9 Major Tools Used in Gene Technology

The following points highlight the nine major tools in gene technology. The tools are: 1. Restriction Enzymes 2. DNA Ligase 3. DNA polymerase 4. RNA polymerase 5. Nucleases 6. Reverse Transcriptase 7. Molecular Cloning Vectors 8. Purification of DNA from Living Cells 9. Isolation of DNA from Plant Material.

Gene Technology: Tool # 1.

Restriction Enzymes:

Restriction endonucleases are found in a wide range of bacteria. Werner Arber discovered that their function is to recognize and cleave foreign DNA (i.e., viral DNA), which is said to be restricted.

The restriction enzymes are grouped as restriction endonucleases and exonucleases. The endonucleases cut the intact DNA strand at a specific recognition site, whereas the exonucleases digest the DNA strand sequentially from the cut ends of specific base sequence.

These recognition sites fall between four to six base pairs of palindromic sequence (i.e., identical when read forward or backward). Generally, each enzyme recognizes only one specific sequence. However, the same specific sequence may be recognized by two or more different enzymes, which are called isochizomers.

Restriction endonucleases are of three types — type-I, II and III. Type-I endonucleases recognize specific sequence of DNA but cut the molecule far away (about 1000 bp) from the recognition site using Mg^{2 +} and ATP.

So, its use in gene technology is restricted. The type-II restriction enzymes, on the other hand, can cut at the specific recognition site requiring Mg²⁺ as cofactor. This class of restriction enzymes, first isolated by Hamilton Smith, is simpler and requires no ATP.

The extraordinary utility of the type-II enzymes was first demonstrated by Daniel Nathans and since then these enzymes are mainly used in genetic engineering. The classical example for class-II type restriction enzyme is EcoR I.

The cell’s own DNA is not cleaved by the restriction enzymes because the sequence recognized by them remains protected through methylation by a specific DNA methylase. These two types of enzymes in bacteria form a restriction-modification system.

Type-I and III are generally large, multi-subunit complexes containing both the endonucleases and methylase activities. Type-III enzymes cleave the DNA about 25 bp from the recognition sequence. Both types of enzyme move along the DNA in a reaction requiring the energy of ATP.

The naming protocol for restriction endonucleases is as follows:

(a) The three-letter code is given for each restriction endonuclease.

(b) The first letter code is always a genus name and written in capital.

(c) The subsequent letters are represented by species name.

(d) The next letter designates the strain, and Roman numbers indicate different endonucleases from the same organism.

EcoRI enzyme recognizes the particular sequence as follows:

This segment has two-fold rational symmetry because it can be rotated 180° without change in base sequence (a palindrome). When the enzyme EcoRl attacks this palindrome, it breaks each strand at the same site in the sequence. Restriction enzymes generate staggered and blunt ends.

EcoRl cuts the DNA producing staggered ends having 3′ and 5′ phosphate extensions (Fig. 17.1). These ends are sticky and known as cohesive ends and are very useful in recombinant DNA technology.

Gene Technology: Tool # 2.

DNA Ligase:

DNA ligase is a key enzyme in gene technology and acts as a molecular suture stitching two different DNA pieces requiring ATP. During joining the enzyme forms a covalent bond between the 5′ phosphoryl group of one strand and the 3′ hydroxyl group of the adjacent strand.

Thus, two phosphodiester bonds are formed between two cohesive ends. The blunt ends produced by certain enzymes hinder the joining process. In such cases joining needs very high concentration of the enzyme. The main source of this enzyme is the T₄ phage virus.

Gene Technology: Tool # 3.

DNA Polymerase:

DNA polymerase is an enzyme that catalyzes the addition of deoxynucleotide residues to a polynucleotide through nucleophilic attack of the chain’s 3′-OH group on the a-phosphoryl group of the incoming deoxynucleoside triphosphate on the DNA template in 5’→3′ direction.

DNA polymerase is a holoenzyme. The larger fragment of this enzyme is known as Klinow fragment. It is the DNA polymerase a occurs only in the nucleus with a tightly associated polymerase activity and lacks 5’→3′ exonuclease activity.

Other polymerases used in gene technology are:

(a) T4 DNA polymerase,

(b) Taq DNA polymerase,

(c) Tth DNA polymerase and T₇DNA polymerase.

T4 DNA pol lacks 5′ → 3′ exonuclease activity. Taq DNA pol is thermo-stable and used in polymerase chain reaction (PCR). It is extracted from the bacterium Thermus aquaticus living in hot spring and its optimum temperature is 72°C. Tth DNA pol is also thermo-stable having reverse transcriptase (RT) activity. T₇ DNA pol is used in DNA sequencing.

Gene Technology: Tool # 4.

RNA Polymerase:

RNA polymerase is the enzyme responsible for the DNA-directed synthesis of RNA. The enzyme couples together the ribonucleoside triphosphates ATP, CTP, GTP, and UTP on DNA templates. RNA polymerases used in gene technology are — (a) T7 RNA polymerase, (b) T3 RNA polymerase, and (c) Qβ replicase.

Both the enzymes T7 and T3 RNA pol are DNA dependent RNA polymerases. Qβ replicase is a RNA dependent RNA polymerase and is used in RNA amplification.

Gene Technology: Tool # 5.

Nucleases:

i. DNases:

These enzymes can digest both the strands of DNA. It can hydrolyse each DNA strand independently in presence of Mg²⁺ ions. The enzyme is used in RNA purification by digesting the contaminated DNA. It is also used in foot printing and nick translation.

The commonly used DNases in genetic engineering are — (a) DNase I, which is a non-specific endonuclease that cleaves DNA, and (b) Exonuclease III, which is used in DNA-dependent 3’→5′ stepwise removal of nucleotides.

ii. RNases:

RNases digest RNA into its component nucleotides. It cleaves the bond next to uracil and guanine. The enzyme is used in DNA purification where it removes contaminated RNA. It is also used in removing poly A tail from mRNA. The main source of the enzyme is Aspergillus and bovine pancreas.

RNases used in gene manipulation are:

(a) RNase A, which is used in mapping studies,

(b) RNase H, which is widely used in the synthesis of the second strand in cDNA preparation and also in removing mRNA from RNA-DNA hybrid, and

(c) S 1 nuclease, which is single-strand-specific nuclease.

Gene Technology: Tool # 6.

Reverse Transcriptase:

It is an RNA dependent DNA polymerase obtained from avian myeloblast virus. This is abbreviated as AMV-RT. The enzyme requires DNA primer complementary to RNA template. AMV-RT consists of two important subunits. In genetic engineering reverse transcriptase is used in cDNA synthesis. It converts mRNA to single strand DNA.

Gene Technology: Tool # 7.

Molecular Cloning Vectors:

A vector is a carrier of DNA molecule to which the fragment of DNA of interest is attached for cloning.

The essential properties of a vector include its ability for autonomous replication, the presence of a specific nucleotide sequence recognized by a restriction endonuclease and a gene for identification. Generally three types of vectors are used in genetic engineering. These are — (a) plasmids, (b) phagemids and (c) cosmids.

(a) Plasmids:

Plasmid is an extra-chromosomal, independently replicating, small circular DNA molecule present in bacteria. It mainly gives the bacteria resistance to antibiotics. The size of the plasmid ranges from 1 to more than 500 kilo base pairs.

If the number of plasmids ranges from 10- 100 per cell it is called high-copy-number plasmids or relaxed plasmids and if the number is 1 to 4 copies per cell it is called low-copy-number or stringent plasmids.

Some of the plasmid vectors are as follows:

i. pBR 322:

It is an ideal cloning vector commonly used in recombinant DNA technology. The source of this vector is E. coli. It has been engineered into a cloning vector. This plasmid consists of 4,363 base pairs and carries genes for tetracycline and ampicillin resistance.

The tetracycline gene has recognition sites for Hind III, Bam HI and Sal I. Similarly, ampicillin resistance gene contains pst 1 site. The first letter p stands for plasmid and BR denotes the names of two scientists, Bolivar and Rodriguez, who engineered the plasmid into a cloning vector. The number 322 is the chronological order of discovery.

The recombinant DNA can be screened with the help of the antibiotic resistance genes. For the first time the transformed cells are grown in a medium containing ampicillin. The transformed cells with ampicillin resistance gene only can grow in that medium. In the subsequent step the cells grown in the ampicillin-containing medium are transferred to a tetracycline-containing medium.

The cells that do not grow in the tetracycline medium carry the cloned DNA, because the recognition site lies within the tetracycline resistance gene and the target DNA has been inserted within that gene (Fig. 17.3).

ii. pUC 19:

This plasmid contains 2,860 base pairs carrying genes for ampicillin resistance and P-galactosidase (Lac Z). The Lac Z gene contains cloning sites for Eco Rl, Sac I, Xma I, Sma I, Bam HI, Pst I, Hnd III, etc. Use of this plasmid is advantageous because the recombinant DNA can be screened within a very short time in comparison to pBR 322.

Moreover, this plasmid exists as high copy number, i.e., up to 500 per cell. It is easy to isolate and contains a variety of restriction enzyme recognition sites.

The first letter p denotes plasmid, the letters U and Care referred to the University of California, where it was constructed by J. Messing and J. Vieria. The number 19 is the chronological order of discovery, by which it can be distinguished from other plasmids.

(b) Phage λ Vectors:

Bacteriophages are linear DNA molecules, containing several restriction enzyme recognition sites. Bacteriophage A. is of wide use in the recombinant DNA technology because it can accept large foreign DNA fragments of 10-20 kb in length. Plasmid vectors, on the other hand, can carry only up to 10 kb of inserted foreign DNA.

During lytic cycle phage DNA attaches and enters the bacterial cell followed by replication. Then newly formed phage particles are released after the lysis of bacteria. In lysogenic cycle phage DNA for the first time is inserted into the bacterial DNA and remain silent for a period of time. In favourable conditions DNA is excised and enter a lytic cycle.

Phage DNA is linear and double stranded and is of approximately 49kb in length, of which 20 kb is responsible for integration and excision during lysogenic cycle.

This intercalary 20 kb region of DNA can be removed and replaced by 20 kb of foreign DNA properly excised from source DNA by restriction enzyme. This vector construction is done in presence of T₄ DNA ligase. The recombinant phages are maintained by continual growth on fresh E. coli.

In the wild type phage λ the DNA follows one of the two possible modes of replication. It may become stably integrated into the host chromosome, where it lies dormant until a signal triggers its excision. This mode of replication is called lysogenic cycle.

Alternatively, it may replicate upon entry into the host cell. The head and tail proteins are synthesized rapidly and new functional phages are assembled and packaged and subsequently released from the cell lysing the cell membrane for further infection of host cells.

This is called lytic cycle. At the extreme ends of the phage λ there are 12 bp sequences termed cos (cohesive) sites. These asymmetric sites allow the phage DNA to be circularized. λ phage is largely used in the formation of gene libraries as it can efficiently enter into the E. coli cells and also due to the fact that the larger fragments of DNA may be stably integrated.

For the cloning of the large DNA fragments (up to 25kb), much of the non-essential λ DNA that codes for the lysogenic life cycle is removed and replaced by the foreign DNA insert.

The recombinant phage is then assembled in preformed protein coats, a process termed in vitro packaging. These newly formed phages are used to infect bacterial cells on agar plates. Inside the host cells the recombinant viral DNA is replicated. The viral DNA including the cloned foreign DNA can be recovered from the viruses from the plaques and analysed further by restriction mapping and agarose gel electrophoresis.

In general two types of λ phage vectors have been developed, λ insertion vectors and λ replacement vectors. The former accept less DNA than the latter, since the foreign DNA is merely inserted into a region of the phage genome with appropriate restriction sites (e.g., λ gt 10 and λ charon 16A).

In a replacement vector, a central region of DNA not essential for lytic growth (stuffer fragment), is removed by a double digestion with EcoRI and BamHI, leaving two DNA fragments termed left and right arms. The notable examples of λ replacement vectors are λEMBL and λZap.

(c) M13 and Phagemid-Based Vectors:

M13 is single-stranded circular DNA containing coli phage. It is filamentous in nature. The protein coat contains three kinds of capsomers. These phages infect cells by adsorbing with the F pili. Upon infection the DNA replicates initially as a double-stranded molecule but subsequently produces single-stranded virions for infection of further bacterial cells.

The nature of these vectors is very suitable for chain termination sequencing and in vitro mutagenesis, since both require single-stranded DNA. In addition to that the double-stranded RF DNA intermediate formed in the coliphage vectors helps a number of regular DNA manipulations like restriction digestion, mapping and DNA ligation.

The bacteriophage may thus act as a plasmid under certain circumstances and at other times produce DNA in the fashion of a virus (Fig. 17.7).

Only the F⁺ E. coli cells are susceptible to M13 infection. After entry through the F pilus the phage DNA is converted to a double-stranded replicative form or RF DNA followed by rapid replication until some 100 RF molecules are produced in a host cell. After that single-stranded DNA synthesis takes place, which are then packaged into the capsid at the bacterial periplasm.

The release of bacteriophage through periplasm results in decreased growth rate of host cell rather than lysis and is visible on a bacterial background or lawn as a clear area (plaque). Approximately 1000 packaged phage particles may be released into the medium in one cell division. M13 derived vectors are termed as M13 mp8/9, .mp 18/19, etc.

These vectors contain a synthetic multiple cloning site (MCS), which is located in the lacZ gene without disturbing the reading frame. In the screening procedure the introduced MCS produces blue plaques on X gal agar plates.

A series of vectors has been developed in which the number of restriction sites was increased in an asymmetric fashion. These vectors are more useful since there is a greater choice of restriction enzymes. However, the inserts greater than 6kb are unstable due to spontaneous loss.

(d) Cosmids:

Cosmids are larger insert cloning vectors, used for cloning large DNA fragments. These vectors contain segments derived from bacteriophages and plasmids. These are specially useful for the analysis of highly complex genomes and are important components of various genome mapping projects. Cosmid has two cos sites derived from λ phage and tetracycline resistance gene from plasmid.

It can be easily maintained in E. coli cells. Cosmid vectors are constructed that incorporate the cos sites from bacteriophage λ and also the plasmid origin of replication, a gene for drug resistance and several restriction sites for insertion of the DNA to be cloned.

It has an MCS with six restriction sites . For insertion, the foreign DNA and cosmid vector are digested with the same restriction endonuclease. Subsequently, source DNA and cosmid DNA are mixed and incubated in presence of T₄ DNA ligase. In such preparation the products include concatamers of alternating cosmid vector and insert.

For re-circularization and packaging into viral heads it should contain cos sites at 37 to 52kb distance. For in vitro packaging phage head precursors, tails and packaging proteins are to be provided as the cosmid is very small, inserts of about 40kb in length can readily be packaged.

(e) Shuttle Vectors:

The vectors, which are capable of replicating in different host systems, are called shuttle vectors. These vectors carry different origins of replication, viz., yeast and bacteria such as E. coli. For that reason they can be easily maintained in different host systems. Plasmids used for cloning DNA in eukaryotic cells require a eukaryotic origin of replication and marker genes that will be expressed in eukaryotic cells.

Yeast has a plasmid called the 2µ-circle, which is too large for use in cloning. Yeast, like bacteria, can be grown rapidly, and is, therefore, well suited for use in cloning.

Shuttle vectors have been created from yeast and E. coli with objective that the constructs may be prepared rapidly in the bacteria and delivered into yeast for expression studies. Eukaryotic yeast cells are better equipped for post-translational modifications, which is essential for the formation of functional eukaryotic proteins. Hence, most eukaryotic vectors are designed to be shuttle vectors.

Yeast episomal plasmids (YEp) are shuttle vectors such as PJDB 219. It is an artificial plasmid found in strains of Saccharomyces cerevisiae with copy number up to 250.

Yeast artificial chromosome (YAC) is also a linear shuttle vector designed to clone more than 100 kb of DNA, which is then maintained as a separate chromosome in the yeast cell. YAC also contains an origin of replication termed ARS (autonomous replicating sequence), an E. coli selectable marker gene and certain sequences such as URA, CEN (centromere), TRP as well as telomeres.

The URA and TRP genes are responsible for uracil and tryptophan biosynthesis respectively. The YAC is digested with restriction enzymes at the SUP4 site (a suppressor tRNA gene marker) and BamHl sites separating the telomere sequences producing two arms, and the foreign DNA is ligated to form a functional YAC construct.

Following the production of recombinant molecule (construct), it is subsequently introduced into the E. coli cells through bacteriophage to get a large copy number. The recombinant DNA is introduced into the eukaryotic cells by transfection.

(f) Viral Vectors:

Viruses can be used as vectors for introducing genes into plants, such as Gemini viruses. The viruses are used as they can distribute their own genome throughout the infected plant body.

A recombinant DNA of tomato golden mosaic virus containing NPT II gene in place of viral coat protein gene is introduced into Agrobacterium system. When those bacteria in turn are injected directly into the stems of transgenic tobacco, the viral DNA was spread throughout the plant body with the expression of NPT II gene.

Cauliflower mosaic virus (CaMv) has a double-stranded DNA, which infects the plants of the family Cruciferae. The host plant can be infected by naked CaMv DNA constructs. The bacterial dihydrofolate reductase gene has been successfully expressed in plants.

Gene Technology: Tool # 8.

Purification of DNA from Living Cells:

Isolation and purification of DNA from living cells is central to molecular biology and biotechnology as its analysis and manipulation requires purification to some extent. From the cell-free extract the carbohydrates, proteins, lipids, and RNAs are removed from the DNA fraction.

The cells are gently ruptured to prevent the fragmentation of DNA by mechanical shearing in presence of EDTA that chelate the Mg²⁺ needed for deoxyribonucleases (DNases) for DNA digestion.

This digestion can also be overcome by using liquid nitrogen. The laboratory contamination of DNases can be minimized by autoclaving all the glassware’s and solutions to be used. The cell walls, if present, are digested enzymatically. For bacterial cell, the walls are removed by lysozyme treatment and the cell membrane is dissolved using anionic detergent such as sodium dodecyl sulphate (SDS).

The plant cells are treated with the enzyme celluloses and hemicelluloses or certain cell wall weakening chemicals. The animal cells do not possess cell wall. The cell membrane is removed by SDS treatment. The DNA in the lysate can be extracted by suitable extraction procedure.

Extraction of Bacterial DNA:

Bacterial DNA is extracted through the following steps:

1. A known amount of bacterial cells is taken in saline EDTA. The high concentration of NaCl prevents strand separation of the DNA double helix. A small amount of lysozyme is supplemented and the suspension is kept at 37°C for 20 min in water bath. Then 10% SDS is added and incubated at 60°C for 10 min.

The mixture is then cooled to room temperature. A small amount of sodium chlorate solution is then added to denature the proteins. To it an organic solvent mixture containing chloroform and isoamyl alcohol is added. Chloroform is the most important solvent for DNA extraction, as it can denature proteins also. The isoamyl alcohol acts as an anti-foaming agent.

2. The resulting mixture is then centrifuged at 10,000 rpm. The supernatant is taken in a beaker and then 80% ethanol is slowly poured down the side of the beaker. The two layers are shaken with the help of a glass rod and the DNA fibres are spooled round the glass rod. Finally, fibrous DNA is blotted on the filter paper.

3. The DNA is dissolved by saline sodium citrate solution that can also check the activity of DNases. The mixture is then incubated at 37°C for 30 min with the enzyme RNase with occasional stirring.

RNase activity is stopped by the addition of sodium chlorate solution followed by the addition of chloroform and isoamyl alcohol. The DNA is precipitated again by the addition of 80% ethanol in the same way and is spooled out by glass rod and can be stored at 4°C.

4. In order to precipitate residual DNA more amount of isopropyl alcohol is added. Precipitated DNA is removed, blotted with filter paper, re-dissolved in saline sodium citrate and stored at 4°C.

Gene Technology: Tool # 9.

Isolation of DNA from Plant Material:

Extraction of DNA from plant is more difficult than from bacterial system as they contain a variety of secondary metabolites like phenols and flavonoids that interfere DNA extraction.

A general outline of the extraction procedure is described in the following steps:

1. Required amount of plant tissues are dried and powdered quickly with the help of liquid nitrogen. Alternatively, air-dried or oven-dried or heat-dried tissues at 40°C for 48 h can be used.

The powdered sample is transferred to EDTA buffer and incubated at 65 °C for 1 h containing SDS that weakens the rigid cell wall. High ionic strength of buffer helps to dissociate the histone proteins. To cleave disulphide linkages in proteins mercapta-ethanol is added.

2. After incubation, the solution is cooled to room temperature, chloroform or phenol is added and the resulting suspension is centrifuged at 10,000 rpm for 10 min.

3. The aqueous supernatant is transferred into a clean beaker. Nearly 2/3 volume of isopropyl alcohol or ice cold ethanol is slowly poured down the side of glass wall of the beaker to precipitate DNA. The precipitated DNA fibres are spooled round a glass rod and blotted on a filter paper.

4. The blotted DNA is dissolved in TE buffer and stored at 4°C for further use.

5. The sedimented DNA fibres are then treated with RNase to digest contaminated RNA.