Chapter 10. Recombinant DNA technology

by Mihály Kovács

Table of Contents

10.1. Recombinant DNA techniques and molecular cloning
10.2. Plasmid vectors
10.3. Creation of recombinant DNA constructs
10.4. Introduction of recombinant DNA constructs into host cells and the identification of recombinant colonies
10.5. Isolation of plasmid DNA
10.6. Analysis of plasmid DNA by gel electrophoresis
10.7. Polymerase chain reaction (PCR)
10.8. Site directed in vitro mutagenesis
10.9. DNA sequencing

10.1. Recombinant DNA techniques and molecular cloning

Recombinant DNA (molecular cloning) techniques enable the amplification, sequence determination, manipulation and functional investigation of DNA segments of interest (e.g. genes) as well as the production, manipulation and investigation of products (RNA, proteins) encoded by these DNA segments. The amplification of DNA molecules of interest can take place by two principal means:

  1. By creating recombinant DNA constructs that enable the in vivo amplification of a certain DNA segment in a host organism. During the process of cloning, one (or a small number of) DNA molecule(s) taken up by a single cell are amplified by the proliferation (division) of the host cell. Recombinant DNA constructs are created by linking the DNA segment to be cloned (the so-called insert) into a vector (carrier) DNA fragment. The vector DNA is necessary for DNA propagation in the host cell. Chapters 10.2-10.6 below will summarise the principles of the design, production, amplification and investigation of recombinant DNA constructs.

  2. Via in vitro amplification of the DNA segment of interest by applying polymerase chain reaction (PCR). PCR will be described in detail in Chapter 10.7.

Recombinant DNA technology is an essential and indispensable tool in practically all lines of modern biological and biomedical research as well as in biotechnology-based industry. The following list contains a few examples for the areas of utilisation of these techniques.

  1. Investigation, alteration and manipulation of the sequence and structure of DNA and RNA molecules, individual genes or entire genomes;

  2. Investigation, alteration and manipulation of the sequence and structure of protein molecules encoded by genes (protein engineering), or the metabolic products produced in reactions catalysed by these proteins;

  3. Investigation of spatial (cell-specific or tissue-specific) and temporal patterns of gene expression;

  4. Elucidation of the mechanisms of action of signal transduction pathways, cancerous (malignant) transformation, physiological and pathological processes, hereditary and infectious diseases, and diagnosis of these conditions;

  5. Research in the areas of developmental biology, evolution, ecology and environmental science;

  6. Applications in agriculture and power industry;

  7. Industrial-scale production of proteins, drug molecules, vaccines, hormones;

  8. Creation of transgenic organisms for research or industrial purposes;

  9. Gene therapy (replacement of missing gene functions);

  10. Legal disputes and criminal investigations; identification of samples, origins and individuals.