Recombinant DNA Technology

1. Methods of Gene
Isolation
Promila Sheoran
Ph.D. Biotechnology
GJU S&T Hisar

2. There are three main methods for obtaining genes
1. Synthesising the gene using an automated gene machine
2. Gene Cloning
3. Using the PCR

3. Gene Machine
•Recently, fully automated commercial instrument called automated polynucleotide
synthesizer or gene machine is available in market which synthesizes predetermined
polynucleotide sequence.
• Therefore, the genes can be synthesized rapidly and in high amount. For example, a
gene for tRNA can be synthesized within a few days through gene machine.
• It automatically synthesizes the short segments of single stranded DNA under the
control of microprocessor.
•The working principle of a gene machine includes (i) development of insoluble silica
based support in the form of beads which provides support for solid phase synthesis
of DNA chain, and (ii) development of stable deoxyribonucleoside phosphoramidites
as synthons which are stable to oxidation and hydrolysis, and ideal for DNA synthesis.

4. •Four separate reservoirs
containing nucleotides (A,T,C
and G) are connected with a
tube to a cylinder (synthesizer
column) packed with small
silica beads.
• These beads provide support
for assembly of DNA
molecules. Reservoirs for
reagent and solvent are also
attached.
•The whole procedure of
adding or removing the
chemicals from the reagent
reservoir in time is controlled
by microcomputer control
system i.e. microprocessor.

5. •If one desires to synthesize a short polynucleotide with a sequence of nucleotides
T,G,C, the cylinder is first filled with beads with a single 'T' attached.
• Thereafter, it is flooded with 'G' from the reservoir. The right hand side of each G
is blocked by using chemicals from the reservoir so that its attachment with any
other Gs can be prevented.
•The remaining Gs which could not join with Ts are flushed from the cylinder. The
other chemicals are passed from the reagent and solvent reservoirs so that these
can remove the blocks from G which is attached with the T.
•In the same way this cycle is repeated by flooding with C from reservoir into the
cylinder. Finally the sequence T.G.C is synthesized on the silica beads which is
removed chemically later on.

6. •The desired sequence is entered on a key board and the microprocessor
automatically opens the valve of nucleotide reservoir, and chemical and solvent
reservoir.
• In the gene machine the nucleotides are added into a polynucleotide chain at the
rate of two nucleotides per hour.
• By feeding the instructions of human insulin gene in gene machine, human insulin
has been synthesized.

7. Gene Cloning
•Gene cloning is a common practice in molecular biology labs that is used by
researchers to create copies of a particular gene for downstream
applications, such as sequencing, mutagenesis, genotyping or heterologous
expression of a protein.
• The traditional technique for gene cloning involves the transfer of a DNA
fragment of interest from one organism to a self-replicating genetic element,
such as a bacterial plasmid.
•This technique is commonly used today for isolating long or unstudied
genes and protein expression.

8. Steps in a gene cloning experiment are:
1 A fragment of DNA, containing the gene
to be cloned, is inserted into a circular
DNA molecule called a vector, to produce
a recombinant DNA molecule.
2 The vector transports the gene into a
host cell, which is usually a bacterium,
although other types of living cell can be
used.
3 Within the host cell the vector multiplies,
producing numerous identical copies, not
only of itself but also of the gene that it
carries.
4 When the host cell divides, copies of the
recombinant DNA molecule are passed to
the progeny and further vector replication
takes place.
5 After a large number of cell divisions, a
colony, or clone, of identical host cells is
produced. Each cell in the clone contains one
or more copies of the recombinant
DNA molecule; the gene carried by the
recombinant molecule is now said to be
cloned.

9. Polymerase Chain Reaction
•The polymerase chain reaction can also be used to obtain a pure sample of a gene.
•This is because the region of the starting DNA molecule that is copied during PCR is
the
segment whose boundaries are marked by the annealing positions of the two
oligonucleotide primers.
• If the primers anneal either side of the gene of interest, many copies of that gene
will be synthesized. The outcome is the same as with a gene cloning experiment,
although the problem of selection does not arise because the desired gene is
automatically “selected” as a result of the positions at which the primers anneal.
•An alternative to cloning, polymerase chain reaction(PCR), can be used to directly
amplify rare specific DNA sequences in a complex mixture when the ends of the
sequence are known.

11. Genomic DNA is digested into large fragments using a restriction enzyme and then is
heat-denatured into single strands. Two synthetic oligonucleotides complementary to
the 3′ ends of the target DNA segment of interest are added in great excess to the
denatured DNA, and the temperature is lowered to 50 – 60 °C. The genomic DNA
remains denatured, because the complementary strands are at too low a concentration
to encounter each other during the period of incubation, but the specific
oligonucleotides, which are at a very high concentration, hybridize with their
complementary sequences in the genomic DNA. The hybridized oligonucleotides then
serve as primers for DNA chain synthesis, which begins upon addition of a supply of
deoxynucleotides and a temperature-resistant DNA polymerase such as that
from Thermus aquaticus (a bacterium that lives in hot springs). This enzyme, called Taq
polymerase, can extend the primers at temperatures up to 72 °C. When synthesis is
complete, the whole mixture is heated further (to 95 °C) to melt the newly formed DNA
duplexes. When the temperature is lowered again, another round of synthesis takes
place because excess primer is still present. Repeated cycles of synthesis (cooling) and
melting (heating) quickly amplify the sequence of interest. At each round, the number
of copies of the sequence between the primer sites is doubled; therefore, the desired
sequence increases exponentially.

12. Limitations of PCR:
•In order for the primers to anneal to the correct positions, either side of the gene
of interest, the sequences of these annealing sites must be known. It is easy to
synthesize a primer with a predetermined sequence, but if the sequences of the
annealing sites are unknown then the appropriate primers cannot be made. This
means that PCR cannot be used to isolate genes that have not been studied before—
that has to be done by cloning.
•There is a limit to the length of DNA sequence that can be copied by PCR.
Five kilobases (kb) can be copied fairly easily, and segments up to forty kb can be
dealt with by using specialized techniques, but this is shorter than the lengths of
many genes, especially those of humans and other vertebrates. Cloning must be
used if an intact version of a long gene is required.

13. Thank you