TOOLS OF RECOMBINANT DNA TECHNOLOGY

The technology or genetic engineering involves restriction enzymes, ligase enzymes, polymerase enzymes, vectors and the host organism.

Restriction Enzymes

In the late 1960's, scientists Stewart Linn and Werner Arber isolated samples of the two types of enzymes responsible for phage growth restriction in Escherichia coli (E. coli) bacteria.

One of these enzymes methylated DNA, while the other cleaved unmethylated DNA at a wide variety of locations along the length of the molecule. The first type of enzyme was called a "methylase" while the other was called a "restriction nuclease".

These enzymatic tools were important to scientists who were gathering the tools needed to "cut and paste" DNA molecules.

What was needed now was a tool that would cut DNA at specific sites, rather than at random sites along the length of the molecule, so that scientists could cut DNA-molecules in a predictable and reproducible way.

Site-specific Nuclease

This important development came when H.O. Smith, K.W. Wilcox, and T.J. Kelley isolated and characterized the first restriction nuclease whose functioning depended on a specific DNA nucleotide sequence.

Working, with Haemophilus influenzae bacteria, this group isolated an enzyme, called Hind II, that always cut DNA molecules at a particular point within a specific sequence of six base pair.

This sequence is :

5' G T (pyrimidine : Tor C) (purine : A or G) A C 3'

3' C A (purine : A or G) (pyrimidine : T or C) T G 5'

They found that the Hind II enzyme always cuts directly in the center of this sequence.

Wherever this particular sequence of six base pairs occurs unmodified in a DNA molecule, Hind II will cleave both DNA strands or backbones between the 3rd and 4th base pairs of the sequence.

Moreover, Hind II will only cleave a DNA molecule at this particular site. For this reason, this specific base sequence is known as the "recognition sequence" for Hind II.

Hind II is just one example of the class of enzymes known as restriction nucleases.

In fact, more than 900 restriction enzymes, some sequences specific and some not, have been isolated from over 230 strains of bacteria since the initial discovery of Hind II.

These restriction enzymes generally have names that reflect their origin.

The first letter (in italics) of the name comes from the genus and the second two letters (in italics) come from the species of the prokaryotic cell from which they were isolated.

Next is the strain of the organism and last is the roman numeral indicating the order of discovery.

For example, EcoRI comes from Escherichia coli RY strain and was the first endonuclease isolated from bacteria, while Hind II comes from Haemophilus influenzae strain Rd. Numbers following the nuclease names indicate the order in which the enzymes were isolated from single strains of bacteria.

Nucleases are further described by addition of the prefix "endo" or "exo" to the name: The term "endonuclease" applies to sequence specific nucleases that break nucleic acid chains somewhere in the interior, rather than at the ends of the molecule.

Nuclease that function by removing nucleotides from the ends of the molecules are called "exonucleases".

Three main classes of restriction endonucleases type-I, type-II and type-III have been described, each distinguished by a slightly different mode of action.

Out of these three types, type-I and type-III restriction enzymes are not used in recombinant DNA technology.

Type-II restriction enzymes are used in recombinant DNA technology, because they can be used In vitro to recognise and cut within the specific DNA sequence typically consisting of 4-8 nucleotide.

Type-I enzymes recognize specific sites within the DNA but do not cut at these sites.

Hence heterogeneous population of DNA fragments is produced, and therefore, type I enzymes do not take part in the technology. Type-III enzymes recognise a specific sequence of DNA molecule. Thus, products of type-III enzymes are homogeneous population of DNA fragments, so they cannot be used for genetic engineering experiments.

­Table-I : Recognition Sequences of Several Restriction Endonucleases

(arrow indicates the site of cleavage)

The DNA segments cut by restriction enzymes are palindromic i.e., the nucleotide sequence of these DNA pieces read the same both, backwards and forward when orientation of reading is kept same, e.g., madam.

Blunt or flush ends are produced by many restriction enzymes which cleave both stands of DNA at exactly the same nucleotide position, in the centre of recognition site. For example, Sma I recognises 6 nucleotide palindromic sequence.

5'-C-C-C-G-G-G-3'

3'-G-G-G-C-C-C-5'

It cuts both DNA strands producing blunt ends.

Action of restriction enzymes : The nucleotide sequences recognised and

cut by widely used restriction endonucleases A. Hae III

Sticky or cohesive ends are produced when restriction enzymes do not cut DNA at the same nucleotide position but cut the recognition sequence unequally. This produces short, single-stranded overhangs at each end. These are known as sticky ends. For example, Eco RI recognises 6 nucleotide palindromic sequence.

Action of restriction enzymes : The nucleotide sequences recognised and

cut by widely used restriction endonucleases B. Eco RI, C. Hind IIIC

Action of restriction enzyme Eco RI and formation of recombinant DNA.

This restriction endonuclease cuts both DNA strands unequally, producing 5' overhangs of 4 nucleotides.

The stickiness helps enzyme ligase to make the DNA pieces join.

Other Enzymes used in Recombinant DNA Technology

In addition to restriction enzymes, there are several other enzymes that play an important role in DNA technology.

Three of the important ones are DNA ligase, alkaline phosphatase and DNA polymerase.

(a) DNA Ligase : This enzyme forms phosphodiester bonds between adjacent nucleotides and covalently links two individual fragments of double-stranded DNA. The action of the ligase enzyme requires a phosphate group at the 5' carbon of one nucleotide and a hydroxyl group at the 3' carbon of the adjacent nucleotide to form the phosphodiester bond between these two nucleotides. The enzyme used most often in the rDNA technology is T4 DNA ligase, which is encoded by phage T4.

(b) Alkaline Phosphatase (AP) : As mentioned above, ligation absolutely requires the presence of 5' phosphate group at the DNA site to be ligated. If this phosphate group is removed, this DNA cannot be ligated. The enzyme alkaline phosphatase is used to remove the phosphate group from the 5' end of a DNA molecule, leaving a free 5' hydroxyl group. This enzyme can be isolated from bacteria (BAP) or calf intestine (CAP). It is used to prevent unwanted self-ligation of vector DNA molecules in procedures of rDNA technology. However, ligation of the vector to the insert can occur as the insert still has its 5' phosphate.

(c) DNA Polymerase : DNA Pol I enzyme polymerizes the DNA synthesis on DNA template or complementary DNA (cDNA). It also catalyses a 5' 3' and 3' 5' exonucleolytic degradation of DNA. The other two enzymes are DNA polymerase II (DNA pol II) and DNA polymerase III (DNA pol III). These have almost similar catalytic activity. DNA pol III is about several times more active than the other two. Where there is preformed DNA template, it produces a parallel strand in the presence of ATP.

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 the opposite charge, viz., positively charged molecules move towards cathode (-ve electrode) and negatively charged molecules travel towards anode (+ve electrode) through a medium/matrix.

This technique was developed by A. Tiselius in 1937.

Now a days, the 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.

Hence, the smaller the fragment size, the farther it moves.

Agarose dissolve in hot water, when this solution is cooled, double helices form and become arranged laterally and produce thick filaments.

These filaments become cross-linked to form the gel.

Pore size depends on agarose concentration.

The separated DNA fragments can be seen only after staining the DNA with a compound known as ethidium bromide 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 is called as elution. Several techniques are used for eluting the DNA from the gel piece. These purified DNA fragments are used in the formation of recombinant DNA by linking them with cloning vectors.

A typical agarose gel electrophoresis showing migration of undigested (lane 1)
and digested set of DNA fragments (lane 2 to 4)

Cloning Vectors : Another important tool for genetic engineering is the vehicle for cloning, called vector. The vector carries a foreign DNA sequence into a given host cell. Bacterial plasmids and bacteriophages are considered the most useful. This is because :

(1) These are independent of the control of chromosomal DNA,

(2) Bacteriophage genomes occur in very large numbers in bacterial cells.

(3) The copies of plasmids per cell range from only a few to hundred or even more.

Certain essential features should be present in a DNA molecule to act as a cloning vector.

1. Origin of replication (ori): This is a DNA sequence which serves as a starting point for replication. When a DNA fragment gets associated with ori, foreign DNA into the vector would also replicate inside the host cell. Some vectors possess origin which favours formation of high copy numbers and hence preferred.

Joining of DNA fragments

2. Selectable marker: Vector should also include a selectable marker. This is a gene which would permit the selection of host cells containing vector from amongst those which do not possess vector. Common selectable markers include genes encoding antibiotic resistance such as ampicillin resistance or enzymes such as -galactosidase (product of lac Z gene of lac operon). These genes can be identified by a colour reaction.

3. Recognition sites : Vector should possess an unique restriction site that would allow particular enzyme to cut the vector only once. This site would be recognised by the commonly used restriction enzymes. If there are more than one recognition site in the vector, several fragments would be produced. Generally the vectors used possess unique recognition sites for several restriction enzymes in a small region of DNA. This is known as polylinker or multiple cloning site (MCS). Such a cloning site offers choice of restriction enzyme.

Unique restriction endonuclease recognition site enables insertion of foreign DNA into the vector for production of recombinant DNA. The foreign DNA is inserted and made to join (ligate) at a specific restriction site generally in antibiotic resistance gene.

pBR322 has genes for resistance against two antibiotics tetracycline and ampicillin, an origin of replication and a variety of restriction sites for cloning of restriction fragments obtained through cleavage with a specific enzyme. Foreign DNA is inserted at a site located in one of the two genes for resistance against antibiotics, so that it will inactivate one of the two resistance genes. The insert bearing plasmid can be selected by their ability to grow in a medium containing only one of the two antibiotics and their failure to grow in a medium containing both antibiotics. The plasmids carrying no insert on the other hand, will be able to grow in a media containing one or both the antibiotics. In this way, the presence of resistance genes against ampicillin and tetracycline allow selection of Escherichia coli colonies transformed with plasmids carrying the desired foreign cloned DNA fragment.

4. Size of the vector : Cloning vector should be small in size. Large molecules have a tendency to breakdown during purification. These are also difficult to manipulate.

DIFFERENT TYPES OF VECTOR

Several types of vectors satisfying the above characters have been developed. The following are some of the commonly used vectors.

1. Plasmids:

These are extra-chromosomal, non-essential self-replicating, usually circular and double stranded DNA molecules occurring in some bacteria and also a few yeasts.

Some of the characters carried by plasmid may not be required for normal bacterial metabolism but may be of great advantage e.g. antibiotic resistance.

pBR322 is one of the standard cloning vectors widely used in gene cloning experiments.

This vector has been restructured by inserting genes for antibiotic resistance.

It is named after Boliver and Rodriguez who prepared this vector, pUC (named after University of California) is another such reconstructed plasmid vector.

The vectors mentioned above are able to replicate only in E. coli.

Therefore, many vectors constructed for eukaryotic cells are also functional in E. coli.

These vectors are called shuttle vectors.

The vectors contain two types of origin of replication and selectable marker genes-one for the eukaryotic cell and another for E. coli.

The common example of this type is yeast episomal plasmid (YEp).

In plants, Ti plasmid of bacterium Agrobacterium tumfaciens has been modified to function as vector.

2. Vectors based on bacteriophages :

Bacteriophages are viruses which infect bacterial cells produce new phages inside the host bacterium, and are released from the host cell to again infect other bacterial cells. M 13 and lambda () phages are in common use.

3. Cosmids:

These combine some features of plasmid and 'cos' (cohesive end sites) of phage lambda (cosmid = cos + plasmid).

4. YAC vectors:

Yeast artificial chromosome contain telomeric sequence, the centromere and autonomously replicating sequence from yeast chromosomes. These also have suitable restriction enzyme sites and genes useful as selectable markers.

5. BAC vectors:

Bacterial artificial chromosome is based on F plasmid (fertility) of E. coli. It contains genes for replication and maintenance of F-factor, selectable marker and cloning sites.

Colour Reaction : Due to inactivation of antibiotics, selection of recombinants becomes burdensome process because it requires simultaneous plating on two plates having different antibiotics. Thus, alternative selectable marker is developed to differentiate recombinants and non-recombinants on the basis of their ability to produce colour in the presence of a chromogenic substance. Now a recombinant DNA is inserted in the coding sequence of an enzyme -galactosidase. This causes inactivation of the enzyme which is called insertional inactivation. If the plasmid in the bacterium does not have an insert, the presence of a chromogenic substrate gives blue coloured colonies. Presence of insert results into insertional inactivation of the -galactosidase and, therefore, the colonies do not produce any colour, these colonies are marked as recombinant colonies.

6. Vectors for Cloning Genes in Plants and Animals:

We know the procedure of transferring genes into plants and animals from bacteria and viruses.

It is also known how to transfer genes to transform eukaryotic cells and force them to do what the bacteria or viruses require.

For example, Agrobacterium tumifaciens, a pathogen (disease causing agent) of several dicot plants is able to transfer a piece of DNA known as 'T-DNA' to convert normal plant cells into tumour and direct these tumour cells to secrete the chemicals required by the pathogen.

Similarly, retroviruses (cause leukosis or sarcoma types of cancer) in animals including humans are able to change normal cells into cancerous cells.

The tumour inducing (Ti) plasmid of Agrobacterium tumifaciens has been modified into cloning vector which is not pathogenic to the plants, however, it is still able to use the procedure to deliver genes of our interest into various plants.

Similarly retroviruses are used to carry desirable genes into animal cells.

Thus once a gene or DNA fragment is joined to a suitable vector it is transferred into a bacterial plant or animal host where it undergoes multiplication.

HOST CELL

Competent host cell is required for transformation with recombinant DNA.

After formation of recombinant DNA, propagation of it must occur inside a living system or a host.

Different type of available host cells are like E. coli, yeast, animal and plant cells.

The type of host cell to be used depends on the aim of cloning experiment.

Eukaryotic cells will be the preferred host for expression of some eukaryotic proteins.

Yeast cells are preferred because these are simplest eukaryotic organisms and like bacteria are single celled, genetically well characterized, easy to grow and manipulate.

Plant and animal cells can be used for protein expression either in tissue culture or as cells in the whole organism to create genetically modified (GM) crops and animals.

As DNA is hydrophilic molecule, it can not pass through cell membrane.

Therefore, the bacterial cells should be capable of uptaking DNA.

This is accomplished by treating them with specific concentration of a divalent cation, e.g., Ca2+ thus making them competent which causes efficient entry of DNA into bacterium through pores in its cell wall.

Recombinant DNA can be forced into such cells by incubating the cells with recombinant DNA on ice, followed by placing them briefly at 42°C (heat shock) and then putting them back on ice. As a result, bacteria gets enabled to pick up recombinant DNA.

There are other methods to introduce foreign DNA into host cells. These are briefly described below.

Microinjection

In this method recombinant DNA is directly injected into the nucleus of animal cell by using micro needles or micro pipettes. It is used in oocytes, eggs and embryo. Jeffey S. Chamberlain et. al. (1993) of Human Genome Centre, Michigan University, USA have cured mice that inherited a neuromuscular disease which is like muscular dystrophy of humans.

Direct DNA Injection

Direct injection of DNA into skeletal muscle led to the possibility of using gene 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.

Gene Gun or Biolistics

New technologies like gene gun are also available for vectorless direct gene transfer. DNA coated onto microscopic pellets is literally shot into target cells. Although, it is developed for plants but is also used for animal cells for promoting tissue repair or reducing healing time. This method made great impact in the field of vaccine development.