The techniques developed in the early 1970s for isolation, cutting and joining of DNA molecules are used for engineering and analysis of DNA and RNA molecules. The basic tools that are required to generate recombinant host cells are described below:
1. Isolation and Purification of DNA:
The host cells that contain genes of desired function (such as Islets of Langerhans of pancreas that secretes insulin) are disrupted mechanically so that intracellular components including nucleic acids may be released.
DNA Is purified and recovered using many techniques such as centrifugation, electrophoresis, precipitation, etc. Comparative properties of viral, bacterial and fungal chromosomes are given in.
The ‘restriction-modification system’ provides defense in bacteria. There are two components present in this system:
1. The first component is the restriction enzyme which recognises a specific DNA sequence and cut any strand which possesses that sequence. The term ‘restriction’ refers to its function because it restricts the foreign DNA (of bacteriophages) and degrades them.
2. The second component is the modification system which modifies the bacterial DNA sequence recognised by phage restriction enzymes, by adding a methyl group to one or two nucleotide(s).
Therefore the restriction enzymes fail to recognise and degrade the modified bacterial DNA. Following these’ mechanisms the bacteria protect its own chromosome from degradation by restriction enzymes of bacteriophage origin.
Thus different bacteria consist of different restriction end nucleases and in correspondence to these, the methylases too are produced.
Mb= million base pairs; K= thousand; nt= nucleotides
2. Restriction Enzymes – The Molecular Scissors:
The restriction enzymes are called as ‘molecular scissors’. These acts as foundation of rDNA technology. These enzymes are present in bacteria and provide a type of defense mechanism called the ‘restriction-modification system’. Molecular basis of these systems was elucidated first by Werner Arber in 1965.
(a) Types of Restriction Endonuclease:
There are three main types of restriction endonuclease. These are designated as Type I, Type II and Type III. Each enzyme differs slightly by different mode of action. Type I and Type D are large multi sub-unit complexes which contain both endonuclease and methylase activity.
(i) Type I:
This enzyme cuts DNA at random sites that can be more than 1000 base pairs from the recognition sequence. These move along the DNA in a reaction and require Mg++, S-adenosyl methionine and ATP as co-factor.
(ii) Type II:
This type of restriction enzyme was first isolated by Hamilton Smith. These are simple and require no ATP for degradation of DNA. They cut DNA within the recognition sites. Daniel Nathans first used these enzymes for mapping and analysing genes and genomes.
(iii) Type III:
This group of endonucleases cut the DNA about 25 base pairs from the recognition sequence. In a reaction, it moves along the DNA and requires ATP as source of energy.
The first (I) Type II restriction enzyme isolated and characterised was from Escherichia coli strain RY. Hence, it was named as EcoRl.
It is named by using first letter of genus, two letters of species, one of strain and one Roman number to denote the order of discovery of enzymes. For the discovery of restriction enzymes, W. Arber, H. Smith and D. Nathans were awarded with Nobel Prize in 1978.
(b) Cleavage of Recognition Sequences:
Thousands of restrictions enzymes have been discovered and some of them are commercially available. Out of above three types of restriction endonucleases, Type II are used in gene manipulation.
Because they can be used in vitro to cut the DNA at recognition sites which are usually 4 to 8 base pairs long and palindromic in nature. The length of recognition sites of different enzymes varies. Different types of Type II restriction enzymes, their source and recognition sites are given in Table 3.2.
(i) EcoRI enzyme binds to a region of having specific palindromic sequence (where two strands are identical when both are read in the same polarity i.e. in 5′-8′ direction). The length of this region is 6 base pairs i.e., hexanucleotide palindrome.
It cuts between G and A residues of each strand and produces two single stranded complementary cut ends which are asymmetrical having 5′ overhangs of 4 nucleotides. These ends are called sticky ends or cohesive ends. Because nucleotide bases of this region can pair and stick the
(ii) On the other hand there are some other Type II restriction enzymes which cleave both strands of DNA at the same base pairs but in the centre of recognition sequence, and results in DNA fragments with blunt ends or flush ends. For example Hae III (isolated from Haemophilus aegypticus, the order of enzyme
(III) Four nucleotide long palindromic sequence and cuts symmetrically the both DNA strands and forms blunt ends as below:
(c) Construction of Recombinant DNA Molecules:
A recombinant DNA molecule can be generated by cutting two different DNA fragments with the same restriction enzyme and mixing the fragments together.
The two different DNA fragments join together due to the presence of sticky ends (identical single stranded nucleotides) on both different fragments.
(i) Restriction Fragment Length Polymorphism (RFLP):
DNA of each organism has specific sequence which can be cleared by several restriction enzymes and fragments of different length can be produced. These fragments are called restriction fragments.
Restriction fragments of different individuals and species vary; these are of different length because of variation in DNA sequence of restriction sites. Therefore, these variations are referred to as restriction fragment length polymorphism (RFLP).
Assume that there are two individuals A and B. Isolate separately their genomic DNA. Digest their DNA by using a restriction endonuclease ‘A’. This enzyme cleaves the DNA material into 4 different fragments of varying length because recognition site of enzyme A varies on the DNA.
These fragments are separated on agarose gel. Different DNA fragments are formed by RFLPs on the gel. The RFLP technique is used to distinguish between different DNA fragments digested by a restriction enzyme. It is very useful because DNA fingerprinting (cDNA profiling or DNA typing) is based on RFLP technique.
3. Use of Other Enzymes in rDNA Technology :
There are many other enzymes too which are used in rDNA technology. Some of the important enzymes are given below:
(i) DNA polymerase I:
It synthesises DNA complementary to DNA template in 5′-8′ direction. It lacks 5′-3′ exocatalytic activity.
(ii) Exonuclease III:
It cleaves from the end of a linear DNA and digests double stranded DNA from the 3′-end only.
(iii) DNA Ligases:
DNA ligase has the physiological role in sealing single strand nicks in the double stranded DNA. It forms phosphodiester bond between two adjacent nucleotides. There are two types of DNA ligase: E. coli DNA ligase (which uses NAD as source of energy) and T4 DNA ligase (which uses ATP as source of energy).
(iv) Alkaline Phosphatase (AP):
The 5’phosphate group is essentially required for ligation of DNA fragments. But DNA cannot be ligated if 5′-phosphate group is removed.
The enzyme AP removes 5’phosphate group from the 5′-end of DNA fragment and makes it free. This enzyme is used to check self-ligation of vector DNA where there is a problem of recircularisation. The sources of AP is bacteria (BAP) or calf intestine (CAP).
(v) Reverse Transcriptase:
It is a RNA dependent DNA polymerase which synthesises DNA complementary to a RNA template in 5′->3′ direction.
(vi) RNase A:
It is a nuclease which digests RNA but not DNA.
(vii) Taq DNA Polymerase:
It is a DNA polymerase isolated from a thermophilic bacterium (Thermus aquaticus). It operates at 72°C but it is stable above 90°C. Taq enzyme is used in PCR.
(viii) Terminal Transferase:
This enzyme adds several nucleotides to 3′-end of a linear or double stranded DNA or RNA.
(ix) SI Nuclease:
SI nuclease acts on single strands of double stranded DNA and results in blunt ends in DNA fragments.
4. Cloning Vectors or Vehicle DNA :
Another major biological tool in recombinant DNA technology is the vector which is used for delivery of desired foeign DNA into a host cell. It acts as a vehicle or carrier. The vectors must possess the following features when acting as cloning vehicle.
(i) It should easily be isolated from the organisms.
(ii) It should be small in size because larger vector DNA molecules often get broken during purification.
Several vectors have been constructed artificially which can fulfil the above criteria or their desired features. The vast majority of vectors are plasmids and remainders are constructed based on phage X and Ml3. Examples of some of the cloning vectors are given in
(a) Bacterial Plasmids:
Plasmids are the extra-chromosomal, self-replicating, circular, doub’ stranded DNA molecules naturally found in all bacteria and some fungi. Plasmids are maintained in bacteria as independent entities.
Usually plasmids do not take part in cell growth but confer some additional traits to bacteria e.g. resistance to certain antibiotics (R plasmids), its own transfer from cell to another plasmid (F plasmid), utilization of unusual metabolites (degradative plasmids), or no apparent functions (cryptic plasmids).
Size of plasmids varies from 1 to 500 kilo base pairs. Numb, of plasmids per cell may be 1 to 4 copies or 10 to 100 copies.
Generally, the naturally occurri plasmids lack several important features which are required for a high quality cloning vectors (described earlier). Therefore, according to requirement plasmids are modified in laboratory so as to act as efficient cloning vector.
There are several plasmid cloning vectors such as pBR322, pSC102, ColEl, pUC, pRP4, pRK2, pRSFlOlO, pEY, pWWO, Ti- and Ri- DNA plasmids, etc.
(i) Plasmid pBR322:
ThepBR322is most often used general purpose plasmid cloning vector. The pBR320 contains 4,361 base pairs.
It carries two antibiotic resistance genes: tef confers resistance to tetracycline and amp confers resistance to ampicilln. Several restriction sites for restriction enzymes are present on the plasmid. It has origin for replication (ori) site too that functions only in E. coli.
(ii) Sequence (MCS) is incorporated into lacZ gene without interfering the function of other genes. The MCS has the unique sites for several restriction endonucleases. It also carries one ori gene of E. coli. This plasmid works in prokaryotes.
It is a naturally occurring plasmid of Agro bacterium tumefactions. It is modified to act as a vector. By using Ti-DNA-based plasmids several transgenic plants have been constructed. Ti-plasmids are very large ranging from 150 to 200 kb. Physical map of the octopine plasmid (pTiB806) is given in Fig. 3.4C.
(iv) Shuttle Vectors:
The prokaryotic vectors cannot exist and work in eukaryotic cells. Therefore, several vectors have been constructed which exist both in prokaryotic (E. coli) and eukaryotic cells. Such vectors having two origin for replication i.e. oriE and oriEuk are called ‘shuttle vector’.
The oriE functions in E. coli and oriEuk functions in eukaryotic cells. Besides, these vectors also contain the antibiotic resistance genes (e.g. ampr) that act as selectable marker.
One of the examples of shuttle vectors is the yeast episomal plasmid (YEP). The YEP is a 2 circle plasmid-based vector which contain 2 µ origin of replication, E. coli shuttle sequence and a selectable marker i.e. Ieu2 yeast gene.
(v) Expression Vectors:
In addition to incorporation desired gene into the host cell, the objectives of rDNA technology is to produce high amount of proteins encoded by the DNA insert. To achieve this goal, the introduced novel gene must express.
Hence, expression of cloned genes is carried out by inserting a ‘promotor sequence’ (signal for initiation of transcription), and a ‘terminator sequence’ (that provides signal for termination of transcription). Near cloning site a translation initiation sequence (a ribosome binding site and short codon) is also incorporated into the vector.
The cloning vectors which contain these signals for protein synthesis are called expression vectors. An expression vector pSOMI containing promotor-operator (PO) for production of a chain of somatostatin (soml) is given in.
(b) Bacteriophage Vectors:
Bacteriophages are the viruses that infect bacterial cells after injecting their genetic material (DNA or RNA) and kill them. The viral DNA replicates and expresses inside the bacterial cells, and produces a number of phage particles released after bursting the bacterial cells. This is called lytic cycle of bacteriophage.
The released phages re-infect the live cells. The ability of transferring the viral DNA from phage capsid specific bacterial cell gave insight to the scientists to exploit bacteriophages and design them as cloning vectors.
The two bacteriophages e.g. phage X and Ml3 have been modified and used as cloning vectors.
(i) Phage X:
Phage X infects E. coli. It has a double stranded DNA genome of 48.514 kb. It exists as linear molecule with complementary 12 nucleotides long single stranded projections at each end. Therefore, it is circularised after introduction into E. coli cells.
The 12 nucleotide long projections show cohesiveness and form the COS site (cohesive site). Phage genome has a large non-essential region which is not involved in cell lysis.
Taking advantage of it two types of cloning vectors can be produced, either by inserting foreign DNA (insertion vectors) or replacing by a foreign DNA (replacement vectors). The upper limit of foreign DNA to be packed is about 23 kb.
M13 is a filamentous phage of E. coli which infects only such cells which contain sex pili. M13 consists of single stranded circular DNA molecule having 6,407 bases. It lands on sex pilus and delivers its DNA into the cell through the lumen of pilus.
After delivery, the DNA is converted from single stranded molecule to double stranded molecule. It is called replicative form (RF) of DNA. The RF of M13 replicates and forms 50-100 RF) molecules per cell.
Finally, single stranded DNA molecules are produced from RF DNA and extrude from the cell as M13 particles. The progeny molecules are packed upon extrusion through the cell wall. The cells are not killed but grow slowly.
Foreign DNA of about 500 bp can be cloned into a multiple cloning sequence that form part of a cloned modified lacZ gene on the double stranded RF of Ml3. Different strategies are followed when large sized foreign DNA is to be cloned.
For the development of M13-based vector, the RF can be purified and manipulated. Foreign DNA inserted into M13 vectors can be procured as single stranded DNA. This form of cloned genes is very useful for DNA sequencing and site-directed mutagenesis as discussed in preceding sections.
(ii) Cosmid Vectors:
For the first time J. Collins and B. Hohn first developed the cosmid in 1978. Cosmids are such vectors that are constructed by uniting some part of plasmid DNA with COS site of phage X (COS site + plasmid = cosmid).
Cosmid vector possesses an ori (origin for replication) gene, a selectable genetic marker (for antibiotic resistance), suitable recognition sites for restriction enzymes (i.e. cloning sites) and cohesive COS site.
Cosmid verctor circularises through COS site and replicates as a plasmid. The upper limit of foreign DNA to be packed into cosmid cosmid for cloning is about 45 kb.
(iii) YAC Vectors:
Yeast artificial chromosomes (YAC) have been developed as high capacity vector which can clone very large DNA segment of about 1 Mb in length. It is maintained as a separate chromosome in the cell.
It has been used for physical mapping of human chromosome in ‘Human Genome Project’.
The pYAC plasmid contains the E. coli origin of replication (oriE) and a selectable marker (ampr), a yeast DNA sequence, genes each for uracil biosynthesis pathway (URA3) providing centromeric function (CEN), automatic replication sequence (ARS), tryptophan synthesis pathway (TRP) and telomeric (T) sequence. There are recognition sites for restriction enzymes such as Sma\ and BamHl.
(iv) BAC Vectors:
Bacterial artificial chromosomes (BAC) are used as cloning vectors. BAC are constructed by using Fertility or F factors (present on F plasmid) of E. coli.
BAC vectors contain ori gene, a gene for maintenance of F factor, a selectable marker (an antibiotic resistance gene) and many restriction sites for insertion of foreign DNA. The upper limit of foreign DNA to be inserted in BAC is about 300-3500 kb. It is used in geneome sequencing projects.
(c) Animal Viral Vectors:
In nature there are several viruses which cause diseases. A virus gets adsorbed to the body surface of suitable host and infects the cell. This ability of animal viruses has been exploited and virus-based vectors have been designed to introduce foreign DNA of known function into cultured eukaryotic cell.
In 1979, for the first time a simivian virus 40 (SV40)-based cloning vector was constructed and used in cloning experiment using mammalian cells. Since then several vectors were constructed using adenovirus, papillomaviruses, retroviruses to clone foreign DNA into the mammalian cells, and Baculoviruses in insect cells.
In recent years, retrovirus-based vectors are commonly used for gene cloning. The gag, pol and env genes of retroviruses (which are required for replication and assembly of viral particles) can be replaced with foreign DNA. The rDNA is introduced into mammalian cells in tissue culture.
(d) Plant Viral Vectors:
There are several plant viruses which cause serious loss to the crop plants. Some of the viruses [e.g. tobacco mosaic virus (TMV), cauliflower mosaic virus (CaMV), potyvirus, gemnivirus, etc.] have been used as vectors but success have been achieved to a limited extent. Some of the features of CaMV is attributed to use it as a vector.
One of the features is that its naked DNA is ineffective and enters the plant cells directly if rubbed onto a leaf after mild abrasion. In addition, size of CaMV genome is small; hence all genome is essential which cannot be detected to insert foreign DNA. It consists of a very strong promoter but its limited host ranges warrants its possible use as efficient vector.
A second approach is the transfer of pathogen-derived resistance (PDR) gene into the host plant. For PDR, a part of complete viral gene is introduced into the host which interfere one or more essential steps in the life cycle of the virus.
For the first time Roger Beachy introduced coat protein (CP) of TMV into tobacco and observed TMV resistance in the transgenic tobacco plant.
5. Host Cells :
For the multiplication of foreign DNA efficient host cells are required. Different types of host cells such as E. coli, yeast, plant and animal cells are available for gene cloning. These cells are used according to the aim of the experiment.
In spite of many types of cells of different groups of organisms, E. coli has been most successfully used because (i) it is easy to handle, (ii) it doubles its cell number in each 20 minutes, (iii) the rDNA also reproduce along with doubling of bacteria, (iv) within hours thousands of bacteria cells and that much number of foreign genes are produced and is recombinant proteins are expressed. The proteins are isolated and purified from the cells.
In addition, for expression of eukaryotic gene the eukaryotic hosts like yeast cells are exploited. Like E. coli, yeasts also have several advantages such as (i) easy to grow and manipulate, (ii) simplest unicellular eukaryote, (iii) well characterised, (iv) requirement of no complex medium, etc., and (v) their multiplication in laboratory in a small vessel or a large sized fermentor.
The eukaryotic foreign gene requires eukaryoteic cell for its expression because (i) the eukaryotic host cell contains splicing enzymes for removal of introns but not the prokaryotic cells, (ii) the enzyme system of the eukaryotic host facilitates the proper folding of protein into 3D structure and their modification (addition of methyl group to protein, etc.).
The cultured plant and animal cells or their whole body is used for introduction of foreign gene. Following these strategies many plant and animals have been genetically modified and transgenic plants and animals have been produced.
6. Construction of Recombinant DNA Molecules :
The strategy of making recombinant DNA has earlier been discussed in Section 1. Its first step is to isolate or procure foreign DNA of desired function and efficient cloning vector. The DNA to be cloned and cloning vectors contain recognition sites for the cleavage by restriction endonucleases.
These two types of DNA molecules are separately digested with the same restriction enzyme so as to generate sticky ends. The sticky ends of foreign DNA and that of plasmid vector are mixed and incubated in the presence of DNA ligase. Consequently, hydrogen bonds are formed between complementary nucleotides.
DNA ligase forms phosphodiester bonds between two sticky ends of target DNA and vector DNA. This results in formation of covalently joined recombinant DNA (rDNA). There is another possibility of combining sticky ends and being circularised plasmid vector without insertion of target DNA. This is called non-recombinant DNA.
If the digested vector is treated with alkaline phosphatase or other restriction enzymes, the phosphate group is removed. Therefore, there remains less possibility for self-ligation of cloning vector.