BIO732 Official Handouts

Topic No. 1

Introduction to Gene manipulation and genetic engineering

“Gene manipulation is also called as genetic engineering and it refers to the alteration of genes of an organism. It involves manually adding new DNA to an organism to introduce novel traits. A variety of microorganisms, plants and animals has been generated with novel traits by using this technique. Overall, in this course, we will learn about the basic techniques that are used for the isolation of target genes, their analysis by gel electrophoresis and PCR amplification. It addition to that, cutting and joining of target DNA molecules or vectors is also an important component of this discipline. Transformation of target organism and expression of foreign genes will also be focused. We will also learn different techniques to modify microbes, plants and animals. Finally, we also discuss the applications of genetic engineering to get beneficial end products or to cure different types of diseases.

Topic 2

What is genetic engineering?

Genetic engineering is the process of manually adding new DNA to an organism. The goal is to add one or more new traits that are not already found in that organism. However, there are several other terms that can be used to describe the technology, including gene manipulation, gene cloning, recombinant DNA technology, genetic modification etc. In most western countries there is a precise legal definition of gene manipulation

“the formation of new combinations of heritable material by the insertion of nucleic acid molecules, produced by whatever means outside the cell, into any virus, bacterial plasmid or other vector system so as to allow their incorporation into a host organism in which they are capable of continued propagation”

Genetic engineering has application in both basic and applied research. In basic research, it is used to study mechanisms of gene replication and expression in prokaryotes, eukaryotes and their viruses. In applied research, it is used to develop microbial cultures that produce valuable metabolic products. It is also important to generate transgenic organisms, in environmental biotechnology and to treat genetic disorders (gene therapy).

Topic 3

Genes and genome

A genome is an organism‟s complete set of DNA, including all genes. Genome contains all of the information needed to build that organism and allow it to grow and develop. Instructions in our genome are made up of DNA-unique chemical code. Both DNA and RNA contain two major purine bases i.e. adenine (A) and Guanine (G) and two major pyrimidines. In DNA and RNA, one of the pyrimidine-cytosine (C) is common but second pyrimidine in DNA is thymine (T) and in RNA it is uracil (U). Gene on the other hand, is the nucleic acid sequence that is necessary for the synthesis of a functional gene product i.e. polypeptide or RNA. A cell typically has many thousands of genes and DNA molecules not surprisingly tend to be very large. The storage and transmission of biological information are the only know functions of DNA.

Topic 4

The flow of genetic information

The flow of genetic information in a living system is called central dogma of molecular biology (Fig. 1). Genetic information is encoded in DNA that is replicated before cell division. Genetic information flows from DNA to RNA and then to protein. This flow of information is dependent on the genetic code (a sequence of triplet bases), which defines the relation between the sequence of bases in DNA or mRNA and the sequence of amino acids in a protein. The code is nearly the same in all organisms: a sequence of three bases, called a codon, specifies an amino acid. Codons in mRNA are read sequentially by respective tRNA molecules, which serve as adaptors in protein synthesis. Protein synthesis takes place on ribosomes, which are complex assemblies of rRNAs and more than 50 kinds of proteins.

Figure 1. Flow of genetic information in living system.

Topic 5

Molecular cloning

Molecular cloning is at the base of most genetic engineering procedures. The basic strategy of molecular cloning is to move the desired gene from a large complex genome to a small, simple one. Fortunately, our knowledge of DNA chemistry and enzymology allows us to break and join DNA molecules in vitro. This process is known as in vitro recombination. Restriction enzymes, DNA ligase and synthetic DNA are important tools used for in vitro recombination. Molecular cloning can be divided into several steps as elaborated in figure 2. i). Isolation and fragmentation of source DNA. This can be total genomic DNA from an organism of interest, DNA synthesized from an RNA template by reverse transcriptase, DNA synthesized by the polymerase chain reaction (PCR) and even DNA synthesized in vitro. If genomic DNA is the source, it is generally cut with restriction enzymes to give a mixture of fragments. ii). Joining of DNA fragments to a cloning vector with DNA ligase. The small, independently replicating genetic elements used to replicate genes are known as cloning vectors. Cloning vectors are generally designed to allow recombination of foreign DNA at a restriction site that cut the vector in a way that does not affect its replication. If the source DNA and the vector are cut with the same restriction enzyme, joining can be mediated by annealing of the single stranded regions called “sticky ends”. iii). Introduction and maintenance of a host organisms. The recombinant DNA molecule made in a test tube is introduced into a host organism, for example, by DNA transformation, where it can replicate. Transfer of the DNA into the host usually yields a mixture of clones. Some cells contain the desired cloned gene, whereas, other cells contain other clones generated by joining the source DNA to the vector. Such a mixture is known as a DNA library or a gene library because many different clones can be purified from the mixture, each containing different cloned DNA segments from the source organism. iv). Detection and purification of the desired clone. Often one of the most difficult tasks is finding the right clone in a mixture that may contain thousands of others. v). Production of large numbers of cells or bacteriophage containing the desired clone for isolation and study of the cloned DNA.