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Techniques of Recombinant DNA Technology
Essay Contents:
- Essay on Isolation of Genetic Material
- Essay on the Amplification of Gene of Interest Using PCR
- Essay on the Preparation of the Gene
- Essay on the Detection of the Cloned Gene (Recombinants)
- Essay on Obtaining the Foreign Gene Product
- Essay on Down Streaming Processing
Essay # 1. Isolation of Genetic Material:
Following steps are required for isolation of genetic material DNA in pure form:
(i) Bacterial cells or plant cells or animal cells are treated with enzymes like lysozyme for bacteria cellulose for plant cells and chitinase for fungus. This will break the cell envelope open and like DNA, RNA, proteins, polysaccharides and lipids will be released.
(ii) As in eukaryotic cells DNA is interwined with protein molecules like histones. Additional Protein can be removed by treating with enzyme protease. RNA can be removed by treating with enzyme ribonuclease.
(iii) DNA sample can be further purified by using additional extraction techniques.
(iv) By addition of chilled ethanol, DNA Precipitates out and can be observed in the form of fine threads m suspension.
Agarose gel electrophoresis is employed to check the progression of restriction enzyme digestion. DNA being negatively charged moves towards anode (positive electrode). Same technique is used for vector DNA.
Essay # 2. Amplification of Gene of Interest Using PCR:
In PCR (Polymerase Chain Reaction) multiple copies of desired DNA (gene) can be formed in vitro.
Polymerase Chain Reaction (PCR) (Specific Sequences can be Amplified):
PCR technique was developed by Kary Mullis in 1985. If one knows the sequence of at least part of a DNA segment to be cloned, a number of copies of that DNA Sent can be hugely amplified using polymerase chain reaction. It is able to generate microgram (µg) quantities of DNA copies (up to billion copies) of desired DNA segment, present even as a single copy with in short time.
The technique is based on principle that when a DNA molecule is subjected to high temperature due to denaturation the two DNA strands separate. As a result two single stranded DNA molecules appear. DNA polymerase can copy these single stranded molecules. This leads to the formation of original DNA double stranded molecule. Due to repetition of this process several copies of DNA sequences can be formed.
Steps involved in PCR reaction:
Basic requirements for PCR reaction are:
(i) DNA template (desired segment) to be amplified,
(ii) Two nucleotide primers (usually 10-18 nucleotides long) specific i.e., complementary to the sequences present at the 3 ends of the desired DNA segment. These primers are oriented with their ends facing each other permitting formation of DNA between them.
(iii) High temperature (790°C) stable DNA polymerase. It is needed for the formation of new DNA Usually used DNA polymerase for PCR reactions is Taq polymerase.
a. Isolated target DNA segment to be amplified is heated to high temperature (94°C) for denaturation. It leads to separation of two DNA strands.
b. Next step is of annealing. Here each single strand of target DNA acts as template for DNA synthesis. It is cooled (40°-60C) in presence of large excess of synthetic oligonucleotide primers. In annealing two oligonucleotide primers anneal or hybridize to each of single stranded template DNA. Annealing sequences are located at 3′ end of two strands of desired segment.
c. It is followed by extension step. Here Taq DNA polymerase synthesizes the complementary strand by using, 3’-OH of primer. The primers extend towards each other so that DNA segment lying between two primers is copied. This step requires dNTPs (deoxynucleoside triphosphates) and Mg2+. Temperature required for this step is 72°C.
The enzyme extends the primers using the genomic DNA as template and nucleotides made available in reaction. Here DNA replication occurs several times. Segment DNA gets amplified to approximately billion times.
As a result 9 billion copies can be formed. Repeated amplification is possible by use of thermostable DNA polymerase. It is isolated from bacterium, Thermus aquaticus. This enzyme remains active during high temperature induced denaturation of double helix DNA.
Essay # 3. Preparation of the Gene:
Gene coining in bacteria is achieved by cleaving the purified DNA with enzyme restriction endonuclease which produces small fragments (approximately 4 kilobase pairs). Each fragment has a stickly’ complementary single-stranded end. Eukaryotic genes contain introns that are not processed in bacteria therefore; DNA for cloning is usually obtained as a reverse transcriptase generated copy DNA (cDNA) of the relevant mRNA. In cases where nucleotide or amino acid sequences are known, synthetic DNA may be produced.
The vector is an agent which is used to transfer DNA into a host cell e.g., plasmid, bacteriophage. The vector is cut with the same enzyme (restriction endonuclease) as that used to generate the chromosomal DNA fragments. The chromosomal fragments and linearised vector are incubated with DNA ligase which covalently joins the DNA molecules (Fig. 11.21). Those plasmids which contain an inserted fragment are called recombinant plasmid.
Transformation of Host Cell:
The ligated plasmid mixture is introduced into the bacterial cell where they take up DNA through transformation process. Transformation is generally carried out by placing actively growing cells of a bacterium in cold, dilute solution of CaCl2 which enhances the ability of bacterial cells to take up foreign DNA.
In majority of the cases, E. coli is the most preferred host because:
(i) Molecular biology of this bacterium is well understood,
(ii) Calcium chloride treated cells are highly transformable, and
(iii) E. coli transcribes and translates most Gram-positive and Gram-negative genes except some actinomycete genes.
There are many methods to introduce the ligated DNA into recipient competent cell. Suppose a recombinant DNA having ampicillin antibiotic resistant gene is transferred to E. coli cells, the host cells become transformed into ampicillin resistant cells. When such cells are spread over agar plates containing ampicillin on transformants will grow there. Ampicillin resistance gene is called ‘selectable marker’ as due to this transformed cell can be selected in presence of ampicillin.
Essay # 4. Detection of the Cloned Gene (Recombinants):
Cells with recombinant DNA (rDNA) are selected on the expression or non-expression of some traits like resistance to antibiotic chloramphenicol. Direct selection of recombinants is made due to encoding of these traits by vector or cloned DNA sequence.
Various methods for identification of recombinants are:
1. Transformants (host cells with foreign DNA) can be selected by:
(i) Host cells transformed with plasmid having ampicillin resistant gene are grown on medium having antibiotic ampicillin, only those cells bearing the above plasmid will be able to grow on it.
(ii) But one is not able to know that which colony bear recombinant plasmid and which bear relegated vector plasmid.
2. Insertional Inactivation Method:
It is based on basic principle that cloned DNA fragment disrupts the coding sequence of gene.
To identify recombinants, one of the important approaches is to use DNA probe. In a DNA molecule, the two complementary strands are held together by hydrogen bonds. If two similar DNA pieces are mixed together and hydrogen bonds broken (by heating) the strands will separate.
Upon lowering the temperature, the hydrogen bonds are formed again. Some of the resultant double-stranded DNA will be hybrids i.e., composed of one strand of one type and one strand of the other type. This concept of DNA hybridization has been exploited for utilizing the DNA molecules as probes (Fig. 11.22).
The transformed colonies are replica plated to a nitrocellulose filter and are lysed to release the DNA. This DNA is denatured (by raising the temperature) and fixed to the nitrocellulose so as to produce a DNA print corresponding exactly to the position of the colonies on the original plate.
The DNA print is then hybridized with the probe which has been previously radioactively labelled. After washing off unhybridized DNA, the position of the radioactive spots on the filter is indicated by autoradiography in order to identify the presence of the required DNA.
Essay # 5. Obtaining the Foreign Gene Product:
When alien DNA is inserted into cloning vector and then transferred to host cell, DNA gets multiplied. Due to expression of this foreign gene, proteins can be formed.
Such target proteins (recombinant proteins) are to be produced in large scale. The cells having cloned genes may be grown on small scale in laboratory. Such cultures are used to make proteins with required characters. For multiplication of cells continuous culture for system is used. Here medium is drained out and fresh medium is added. This helps in active growth of cells during log/exponential phase.
To produce this product in large quantities bioreactors are needed. In such bioreactors 100-1000 litres of culture can be processed.
Most commonly used in stirring type bioreactor, whose details are given below?
Fermenter (Bioreactor):
Structure:
The basic design of a stirred-tank fermenter is shown in Fig. 11.23. It consists of a large stainless steel vessel with a capacity of upto 500,000 dm3 around which there is a jacket of circulatory water used to control the temperature within the fermenter. There is also an agitator, comprising of a series of flat blades, which can be rotated with the help of a motor. This ensures the thoroughly mixing of the contents so that nutrients come in close with the micro-organisms. The agitator also prevents settling out of the cells at the bottom.
Fermenter also has adequate arrangement for aeration, temperature and pH control. For proper aeration, air can be forced in at the bottom of the tank through a porous ring, called sparger, by the process called sparging, while there is an outlet to remove air and waste gases at the top of the tank.
The top of the tank also a number of inlet tubes called ports, through which materials can be introduced or withdrawn e.g:
i. Inoculation port for introducing initial inoculum;
ii. Nutrient port for introducing more nutrients;
iii. Antifoam port for introducing antifoaming agents; and
iv. pH port for introducing acid or alkali to maintain optimal pH.
At the base of the tank, there is a harvest line to extract culture medium and microbial products. To regularly detect the pH and temperature changes, tank is fitted with certain probes.
Significance:
The stirred-tank fermenter is a well- tried and tested design for large-scale production of micro-organisms under aseptic and controlled environment for a number of days. Small-scale fermenters of 10-100 litres capacity are used in research laboratories. It is also provided with many controls for the monitoring of physical, chemical and biological parameters that affect the growth of cells.
Drawbacks:
It is relatively costly to run largely due to high energy requirements to drive the agitators and introduce the compressed air.
Essay # 6. Down Streaming Processing:
Products formed are separated and purified. Steps are collectively called as down streaming processing. Suitable preservatives are used. For medicinal purposes, clinical trials are carried out. Quality control is also maintained.