DNA and Recombinant molecular biology techniques

Introduction:-

Recombinant DNA technology was only a dream a century ago, when it was thought that by regulating the expressions of target genes, desired traits might be improved in live organisms. However, in the recent age, this sector has exhibited distinctive impacts in terms of human life improvement. Crucial proteins necessary for health issues and nutritional needs may be generated securely, inexpensively, and in adequate quantities thanks to this technology.

This technique offers a wide range of uses and has the potential to improve essential elements of life, such as health, food security, and resistance to a variety of harmful environmental impacts. Genetically engineered plants, particularly in agriculture, have boosted resistance to hazardous agents, increased product production, and demonstrated higher flexibility for better survival. (Taylor Robinson AW, December 29, 2016)

Furthermore, recombinant medicines are now being utilized with confidence, and commercial clearances are being obtained quickly. Bioremediation and the treatment of severe illnesses are additional common uses of recombinant DNA technology, gene therapy, and genetic alterations. Because of the rapid progress and wide range of applications in the field of recombinant DNA technology, I've chosen this topic and will attempt to discuss some key approaches.

Applications:-

Techniques:-

There are various techniques of molecular biology but some of the Techniques used in rDNA technology are as follows:-

• Gel electrophoresis

• Cloning libraries

• Restriction enzyme mapping

• PCR

• Nucleic Acid Hybridization

• DNA Microarrays

 

Gel electrophoresis: - DNA fragments of different sizes can be separated by an electrical field applied to a “gel”. The negatively charged DNA migrates away from the negative electrode and to the positive electrode. The smaller the fragment the faster it migrates. For separation of these cut fragments and isolation of desired DNA fragment, the technique of gel electrophoresis is employed. Gel electrophoresis may be of horizontal or vertical type. Usually agarose gel is used for separation of large segments of DNA while the polyacrylamide gel is used for the separation of small DNA fragments which are only a few base pairs long.

Gel electrophoresis employs a buffer system, a medium which is a gel and a source of direct current. Samples having DNA fragments are applied on the gel and current is passed through the system for an appropriate time. Different DNA fragments move up to different distances on the gel depending on their charge to mass ratio.

Cloning libraries: - Libraries are collection of DNA clones in a certain vector. The goal is to have each gene represented in the library at least once. Genomic made from R-DNA fragments of total genomic DNA. cDNA (complementary DNA) made from DNA synthesized from mRNA.

PCR: - Using DNA primers at the ends of the segment of interest, it is possible to isolate a specific segment of DNA from a tiny DNA. (Suliman Khan, 2016)

 PCR is a technique which results in selective amplification of a selected DNA molecule. One limitation of PCR is that the border region sequences of the DNA must be known in order to select the appropriate primers which anneal at its 3′ ends. Primer annealing is important due to the fact that enzyme DNA polymerases require double stranded primer regions for initiating the DNA synthesis.

The whole reaction of PCR takes place in a tube called eppendorf tube. Scientists are using PCR in a number of disciplines due to the advantages like it is a quick, simple and extremely accurate technique. Major limitation of PCR is that due to its extreme sensitivity it may produce erroneous results caused by several inhibitors or contaminating DNA segments present in the sample DNA preparation.

Restriction enzyme mapping: - A restriction enzyme site map of a cloned gene is usually required for further gene modifications. This is achieved by digesting the gene individually with multiple enzymes before combining them. The fragments are separated by size using gel electrophoresis, and the sites are calculated based on the sizes of the fragments.

Nucleic Acid Hybridization: - A Southern allows the detection of a gene of interest by probing DNA fragments that have been separated by electrophoresis with a “labeled” probe. Northern Blot (probe RNA on a gel with a DNA probe) Western Blot (probe proteins on a gel with an antibody)

DNA Microarrays: - In light of the DNA silicon chip business, the great majority of protein-encoding characteristics will be encoded on a microarray chip. The chip may be used to hybridize to cell RNA and assess the rate of expression of a large number of different characteristics in a cell. (BASHIR, 2015)

CONCLUSION:-

Recombinant DNA technology is an important development in science that has made the human life much easier. In recent years, it has advanced strategies for biomedical applications such as cancer treatment, genetic diseases, diabetes, and several plants disorders especially viral and fungal resistance. The role of recombinant DNA technology in making environment clean and enhanced resistance of plants to different adverse acting factors has been recognized widely. The improvements it brought not only in humans but also in plants and microorganisms are very significant. The challenges in improving the products at gene level sometimes face serious difficulties which are needed to be dealt for the betterment of the recombinant DNA technology future.

                                                                                   ARTICLE BY:-SEHRISH ZAHID

Bibliography

1) BASHIR, N. (2015, MAY). RECOMBINANT DNA TECHNOLOGY. Retrieved from Slideshare: https://www.slideshare.net/nasira55/recombinant-dna-technology-47715143

2)Suliman Khan, 1. M. (2016). Role of Recombinant DNA Technology to Improve Life. INTERNATIONAL JOURNAL OF GENOMICS, 1110-1127.

3)Taylor Robinson AW, 1. R. ( December 29, 2016). Application of recombinant DNA technology. Applied Biotechnology & Bioengineering, 35-46.