Methods of Gene Isolation

Promila SheoranPh.D. BiotechnologyGJU S&T Hisar

There are three main methods for obtaining genes

1. Synthesising the gene using an automated gene machine

2. Gene Cloning

3. Using the PCR

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.

•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.

•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.

•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.

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.

Steps in a gene cloning experiment are:

1 A fragment of DNA, containing the gene to be cloned, is inserted into a circularDNA 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 tothe progeny and further vector replication takes place.

5 After a large number of cell divisions, a colony, or clone, of identical host cells isproduced. Each cell in the clone contains one or more copies of the recombinantDNA molecule; the gene carried by the recombinant molecule is now said to becloned.

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 thesegment 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.

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.

Limitations of PCR:

•In order for the primers to anneal to the correct positions, either side of the geneof interest, the sequences of these annealing sites must be known. It is easy tosynthesize 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 bedealt with by using specialized techniques, but this is shorter than the lengths ofmany genes, especially those of humans and other vertebrates. Cloning must beused if an intact version of a long gene is required.

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