TOOLS OF RECOMBINANT DNA TECHNOLOGY
Three types of biological tools are used in the synthesis of recombinant DNA:
- Enzymes
- Cloning vectors (vehicle DNA)
- Competent host (for transformation with recombinant (DNA).
Enzymes
Different kinds of specific enzymes are used in genetic engineering (recombinant DNA technology). These include lysing enzymes, restriction (cleaving) enzymes, synthesizing enzymes, Joining enzymes and alkaline phosphatase.
Lysing enzyme
They are used to open up the cells to get DNA for genetic experiments. Lysozyme is usually used to dissolve the bacterial cell wall.
Restriction enzymes
Restriction enzymes (cleaving enzymes) are used to break DNA molecules. They belong to a larger class of enzymes called nucleases. Restriction enzymes are of three types: exonucleases, endonucleases and restriction endonucleases. Exonucleases remove nucleotides from the terminal ends (either 5 or 3') of DNA in one strand of duplex. Endonucleases make cuts at specific positions within theDNA. These enzymes do not cleave the ends and involve only one strand of the DNA duplex.Restriction endonucleases were found by Arber in 1962 in bacteria. They act as "molecular scissors or chemical scalpels.They recognize the specific base sequence at palindromes in DNA duplex and cut its strands.]
The palindromes in DNA are base pair sequences that are the same when read forward (left to right) or backward (right to left) from a central axis of symmetry.
for example, the following sequence read the same on the two strands in 5'
3 directions. 3 direction and in the 3'.
Restriction endonuclease EcoRI found in the colon bacteria E.coli, recognizes the base sequence GAATTC in DNA duplex and cuts its strands between G and A as shown below:
three main types of restriction endonucleases are typeI, type II and type III
Type I restriction endonucleases consist of 3 different subunits. They require ATP, Mg 2+ and S-adenosyl methionine for restriction. Type I restriction endonucleases recognize specific sites within the DNA but do not cut these sites
Type II restriction endonucleases are simple and requireMg ions for restriction. Only type II restriction enzymes are used in recombinant DNA technology because they can be used in vitro to recognize and cut within specific DNA sequence typically consisting of 4 to 8 nucleotides and cause cleavage at unmethylated sites within their recognition.
Type IlI restriction endonucleases are intermediate between type I and type II. They possess both the activities of restriction as well as methylation. Therefore, they are not used in recombinant DNA technology.The first restriction endonuclease was Hin d II (hin-dee-two). Its functioning depends on a specific DNA nucleotide sequence. It was isolated from Haemophilus influenzae.
Hin d always cut DNA molecules at a particular point by recognising a specific sequence of six base pairs. It produces blunt ends, Restriction enzymes cut the strand of DNA a little away
from the centre of the palindrome sites but between the same two bases of the opposite strands. This leaves single stranded unpaired bases at cut ends These ends with unpaired bases are called sticky ends or cohesive ends. They are named so because they form
hydrogen bonds with their complementary cut counter Parts The sticky ends facilitate the action of the enzyme DNA Ligase. Some restriction enzymes cut both the strands of a DNA
molecule at the same site so that the resulting ends have blunt or flush ends in which the two strands end at the same point Specific name (nomenclature) of enzyme is derived from
the name of the prokaryotic cell from which the enzyme is isolated. The first letter of the genus becomes the first letter of the name of enzyme which is written in capital letter The first two letters of species make second and third letters of the name of enzyme, which is written in small letters. All these three letters are written in italics. The fourth letter of the name of enzyme is the first letter of strain, so written in capital. The Roman number written at the end of the name indicates the order in which the enzyme was isolated from that strain of the prokaryotic cell.
Synthesizing enzymes
They play a role in the synthesis of DNA strands on suitable templates.They are further of 2 types; reverse transcriptases, which help in the synthesis of complementary DNA strands
on RNA templates and DNA polymerases which aid in the synthesis of complementary DNA strands on DNA templates.
Joining enzymes
Joining enzymes (DNA ligases or sealing enzymes) help in sealing gaps in DNA fragments which are otherwise joined by complementary base pairing. Tu ligases are
examples.These act as a molecular glue. They join DNA fragments by forming phosphodiester bonds.
Alkaline phosphatases
These cut phosphate groups from the S end of linearised circular DNA to check its recircularization.
Cloning vectors (Vehicle DNA)
The vectors are DNA molecules that can carry a foreignDNA segment and replicate inside the host cell.Vectors may be plasmids, bacteriophage, cosmids,phagemids, yeast artificial chromosomes (YACS), bacterial artificial chromosomes (BACS), transposons, and virus.
Out of these vectors,plasmid vectors and bacteriophage vectors are commonly used.
Plasmid
Plasmid vectors are extrachromosomal, self-replicating usually circular, double-stranded DNA molecules, found naturally in many bacteria and also in some yeast. It was discovered by William Hays and Joshua Lederberg in 1952.Plasmids are usually not essential for normal cell growth and division, they often confer some traits on the host organism.The plasmid molecules may be present as I or 2 copies or in multiple copies (500-700) inside the host organism.
The naturally occurring plasmids have been modified to serve as vectors in the laboratory. The most widely used, versatile, easily manipulated vector pBR322 is an ideal plasmid vector.
pBR322 vector
This was the first artificial cloning vector constructed in 1977 by Boliver and Rodriguez.
In the name pBR, p signifies plasmid, B is Boliver, and RIS trom Rodriguez, thus B and R represent the initials of the scientists who developed pBR322. The numeral '322' distinguishes, this plasmid from the other plasmids developed n the same laboratory, e.g., pBR325, pBR321,
pBR328, etc.
pBR322 plasmid contains the following regions:
-Origin of replication (Ori): It allows production of multiple copies per cell.
-Antibiotic resistance genes such as ampicillin resistance (amp) gene and tetracycline resistance (ter ) gene
-Unique recognition sites for restriction endonucleases.
pBR322 vector
Two unique sites, Pst I and Pvu I are located within the amp gene and BamH1, Sal 1, etc. are within the tet gene.Some other unique restriction sites are Eco RI, Cla I,Hin d I, Pvu IL. Rop codes for the proteins involved in the replication of the plasmid.
Bacteriophage vector
Bacteriophages are viruses that infect bacterial cells by injecting their DNA into these cells.
The injected DNA is selectively replicated and expressed in the host bacterial cell resulting in a number of phages which burst out of the cell lytic pathway) and reinfect neighbouring cells.Two phages that have been extensively modified for development of cloning vectors are lambda (Lambda) phage and M13 phage.Lambda phage vector has a double-stranded, linear DNA genome of A8, 514bp. in which the 12 bases at each end are unpaired but complementary.
These ends are sticky or cohesive and are referred to as the cos sites (cohesive end sites). These sites are important for packaging DNA into phage heads,An important feature of lambda (Lambda) genome is that a large fragment im the central region of its genome is not essential for lytic infection of E.coli cells.Therefore, vectors have been designed such that this region can be substituted or replaced by a foreign DNA.These vectors allow cloning of DNA fragments upto 23 Ka in size M13 phage vector is filamentous phage which infects coli having F-pili.Its genome is a single stranded, circular DNA of 6407bp Foreign DNA can be inserted into it without disrupting any of the essential genes.After the M13 phage DNA enters the bacterial cell, it is converted to a double-stranded molecule known as the replicative form or RE, which replicates until there are100 copies in the cell.
Cosmid vector
Cosmids have been constructed by combining certain features of plasmid and the cos' sites of phage lambda. The simplest cosmid vector contains a plasmid origin of
replication, selectable marker, suitable restriction enzyme sites and the lambda 'cos site.
Cosmids can be used to clone DNA fragments upto 45 kbp in length They can be packaged into Particles. This is more effñcient than plasmid transformation.
Bacterial artificial chromosome (BAC) vector
The vector is based on the natural, extra- chromosomal plasmid of E. coli. A BAC vector contains genes for replication and maintenance of the F-factor, a selectable marker and cloning site.These vectors can accommodate upto 300-350 kbp (kilo base pairs) of foreign DNA and are also being used in genome sequencing projects.
Yeast artificial chromosome (YAC) vector
These are used to clone DNA fragments of more than 1 Mb in size, therefore, they have been exploited extensively in mapping the large genomes, e.g., in the Human Genome
Project.These vectors contain the telomeric sequence, the centromere and the autonomously replicating sequence from yeast chromosomes They also contain restriction enzyme sites and genes which act as selectable markers in yeast
Phagemid vectors
phagemid vectors is a composite structure made of bacteriophage and plasmid, They are used for carrying larger DNA sequences.
Animal and plant viral vectors
Viruses that infect plant and animal cells can be used to introduce foreign DNA into plant and animal cells in culture.This is Known as plant and animal viral vectors.A Vector based on Simian Virus 40 (SV40) was used in the first cloning experiment involving mammalian cells in 1979 Since 1979, a number of vectors based on other types of viruses like adenovirus and papillomavirus have been used to clone genes in mammals. At Present, retrovirus vectors are the most commonly used vectors for cloning genes in mammalian cells in case of plants, plant viruses like cauliflower mosaic viruses, tobacco mosaic virus and gemini viruses were used but with limited success.
Transposon as vector
DNA sequences which change their location in the genome and hence are said to be mobile are called transposons.They are able to be excised from one locus and become inserted at separate locus. They are used as vectors.The activator (Ac) and dissociation (Ds) are popular
transposons of maize which are also called Ac-Ds Elements.The transposons of Drosophila are known as P-Elements.
Shuttle vectors
The plasmid vectors can replicate only in E.coli. Many of the vectors for use in eukaryotic cells are constructed such that they can exist in both the eukaryotic cells and E.coli Such vectors are known as shuttle vectors.in case of plants, a naturally occurring plasmid of the bacterium Agrobacterium tumefaciens called 1li plasmid has been suitably modified to function as a vector. Most of the eukaryotic vectors are in fact, shuttle vectors.
Characteristics of a cloning vector
Origin of replication
This is a sequence from where replication starts and a piece of foreign DNA is linked to this sequence The replication occurs inside the host cells. This sequena is also responsible for controlling the copy number of linker DNA.Therefore, if any person wants to produce many copie of the target DNA he/she should clone in a vector whose origin gives support for high copy numbers.
Selectable marker
The vector also requires a selectable marker (antibiotic resistance gene) to identify and eliminate non transformants and selectively permit the growth of the transformants.
Transformation is a process through which a piece of DNA is introduced in a host bacterium.
Generally, the genes encoding resistance to antibiotics such as tetracycline, ampicillin, kanamycin or chloramphenicol, etc. are useful in selectable markers for E. coli.
Recognition sites (cloning sites)
The vector must also have one unique restriction endonuclease recognition site to enable foreign DNA to be inserted into the vector during the generation of a
recombinant DNA molecule Presence of a unique restriction site allows the particular
enzyme to cut the vector only once. Most of the commonly used vectors contain unique
recognition sites for several restriction enzymes in a small region of DNA which is referred to as a polylinker or multiple cloning site (MCS).Polylinker provides flexibility in the choice of restriction enzyme(s) that can be used for cloning The ligation of alien DNA is carried out at a restriction site present in one of the two antibiotic resistance genes.
Competent host (For transformation with recombinant DNA)
Competent host bacterium is essential for transformation with recombinant DNA. Transformation is a process by which a cell takes up naked DNA fragment from the environment, incorporates it into its own chromosomal
DNA and finally expresses the trait controlled by the incoming DNA.Since DNA 1S a hydrophilic molecule, it cannot pass through membranes, so the bacterial cells must be made capable to take up DNA.This is done by treating them with a specific concentration of a divalent cation, such as calcium which increases the efficiency with which DNA enters the bacterium through pores in its cell wall.Eticient transformation takes only a few minutes and the cells are plated on a suitable medium for the selection of transformed clones.
Selection of the recombinant clones
When recombinant DNA is constructed and used for transformation of bacterium, the following types of bacterial cells are obtained:
Non-transformed cells (majority of the cells).
Transformed cells containing unaltered vector
Transformed cells having recombinant DNA.
Desired gene is isolated by a two-step indirect selection procedure.The first step of this procedure consists of identification and isolation. following the transformation of host cells, of the small number of cells that contain recombinant DNA from a large number of non transformed cells and those cells that are transformed by the unaltered vector molecules.The next step is to identify the clone having the desired DNA insert from among the large number of clones containing recombinant DNA molecules.This is generally achieved by inserting a selectable marker gene into the vector used for producing the recombinant DNAs.A selectable marker gene or reporter gene produces a phenotype, which permits either an easy selection of quick identification of the cells in which it is present.A selectable marker govems a reatire, which enables only those cells that possess them to survive under the selective conditions e g, an antibiotic like Kanamycin are good selectable markers. When A population of bacteria cells is plated on a kanamycin containing medium, only those cells that have the kanamycin resistance gene (kan)survive and form colonies,The non-transformed bacterial cells are eliminated by plating them on a medium containing the selection agent All the colonies that develop on the selective medium are transformed either by the unaltered vector or the recombinant DNA.The second step consists of identification and isolation of those clones that are transformed by the recombinant DNAs from among those that contain the unaltered vector.In case the vector has two selectable markers, e.g. pBR322, the DNA insert may be placed within one of these markers, say (amp) genes.The other marker, ter', is used for elimination of the non-recombinant cells. The transformed clones are then replica-plated on ampicillin containing medium. The clones containing the recombinant DNA will be sensitive to ampicillin due to the inactivation of ampr by the insertion of the DNA fragment. Such clones are identified and isolated from the master plate.Altemative selectable markers have been developed which differentiate recombinants from non-recombinants on the basis of their ability to produce colour in the presence of chromogenic substrate. In this, a recombinant DNA is inserted within the coding sequence of an enzyme, B-galactosidase. This results in inactivation of the enzyme, which is referred to as insertional inactivation.
The presence of a chromogenic substrate gives bluc coloured colonies if the plasmid in the bacteria does not have an insert. Presence of insert results into insertion inactivation of the B-galactosidase and the colonies do no produce any colour, these are identified as recombinant colonies.In animals the term transformation is replaced by the transfection
Vector less gene transfer
Different alternative methods have been used to introduce the recombinant DNA into recipient cells of animals without involving carrier molecules.
Microinjection
In this method foreign DNA is directly injected into the nucleus of an animal cell or plant cell by using micro needles or micro pipettes. It is used in oocytes, eggs and embryo
Electroporation
In this method the electrical impulses induce transient (temporary) pores in the plant cell membrane through which the DNA molecules are incorporated into the plant cells.
Gene gun or biolistic
DNA coated onto microscopic pellets of gold or tungsten of size 1-2 um is literally shot with high velocity into target cells. Although it is developed for plants, this technique is also used to insert genes into animal cells.
Direct DNA injection
Direct DNA injection on DNA into skeletal muscle led to the possibility of using genes as vaccines. Due to low level of expression therapeutic benefits for the treatment of genetic disorder could not be derived. This method gave birth to the Concept of DNA vaccine or genetic immunization.
Gel electrophoresis
separation and isolation of DNA fragments after the cutting of DNA by restriction enzymes, fragments of DNA are formed. these fragments can be separated by a technique called gel electrophoresis.Electrophoresis is a technique of separation of charged molecules under the influence of an electrical field so that they migrate in the direction of electrode bearing theoPposite charge, through a medium/matrix.he most commonly used matrix is agarose which is a polysaccharide extracted from sea weeds.DNA fragments separate according to size through the Pores of agarose gel.
The separated DNA fragments can be seen only after staining the DNA with a compound known as ethidium bromide (EtBr) followed by exposure to UV.radiation as
bright orange coloured bands. The separated bands of DNA are cut out from the agarose
gel and extracted from the gel piece. This step 1s called as elution
PROCESSES OF RECOMBINANT DNA TECHNOLOGY
(Isolation of the genetic material (DNA)
in order to cut the DNA with restriction enzýnes, 1tneeds to be in pure form, other macromolecules The DNA is enclosed with in DNA membrane so it has to break the cell open to release DNA along with other macromolecules such as RNA ,protein,polysaccharides and also lipid.This can be achieved by treating the bacterial cells plant or animal tissue with enzymes such as lysozyme (bacteria),cellulase (plant cells), chitinase (fungus)
The RNA can be removed by treatment with ribose nucleanse whereas protein can be removed by treatment with proteaseOther molecules are removed by proper treatments.The purified DNA finally precipitates out after the addition of chilled ethanol, This is seen as collection of the trends and suspensions
Cutting of DNA at specific locations
Restriction enzyme digestions are performed by incubating purged DNA molecules with the restriction enzyme, at the optimal conditions for that special enzyme. Agarose gel electrophoresis is employed to check the progression of a restriction enzyme digestion.
The joining of DNAinvolves several processes, After having cut the source DNA as well as the vector DNA with a specific restriction enzyme, the cut out 'gene of
interest trom the soiree DNA and the cut veetor with piace are mixed and ligase is added. 1 his results in the preparation of recombinant DNA
Amplification of gene of interest using PCR
Polymerase chain reaction (PCR) is a technique of synthesizing multiple copies of the desired gene (or DNA) in vitro. This technique was developed by Rary Mullis in 1985
lt is based on the principle that a DNA molecule, when subjected to high temperature. splits into two strands due to denaturation.
These single stranded molecules are then converted to original double stranded molecules by synthesizing new strands in presence of enzyme DNA polymerase.A double stranded molecule of DNA is duplicated and multiple copies of the original DNA sequence can be Generated by repeating the process several times.
The basic requirements of PCR are
-DNA template. 1he desired segment of the target DNA molecule that 1s to be amplified
-Two nucleotide primers. Two nucleotide primers, usually 10-18 nucleotides ions to the sequences present at the 3 borders of the target DNA segment
-Enzymes. High temperature (more than 90°C) stable DNA polymerase (usually taq polymerase) for Synthesis of new DNA molecules.
Procedure of PCR
A the start of PCR, the DNA from which a segment is to be amplified, an excess of 1he two primer molecules, to deoxynucleoside .triphosphates and the DNA.polymerase are mixed together in the reaction mixture that has appropriate quantities of Mg The PCR operation is followed in a sequence where denaturation, primer annealing and primer extension
Occurs.
Denaturation
The reaction mixture is first heated to a temperature between 90-98°C (commonly 94°C) that ensures DNA denaturation i.e The separation of the two strands Each single strand of the target DNA then acts as a template for DNA synthesis.
Primer annealing
The mixture is now cooled to a temperature (generally 40-60°C) that permits annealing
of the primer to the complementary sequences in the DNA; these sequences are located at the 3-ends of the two strands of the desired segment. Since the primer concentration is kept very high relative to that of the template DNA, primer-template hybrid formation is greatly favoured over reannealing of the template strands.
PCR (polymerase chain reaction)
Primer extension
The temperature i1s now so adjusted that the DNApolymerase synthesizes the complementary strands by utilizing 3 OH of the primers.This reaction is the same as that occurs in vivo during
replication of the leading strand of a DNA duplex.The primers are extended towards each other so that the DNA segment lying between the two primers is copied.This is ensured by employing primers complementary to the 3- ends of the segment to be amplified. The duration of primer extension is usually 2 min at 72°C.It has been shown that in case of longer target sequences, best results are obtained when the period of extension is kept at the rate of 1 min per Kb of the target sequence and the extension is carried out at 68 C in the place of usual 72 C.
Taq polymerase (1solated from a bacterium Thermus aquaticus) which remains active during the high temperature, usually amplifies DNA segments of upto 2 Kb.To begin the second cycle, the DNA is again heated to convert all the newly synthesized DNA into single strands,each of which can now serve as a template for synthesis of more new DNA.
Thus, the extension product of one cycle can serve as a template for subsequent cycles and each cycle essentially doubles the amount of DNA from the previous cycle.
As a result, from a single template molecule, it is possible to generate 2 molecules after n number of cycles.
Applications of PCR
Some of the areas of application of PCR are mentioned
here:
-Diagnosis of pathogens.
-Diagnosis of specific mutation.
-DNA fingerprinting
-Detecting specific microorganisms
-In prenatal diagnosis.
-Diagnosis of plant pathogens
-in palaeontology
-Gene therapy
Insertion of recombinant DNA into the host cell organism
The vector DNA (e.g. plasmid DNA) and alien (foreign) DNA carrying gene of interest are cut by the same restriction endonuclease to produce complementary sticky ends. This process of cutting DNA by restriction enzymes is called restriction digestion.
With the help of DNA ligase enzyme, the complementary sticky ends of the two DNAS are joined (annealing) to produce a recombinant (chimaera) DNA (FDNA). The ligase forms new sugar-phosphate bonds to join two DNAS.
Eukaryotic genes do not function properly when cloned into bacterial cells, because of their inability to excise introns of eukaryotic genes and their destruction by bacterial restriction enzymes.In such cases, DNA is made from mRNA by reverse transcription or synthesised artificially. This rDNA is inserted into the host bacterium by transformation using a cold CaCl solution.The bacterial cell containing the desired rDNA is selected using selective antibiotics in the culture medium.
Obtaining desirable gene product
