11. BIOTECHNOLOGY: PRINCIPLES & PROCESSES
TOOLS OF RECOMBINANT DNA TECHNOLOGY
1. Restriction Enzymes (‘molecular scissors’)
-
The enzymes that cut DNA at specific sites
into fragments.
- They
belong to a class of enzymes called nucleases.
- In 1963, two enzymes responsible for
restricting growth of bacteriophage in E. coli were isolated. One enzyme
added methyl groups to DNA. The other (restriction endonuclease) cut
DNA.
-
More than 900 restriction enzymes
have been isolated from over 230 strains of bacteria.
Naming of the restriction enzymes:
- First
letter indicates genus. The second two letters indicate species of prokaryotic
cell from which they were isolated.
E.g. EcoRI comes from E. coli RY
13 (R = the strain. Roman numbers = the order in which the enzymes were
isolated from that strain of bacteria).
Types of Restriction enzymes:
· Exonucleases:
They
remove nucleotides from the ends of the DNA.
·
Endonucleases:
-
They cut at specific positions within the
DNA. E.g. EcoRI.
-
They bind to specific recognition sequence
of the DNA and cut the two strands at specific points.
- The first restriction endonuclease is Hind
II. It cuts DNA molecules by recognizing a specific sequence of 6 base
pairs. This is called the recognition sequence for Hind II.
-
Restriction endonuclease recognizes a
specific palindromic nucleotide sequences in the DNA. It is a sequence
of base pairs that read the same on the two strands in 5' → 3' direction
and in 3' → 5' direction. E.g. Palindromic nucleotide sequence
for EcoRI is
5'
—— GAATTC —— 3'
3' —— CTTAAG —— 5'
Steps in
formation of recombinant DNA by EcoRI
- Restriction enzymes cut the strand a
little away from the centre of the palindrome sites, but between the same two
bases on the opposite strands. This leaves single stranded overhanging
stretches at the ends. They are called sticky ends. They form H-bonds
with their complementary cut counterparts. This stickiness facilitates action
of the enzyme DNA ligase.
-
When cut by the same restriction enzyme,
the resultant DNA fragments have the same kind of sticky-ends and these are
joined together by DNA ligases.
2. Cloning
Vector
-
It is a DNA molecule that can carry a
foreign DNA segment and replicate inside the host cells.
E.g. Plasmids, bacteriophages etc.
- Plasmids are autonomously
replicating circular extra-chromosomal DNA of
bacteria. Some plasmids have only 1-2 copies per cell. Others
have 15-100 copies per cell.
-
Bacteriophages
(high number per cell) have very high copy numbers of their genome within the
bacterial cells.
-
When the cloning vectors are multiplied in
the host, the linked piece of DNA is also multiplied to the numbers equal to
the copy number of the vectors.
Features required
for cloning into a vector
a. Origin of replication (ori)
-
This is a sequence where replication
starts.
-
A piece of DNA linked to ori can
replicate within the host cells. This also controls the copy number of linked
DNA. So, for getting many copies of the target DNA, it should be cloned in a
vector whose origin support high copy number.
b. Selectable marker (marker gene)
-
It is a gene that helps to select the transformants
and eliminate the non-transformants.
-
If a piece of DNA is introduced in a host
bacterium, it is called transformation. Such bacterium is transformant.
If transformation does not take place, it is non-transformant.
-
Selectable markers of E. coli
include the genes encoding resistance to antibiotics like ampicillin,
chloramphenicol, tetracycline, kanamycin etc. Normal E. coli cells
have no resistance against these antibiotics.
c. Cloning sites
-
These are the recognition
sites for restriction enzymes.
-
To link the alien DNA, the vector needs a
single or very few recognition sites.
-
More than one recognition sites generate
several fragments. It complicates the gene cloning.
-
Ligation of alien DNA is carried out at a
restriction site present in one of the two antibiotic resistance genes.
E.g. In vector pBR322, foreign
DNA is ligated at Bam H I site of tetracycline resistance gene. As a result, recombinant
plasmid is formed. If ligation does not occur, it is called non-recombinant
plasmid.
|
- When a foreign DNA is inserted within a
gene of bacteria, that gene is inactivated. It is called insertional
inactivation. Here, the recombinant plasmids lose tetracycline resistance
due to insertion of foreign DNA.
-
When the plasmids are introduced into E.
coli cells, 3 types of cells are obtained:
o
Non-transformants: They have no plasmid. So they are not resistant to either
tetracycline or ampicillin.
o Transformants with non-recombinant
plasmid: They are
resistant to both tetracycline & ampicillin.
o
Transformants with recombinant
plasmid: They are
resistant only to ampicillin.
- Recombinant plasmids can be selected out
from non-recombinant ones by plating transformants on ampicillin medium.
Then the transformants are transferred on tetracycline medium.
- The recombinants grow in ampicillin
medium but not on tetracycline medium. But, non-recombinants grow on
the medium containing both the antibiotics.
- Thus, one antibiotic resistance gene helps
to select the transformants. The inactivated antibiotic resistance gene helps to
select recombinants.
- But this type of selection
of recombinants is a difficult procedure because it needs simultaneous plating on
2 plates having different antibiotics. So, alternative selectable markers
have developed based on their ability to produce colour in presence of a chromogenic
substrate.
-
In this, a recombinant DNA is inserted into
the coding sequence (gene) of an enzyme, b-galactosidase. So,
the gene is inactivated (insertional inactivation). Such colonies do not
produce any colour. These are identified as recombinant colonies.
-
If the plasmid in bacteria have no an
insert, it gives blue coloured colonies in presence of chromogenic
substrate.
d. Vectors for cloning genes in plants & animals
Genetic
tools of some pathogens can be transformed into useful vectors for delivering genes to plants & animals. E.g.
· Agrobacterium
tumefaciens (a pathogen of many dicot plants) can
deliver a piece of DNA (T-DNA) to transform normal plant cells into a tumor.
These tumor cells produce the chemicals required by the pathogen.
The tumor inducing (Ti) plasmid of A. tumefaciens
is modified into a cloning vector which is not pathogenic but can use
mechanisms to deliver genes of interest into plants.
· Retroviruses
in animals can transform normal cells into cancerous cells. So, they are
used to deliver desirable genes into animal cells.
3. Competent
Host (For Transformation with Recombinant DNA)
- Since DNA is a hydrophilic
molecule, it cannot pass through cell membranes. So bacterial cells are made
‘competent’ to take up alien DNA or plasmid as follows:
- Treat bacterial cells with a specific
concentration of a divalent cation (e.g. calcium) → DNA enters the bacterium through
pores in cell wall → Incubate the cells with recombinant DNA on ice → Place
them briefly at 420C (heat shock) → Put them back on
ice → Bacteria take up recombinant DNA.
Other methods to introduce alien DNA into host cells
·
Micro-injection: In
this, recombinant DNA is directly injected into the nucleus of an animal cell.
· Biolistics (gene gun): In
this, cells are bombarded with high velocity micro-particles of gold or
tungsten coated with DNA. This method is suitable for plants.
· ‘Disarmed pathogen’ vectors: They
infect the cell and transfer the recombinant DNA into the host. E.g. A.
tumefaciens.
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Very concise and elucidated explanation.Realy very good
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