Tools of Recombinant DNA Technology

Recombinant DNA technology involves using enzymes and various laboratory techniques to manipulate and isolate DNA segments of interest. This method can be used to combine (or splice) DNA from different species or to create genes with new functions. The resulting copies are often referred to as recombinant DNA. Such work typically involves propagating the recombinant DNA in a bacterial or yeast cell, whose cellular machinery copies the engineered DNA along with its own.

• Genetic engineering : Techniques to alter the chemistry of genetic material (DNA and RNA),  to introduce these into host organisms and thus change the phenotype of the host organism.
• The techniques of genetic engineering which include creation of recombinant DNA, use of gene cloning and gene transfer, overcome this limitation and allow us to isolate and introduce only one or a set of desirable genes without introducing undesirable genes into the target organism. 
• The piece of DNA would not be able to multiply itself in the progeny cells of the organism. 
• But, when it gets integrated into the genome of the recipient, it may multiply and be inherited along with the host DNA. 
• This is because the alien piece of DNA has become part of a chromosome, which has the ability to replicate. In a chromosome there is a specific DNA sequence called the origin of replication, which is responsible for initiating replication. 
• Therefore, for the multiplication of any alien piece of DNA in an organism it needs to be a part of a chromosome(s) which has a specific sequence known as ‘origin of replication’. 
• Thus, an alien DNA is linked with the origin of replication, so that this alien piece of DNA can replicate and multiply itself in the host organism. This can also be called cloning or making multiple identical copies of any template DNA. 
• The construction of the first recombinant DNA emerged from the possibility of linking a gene encoding antibiotic resistance with a native plasmid (autonomously replicating circular extrachromosomal DNA) of Salmonella typhimurium. 
• Stanley Cohen and Herbert Boyer accomplished this in 1972 by isolating the antibiotic resistance gene by cutting out a piece of DNA from a plasmid which was responsible for conferring antibiotic resistance. 
• The cutting of DNA at specific locations became possible with the discovery of the so-called ‘molecular scissors’– restriction enzymes. The cut piece of DNA was then linked with the plasmid DNA.
• These plasmid DNA act as vectors to transfer the piece of DNA attached to it. You probably know that mosquitoes act as an insect vector to transfer the malarial parasite into the human body. In the same way, a plasmid can be used as a vector to deliver an alien piece of DNA into the host organism. 
• The linking of antibiotic resistance genes with the plasmid vector became possible with the enzyme DNA ligase, which acts on cut DNA molecules and joins their ends. This makes a new combination of circular autonomously replicating DNA created in vitro and is known as recombinant DNA. 
• When this DNA is transferred into Escherichia coli, a bacterium closely related to Salmonella, it could replicate using the new host’s DNA polymerase enzyme and make multiple copies. The ability to multiply copies of antibiotic resistance genes in E. coli was called cloning of antibiotic resistance genes in E. coli.
• There are three basic steps in genetically modifying an organism —
• (i) identification of DNA with desirable genes;
• (ii) introduction of the identified DNA into the host;
• (iii) maintenance of introduced DNA in the host and transfer of the DNA to its progeny. 

1.0Restriction Enzymes

• In 1963, the two enzymes responsible for restricting the growth of bacteriophage in Escherichia coli were isolated. One of these added methyl groups to DNA, while the other cut DNA. 
• The later was called restriction endonuclease.
• The first restriction endonuclease–Hind II, whose functioning depended on a specific DNA nucleotide sequence was isolated and characterised five years later.
• It was found that Hind II always cut DNA molecules at a particular point by recognising a specific sequence of six base pairs. 
• This specific base sequence is known as the recognition sequence for Hind II. 
• Besides Hind II, today we know more than 900 restriction enzymes that have been isolated from over 230 strains of bacteria each of which recognise different recognition sequences.

Nomenclature of Restriction Enzyme 

• The convention for naming these enzymes is that the first letter of the name comes from the genus and the second two letters come from the species of the prokaryotic cell from which they were isolated, e.g., EcoRI comes from Escherichia coli RY 13. 
• In EcoRI, the letter ‘R’ is derived from the name of strain. Roman numbers following the names indicate the order in which the enzymes were isolated from that strain of bacteria. 

2.0Cleaving of Fragment 

• Restriction enzymes belong to a larger class of enzymes called nucleases. 
• These are of two kinds; exonucleases and endonucleases. Exonucleases remove nucleotides from the ends of the DNA whereas endonucleases make cuts at specific positions within the DNA. 
• Each restriction endonuclease functions by ‘inspecting’ the length of a DNA sequence. 
• Once it finds its specific recognition sequence, it will bind to the DNA and cut each of the two strands of the double helix at specific points in their sugar -phosphate backbones. 
• Each restriction endonuclease recognises a specific palindromic nucleotide sequence in the DNA.
• Restriction enzymes cut the strand of DNA a little away from the centre of the palindrome sites, but between the same two bases on the opposite strands. 
• This leaves single stranded portions at the ends. There are overhanging stretches called sticky ends on each strand. 
• These are named so because they form hydrogen bonds with their complementary cut counterparts. This stickiness of the ends facilitates the action of the enzyme DNA ligase.

DNA polymerases

DNA polymerases are associated with the synthesis of a new complementary DNA strand of an existing DNA or RNA template. Examples of some DNA polymerases used commonly in genetic engineering are:

• DNA polymerase 1 prepared from E coli. 
• Taq DNA polymerase used in PCR (Polymerase Chain Reaction). 
• Reverse transcriptase that uses RNA as a template for synthesizing a new DNA strand (called cDNA/complementary DNA). Its main use is in the formation of cDNA libraries. 

DNA ligase

• The function of these enzymes is to ligate or join two fragments of DNA. 
• This is achieved by the synthesis of the phosphodiester bond. In recombinant DNA technology, DNA ligases are used for sealing nicks between adjacent nucleotides. 

3.0Vector 

• Plasmids and bacteriophages have the ability to replicate within bacterial cells independent of the control of chromosomal DNA. 
• Bacteriophages, because of their high number per cell, have very high copy numbers of their genome within the bacterial cells. Some plasmids may have only one or two copies per cell whereas others may have 15-100 copies per cell. 
• Their numbers can go even higher. If we are able to link an alien piece of DNA with bacteriophage or plasmid DNA, we can multiply its numbers equal to the copy number of the plasmid or bacteriophage. 
• Vectors used at present, are engineered in such a way that they help easy linking of foreign DNA and selection of recombinants from non-recombinants.

The following are the features that are required to facilitate cloning into a vector.

(i) Origin of replication (ori) : This is a sequence from where replication starts and any piece of DNA when linked to this sequence can be made to replicate within the host cells. This sequence is also responsible for controlling the copy number of the linked DNA. So, if one wants to recover many copies of the target DNA it should be cloned in a vector whose origin supports a high copy number.

(ii) Selectable marker : In addition to ‘ori’, the vector requires a selectable marker, which helps in identifying and eliminating non transformants and selectively permitting the growth of the transformants. Transformation is a procedure through which a piece of DNA is introduced in a host bacterium (you will study the process in subsequent sections). Normally, the genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, tetracycline or kanamycin, etc., are considered useful selectable markers for E. coli. The normal E. coli cells do not carry resistance against any of these antibiotics.

(iii) Cloning sites: In order to link the alien DNA, the vector needs to have very few, preferably single, recognition sites for the commonly used restriction enzymes. Presence of more than one recognition sites within the vector will generate several fragments, which will complicate the gene cloning. The ligation of alien DNA is carried out at a restriction site present in one of the two antibiotic resistance genes.

• For example, you can ligate a foreign DNA at the BamHI site of the tetracycline resistance gene in the vector pBR322. The recombinant plasmids will lose tetracycline resistance due to insertion of foreign DNA but can still be selected out from non-recombinant ones by plating the transformants on tetracycline containing medium. 
• The transformants growing on ampicillin containing medium are then transferred on a medium containing tetracycline. The recombinants will grow in ampicillin containing medium but not on that containing tetracycline. But, non- recombinants will grow on the medium containing both the antibiotics. In this case, one antibiotic resistance gene helps in selecting the transformants, whereas the other antibiotic resistance gene gets ‘inactivated due to insertion’ of alien DNA, and helps in selection of recombinants. 
• Selection of recombinants due to inactivation of antibiotics is a cumbersome procedure because it requires simultaneous plating on two plates having different antibiotics. Therefore, alternative selectable markers have been developed which differentiate recombinants from non-recombinants on the basis of their ability to produce colour in the presence of a chromogenic substrate. In this, a recombinant DNA is inserted within the coding sequence of an enzyme, β-galactosidase. 
• This results in inactivation of the gene for synthesis of this enzyme, which is referred to as insertional inactivation. The presence of a chromogenic substrate gives blue coloured colonies if the plasmid in the bacteria does not have an insert. Presence of insert results into insertional inactivation of the β-galactosidase gene and the colonies do not produce any colour, these are identified as recombinant colonies.

 Vectors for cloning genes in plants and animals 

• We have learnt the lesson of transferring genes into plants and animals from bacteria and viruses which have known this for ages – how to deliver genes to transform eukaryotic cells and force them to do what the bacteria or viruses want. 
• For example, Agrobacterium tumefaciens, a pathogen of several dicot plants is able to deliver a piece of DNA known as ‘T-DNA’ to transform normal plant cells into a tumor and direct these tumor cells to produce the chemicals required by the pathogen. 
• Similarly, retroviruses in animals have the ability to transform normal cells into cancerous cells. A better understanding of the art of delivering genes by pathogens in their eukaryotic hosts has generated knowledge to transform these tools of pathogens into useful vectors for delivering genes of interest to humans. 
• The tumor inducing (Ti) plasmid of Agrobacterium tumefaciens has now been modified into a cloning vector which is no more pathogenic to the plants but is still able to use the mechanisms to deliver genes of our interest into a variety of plants. 
• Similarly, retroviruses have also been disarmed and are now used to deliver desirable genes into animal cells. So, once a gene or a DNA fragment has been ligated into a suitable vector it is transferred into a bacterial, plant or animal host .

4.0Host Cell 

• ANIMAL CELL
• PLANT CELL
• BACTERIAL CELL