Basic Tools Used in Genetic Engineering

Keywords

Recombinant DNA, genetic engineering, genes, gene cloning, genetic manipulations.

Introduction

Basic tools are necessary for the process to generate recombinant DNA molecules which are enzymes, vectors, and host organisms. The most important enzymes required for genetic engineering are the restriction enzymes, DNA ligase and alkaline phosphatase etc. Each DNA strand contains thousands of genes. Genetic engineering is the process of using gene cloning and other genetic manipulations to isolate specific genes and use it for research and other purposes.

Tools Used in Genetic Engineering

The list of ten tools/enzymes that are commonly used in genetic engineering are discussed below.

Polymerase Chain Reaction

PCR is known as the polymerase chain reaction. It is efficient because it multiplies the DNA exponentially for each of the 25 to 75 cycles. A cycle takes only a minute, and each new segment of DNA that is made can serve as a template for new ones.

Restriction Enzymes (Molecular Scissors)

The discovery of enzymes known as restriction endonucleases has been essential to protein engineering. These enzymes cut DNA at specific locations based on the nucleotide sequence. Hundreds of different restriction enzymes, capable of cutting DNA at a distinct site, have been isolated from many different strains of bacteria. DNA cut with a restriction enzyme produces many smaller fragments of varying sizes. These can be separated using gel electrophoresis or chromatography.

Gel Electrophoresis

Purifying DNA from cell culture or cutting it using restriction enzymes wouldn’t be of much use if we couldn’t visualize the DNA-that is, find a way to view whether your extract contains anything, or what size fragments you’ve cut it into. One way to do this is by gel electrophoresis. Gels are used for a variety of purposes, from viewing cut DNA to detecting DNA inserts and knockouts.

DNA Ligase

It is often necessary to link two or more individual strands of DNA, to create a recombinant strand, or close a circular strand that has been cut with restriction enzymes. Enzymes called DNA ligases can create covalent bonds between nucleotide chains. The enzymes DNA polymerase I and polynucleotide kinase are also important in this process, for filling in gaps, or phosphorylating the 5′ ends, respectively.

Alkaline Phosphatase

It is a DNA modifying enzymes and adds or removes specific phosphate group at 5’ terminus of double stranded DNA (dsDNA) or single stranded DNA (ssDNA) or RNA. It prevents self-ligation. This enzyme is purified from bacteria and calf intestine.

Polymerases

The groups of enzymes that catalyze the synthesis of nucleic acid molecules are collectively referred to as polymerases. The three important polymerases are given below.

• DNA-dependent DNA polymerase that replicates DNA from DNA.
• RNA-dependent DNA polymerase (reverse transcriptase) that transcribes DNA from RNA.
• DNA-dependent RNA polymerase that transcribes RNA from DNA

Vectors

Another major component of a gene cloning experiment is a vector such as a plasmid. A Vector is a small DNA molecule capable of self-replication and is used as a carrier and transporter of DNA fragments which is inserted into it for cloning experiments.

Selection of Small Self-Replicating DNA

Small circular pieces of DNA that are not part of a bacterial genome, but are capable of self-replication, are known as plasmids. Plasmids are often used as vectors to transport genes between microorganisms. In biotechnology, once the gene of interest has been amplified and both the gene and plasmid are cut by restriction enzymes, they are ligated together, generating what is known as recombinant DNA. Viral (bacteriophage) DNA can also be used as a vector, as can cosmids, recombinant plasmids containing bacteriophage genes.

Competent Host (For Transformation with Recombinant DNA)

The propagation of the recombinant DNA molecules must occur inside a living system or host. Many types of host cells are available for gene cloning which includes E. coli, yeast, animal, or plant cells based on the experiment. E. coli is the most widely used organism as its genetic make-up has been extensively studied, it is easy to handle and grow, can accept a range of vectors and has also been studied for safety. One more important feature of E. coli to be preferred as a host cell is that under optimal growing conditions the cells divide every 20 minutes. Since DNA is a hydrophilic molecule, it cannot pass through cell membranes, to force bacteria to take up the plasmid, the bacterial cells must first be made competent to take up DNA.

Methods to Select Transgenic Organisms

Not all cells will take up DNA during transformation. Therefore, it is essential to identify the cells that undergo a transformation and those that have not. Generally, plasmids carry genes for antibiotic resistance, and transgenic cells can be selected based on the expression of those genes and their ability to grow on media containing that antibiotic. Alternative methods of selection depend on the presence of other reporter proteins such as the x-gal/lacZ system, or green fluorescence protein, which allow selection based on color and fluorescence, respectively.

Conclusion

Products of genetic engineering are common nowadays in our daily life. Genetic engineering plays a vital role in our day-to-day life, from our food to the medicinal drugs that we use. This is one of the greatest achievements of humankind. Molecular biology is the future of this world. This technology can create a defect less world. Molecular biology creates areas for more research and developmental studies.