(ii) Insertion of the isolated gene in a suitable vector to
obtain recombinant DNA.
(iii) Introduction of the recombinant DNA into a
suitable organism/cell (usually E.coli) called host
(transformation).
(iv) Selection of the transformed host cells, and
identification of the clone containing the desired gene/
DNA fragment.
(v) Multiplication/expression of the introduced gene in the
host.
(vi) Where needed, transfer and expression of the gene
into another organism.
ORDiagram showing various steps of recombinant DNA
technology is as follows:
rDNAFormation of rDNArr- (a) In order to cut the DNA with restriction enzymes, it
needs to be in pure form, free-from other macro-
molecules. Since the DNA is enclosed within the
membrane, It is required to break the cell open to
release DNA along with other macromolecules such
as RNA, proteins or lipids. This can be achieved by
treating the bacterial cells, plant or animal tissue with
enzyme such as lysozyme (bacteria), cellulase (plant
cells), etc. Restriction enzyme digestion is performed
by incubating purified DNA molecules with restriction
enzyme. The cutting of DNA at specific location is done
by restriction enzyme which belongs to larger class
of enzymes called nucleases. They are of two types;
exonucleases (remove nucleotides from the ends of the
DNA), endonucleases (make cuts at specific positions
within the DNA).
(b) In polymerase chain reaction (PCR), multiple copies
of the gene (DNA) of interest are synthesised in vitro
using two set of primers (oligonucleotides that are
complementary to regions of DNA) and the enzyme
DNA polymerase. The segment of DNA can be amplified
to approximately billion times. The amplification is done
performing sequential steps such as DNA denaturation
(at 90 – 98°C), annealing of primer-template DNA
strands, and primer extension (polymerisation carried
out at 72°C). Such repeated amplifications is achieved
by the use of thermostable DNA polymerase (Ta q
polymerase obtained from Thermus aquaticus), which
remains active during high temperature induced
denaturation of double stranded DNA.
OR
(a) Enzyme-linked immunosorbent assay (ELISA) is a
non-isotopic immunoassay. ELISA is based on the
immunochemical principles of antigen antibody
reaction.
ELISA can detect very small amount of protein with the
help of enzymes, e.g., peroxidase, amylase and alkaline
phosphatase. ELISA is widely used for the determination
of small quantities of proteins (hormones, antigens,
antibodies) and other biological substances. The most
commonly used pregnancy test for the detection of
human chorionic gonadotropin (hCG) in urine is based
on ELISA. ELISA is also been used for diagnosis of HIV
viruses in AIDS patient.
(b) (i) Transgenic animals that produce useful biological
products can be created by the introduction of the
DNA segment (or gene) which code for a particular
product such as human protein (a-1-antitrypsin)
used to treat emphysema. Similar attempts are
being made for treatment of phenylketonuria
(PKU) and cystic fibrosis. Human genes encoding