative
selectable markers have been developed which differentiate
recombinants from non-recombinants on the basis of their ability
to produce colour in the presence of a chromogenic substrate.
In this, a recombinant DNA is inserted within the coding sequence of an enzyme, β-galactosidase. This results into inactivation of the
gene for synthesis of this enzyme, which is referred to as insertional
inactivation.
The presence of a chromogenic substrate gives blue coloured colonies if the plasmid in the bacteria does not have an insert. Presence of insert results into insertional inactivation of the
β-galactosidase gene and the colonies do not produce any colour, these are identified as recombinant colonies.
what is the suitable cloning vector for plant cell?
Agrobacterium tumifaciens, a pathogen of several dicot plants is able
to deliver a piece of DNA known as ‘T-DNA’ to transform normal
plant cells into a tumor and direct these tumor cells to produce the
chemicals required by the pathogen.
The tumor inducing (Ti) plasmid of Agrobacterium
tumifaciens has now been modified into a cloning vector which is no
more pathogenic to the plants but is still able to use the mechanisms
to deliver genes of our interest into a variety of plants.
how are cloning vectors delivered into animal cells
Similarly, retroviruses in animals
have the ability to transform normal cells into cancerous cells. Similarly,
retroviruses have also been disarmed and are now used to deliver
desirable genes into animal cells. So, once a gene or a DNA fragment
has been ligated into a suitable vector it is transferred into a bacterial,
plant or animal host (where it multiplies).
what does it mean to make a host competent for transformation
Since DNA is a hydrophilic molecule, it cannot pass through cell
membranes. In order to force bacteria to take up the plasmid, the
bacterial cells must first be made ‘competent’ to take up DNA.
how are bacterial cells made competent
This is
done by treating them with a specific concentration of a divalent cation,
such as calcium, which increases the efficiency with which DNA enters the bacterium through pores in its cell wall. Recombinant DNA can then
be forced into such cells by incubating the cells with recombinant DNA
on ice, followed by placing them briefly at 42 oC (heat shock), and then
putting them back on ice. This enables the bacteria to take up the
recombinant DNA.
ways to make host cells competent ( eukaryotic hosts)
This is not the only way to introduce alien DNA into host cells. In a
method known as micro-injection, recombinant DNA is directly injected
into the nucleus of an animal cell. In another method, suitable for plants,
cells are bombarded with high velocity micro-particles of gold or tungsten
coated with DNA in a method known as biolistics or gene gun. And the
last method uses ‘disarmed pathogen’ vectors, which when allowed to
infect the cell, transfer the recombinant DNA into the host.
