Tools of Genetic Engineering: Techniques and Examples

Tools of Genetic Engineering: Have you heard about molecular scissors? or Restriction Enzymes? All these come under the broader term Biotechnology. Biotechnology is the science of utilising biological systems to benefit humanity. Genetic Engineering that comes under this stream of study is a set of strategies for altering the chemistry of genetic material so that when it is put into a host, the phenotypic of the host changes.

Tools of Genetic Engineering involve many tools such as restriction enzymes, vectors, etc. Apart from these, there are a lot of Genetic engineering tools. For candidates who are wondering what those tools are, we’ve included detailed explanations regarding the same in this article.

Tools Used in Genetic Engineering

Before discussing the tools used in Genetic Engineering, let us first discuss what genetic engineering is.

What is Genetic Engineering?

The manipulation of the genetic make-up of living cells by inserting desired genes through a DNA vector is called Genetic Engineering.

Tools and Techniques of Genetic Engineering

The molecular tools of genetic engineering are as follows:

1. Restriction Enzymes

1. Restriction enzymes are called molecular scissors, as enzymes cut DNA at specific sites.
2. The first restriction endonuclease that was identified was Hind II.
3. The restriction enzymes cut DNA at a specific nitrogen base sequence, and these sequences are referred to as the recognition sequence.
4. The convention for naming restriction enzymes is as follows:
5. The first letter of the name is derived from the genus.
6. The second two letters are derived from the prokaryotic cell species from which they were isolated, e.g., EcoRI is obtained from Escherichia coli \({\rm{RY}}\,{\rm{13}}.\)
7. In EcoRI, the letter ‘\({\rm{R}}\)’ is obtained from the name of the strain.
8. Roman numbers that follow the names indicate how the enzymes were isolated from a particular strain of bacteria.
9. Around \(900\) restriction enzymes have been isolated from around 230 strains of bacteria.
10. The nuclease enzymes are of two types:
    a. Exonucleases – Exonucleases take off nucleotides from the ends of the DNA.
    b. Endonucleases – Endonucleases develop cuts at specific positions within the DNA.
11. Each restriction endonuclease recognises specific sequences (palindromic nucleotide sequences) within the DNA.
12. The palindrome in the DNA sequence of base pairs reads identical on the two strands when the reading direction is kept the same.
13. For Example- the following sequences read the same on the two strands in the \(5\)’ to \(3\)’ direction; this also holds if read in the \(3\)’ to \(5\)’ direction. The EcoRI cutting site is given as:
    \(5\)’ – GAATTC – \(3\)’
    \(3\)’ – CTTAAG – \(5\)’
14. Restriction enzymes cut the strand of DNA a little away from the centre of the palindromic sites but between the same two bases on the opposite strands, which leaves behind single-stranded portions at the ends. The overhanging stretches called on each strand are called sticky ends.
15. When cut by the same restriction enzyme, the DNA fragments obtained have the same kind of ‘sticky-ends,’ and these ends can be linked together using DNA ligases.

Fig: Steps in Formation of Recombinant DNA by Action of Restriction Endonuclease

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2. DNA Ligase

1. DNA ligase is an important enzyme in gene technology because it serves as a molecular suture, joining two strands of DNA that require ATP. During joining, the enzyme establishes a covalent link between one strand’s \(5\)′ phosphoryl group and the neighbouring strand’s \(3\)′ hydroxyl group.
2. Between two cohesive ends, two phosphodiester bonds are generated. The joining process is inhibited by the blunt ends produced by some enzymes. In such instances, joining requires a very high enzyme concentration. The \({{\rm{T}}_4}\) phage virus is the major source of this enzyme.

3. Methylases or Methyl Transferases

1. Methylase is an enzyme that adds a methyl group to the cytosine and adenine of DNA. It is a monomeric enzyme and has a molecular weight of \(62,000\) Daltons.
2. Methylases add a methyl group to the \({\rm{N – 6}}\) position of adenine to form \(6\)-methyl adenine and the \({\rm{N – 5}}\) position of cytosine to form \(5\)-methylcytosine.
3. It adds only one methyl group at a time and immediately dissociates from the DNA. To add a second methyl group, the enzyme once again binds to the DNA.

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4. DNA Polymerase

i. The DNA polymerase that is generally utilised or used is either the DNA Pol I from E. coli or the \({{\rm{T}}_4}\) DNA polymerase, which is enco­ded by the phage gene.

ii. The E. coli enzyme basically functions as a proof-reading and repairing enzyme. It is composed of three subunits, each with a specific function. They are \(5′-3′\) poly­merase, \(3′-5′\) exonuclease and \(5′-3′\) exonuclease.

5. Alkaline Phosphatase

It is a DNA modifying enzyme that adds or removes phosphate groups from the \(5’\) terminal of double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), or RNA. As a result, self-ligation is avoided. This enzyme is isolated from bacteria and the gut of a calf.

6. Reverse Transcriptase

1. It comes from the avian myeloblast virus and is an RNA-dependent DNA polymerase. The abbreviation for this is AMV-RT.
2. The enzyme needs the use of a DNA primer that is complementary to the RNA template. AMV-RT is made up of two key components.
3. Reverse transcriptase is used to make cDNA in genetic engineering. It is responsible for converting mRNA to single-stranded DNA.

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7. Cloning Vectors

1. A cloning vector is a small stretch of DNA obtained from any organism into which foreign DNA fragments can be introduced for cloning purposes.
2. Plasmids and bacteriophages have the potential to replicate within bacterial cells independent of the control of chromosomal DNA.
3. In case of a foreign piece of DNA inserted in a bacteriophage or plasmid DNA (vector), one can multiply its numbers equal to the copy number of the plasmid or bacteriophage.
4. The essential features that are required to facilitate cloning into a vector are as follows:
   a. Origin of replication (ori)
   This is the sequence from which replication begins, and any piece of DNA, when linked to this sequence, can be made to replicate inside the host cells.
   b. Selectable marker
   The vector must also have a selectable marker that permits recombinants to be chosen over non-recombinants. Genes that give resistance to antibiotics such as ampicillin, kanamycin, and chloramphenicol, among others, are effective selectable markers in E. coli. Because regular E. coli cells lack these resistance genes, it’s simple to identify recombinants.
   c. Cloning Sites
   i. Cloning sites are the recognition sites of the restriction enzymes.
   ii. The ligation of foreign DNA is performed at a restriction site located in one of the two antibiotic resistance genes.
   iii. For example: Ligation of a foreign DNA at the BamHI site of tetracycline resistance gene in the plasmid vector \({\rm{pBR}}322.\)

Fig: Plasmid Vector \({\rm{pBR}}322\)

d. Vectors to Clone Genes in Plants and Animals
i. Vector for cloning genes in plants is Agrobacterium tumefaciens, a pathogen of several dicot plants that deliver a piece of DNA known as ‘T-DNA’ to transform normal plant cells into a tumour and direct these tumour cells to produce the chemicals required by the pathogen.
ii. The tumour-inducing \(\left( {{\rm{Ti}}} \right)\) plasmid of Agrobacterium tumefaciens has now been modified into a cloning vector.
iii. Vector for cloning genes in animals is a retrovirus that transforms normal cells into cancerous cells.
iv. Retroviruses have been disarmed and used to deliver desirable genes into animal cells.

8. Competent Host

1. DNA cannot flow through cell membranes since it is a hydrophilic molecule. Why? Bacterial cells must first be considered ‘competent’ to take up DNA before they can be forced to take up the plasmid.
2. This is accomplished by administering a particular quantity of a divalent cation, such as calcium, to the bacteria, which enhances the efficiency with which DNA enters the bacterium through openings or the pores in the cell wall.
3. Recombinant DNA can then be pushed into such cells by incubating the cells with recombinant DNA on ice, then exposing them to \({\rm{42}}\,^\circ {\rm{C}}\) for a brief period of time (heat shock), and then returning them to ice.
4. The bacteria can now take up the recombinant DNA. This is not only the method foreign DNA may enter host cells. Microinjection is a technique that involves injecting recombinant DNA directly into the nucleus of an animal cell.
5. Cells are bombarded or blasted with high-velocity gold or tungsten microparticles coated with DNA in a process called or referred to as biolistics or gene gun, which is ideal for plants.
6. Finally, ‘disarmed pathogen’ vectors are used, which, when permitted to infect a cell, transmit the recombinant DNA into the host.

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Summary

Biotechnology is the use of natural science to organisms, cells, parts of living things, and molecular equivalents to create products and services. Through this article, we understood the importance of genetic engineering. The simple addition, deletion or manipulation of one single trait in an organism to create the desired change. The enzymes useful for cutting DNAs into pieces and joining the cut pieces of DNA into a long DNA are called tailoring enzymes or modifying or enzyme tools of genetic engineering.

Frequently Asked Questions (FAQs) on Tools of Genetic Engineering

Here are the answers to the most commonly asked questions n tools of genetic engineering:

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