The polymerase chain reaction (PCR) is the subject for Chapter 9. The basic principle is outlined, and the standard end-point PCR technique is described to illustrate how DNA amplification from defined primers is achieved. The design of primers for PCR is detailed, and the effect of redundancy of the genetic code noted when working from amino acid sequence data. The use of thermostable DNA polymerases in enabling automation of the PCR process using thermal cycling is outlined. Many different applications have been developed for the PCR, with variants of the basic protocol becoming more complex and sophisticated. PCR from mRNA templates is described, with other variants, including nested PCR, inverse PCR, quantitative and digital PCR, outlined. The extensive range of PCR variants is listed for comparison, and used to illustrate how the original technique of sequential amplification of DNA has become a key technique for the detection, analysis and quantification of DNA.

Chapter 6 outlines the range of methods used to isolate, purify and analyse nucleic acids. Methods to quantify DNA and RNA, and labelling of nucleic acids using radioactive and fluorographic precursors and a range of enzymatic methods, are described. The use of gel electrophoresis to separate DNA fragments is discussed. The principles of first-generation DNA sequencing are outlined, and the Sanger dideoxy method described for manual and automated methods. Next-generation methods for DNA sequencing are covered, to illustrate the range of advanced techniques that have enabled large-scale genome sequencing to become a routine laboratory procedure that is both rapid and cost-effective. Techniques for massively parallel and single-molecule real-time sequencing are described.

Chapter 2 outlines the history of DNA research and the key scientists who made the discoveries that enabled the manipulation of DNA. The scope, nature and ethos of science and the scientific method are described, with models for the scientific method and support for research. The importance of gathering and evaluating data in experimental science is outlined, and some of the key aspects and terminology are discussed.

Chapter 14 describes the biotechnological applications of recombinant DNA technology. The range of disciplines that contribute to biotechnology is outlined to illustrate the scale and scope of the sector. Production of proteins is one key area where cloned genes can be expressed to produce high-value products for use in a variety of applications, and the types of systems used for protein production are discussed. Protein engineering by methods such as rational design and directed evolution has enabled customised proteins to be developed for specific applications. The requirements for transition from laboratory-scale research and development to industrial production at a commercially viable level are outlined, and the contribution of the biotechnology sector in managing the COVID-19 pandemic is discussed.

In Chapter 8, various strategies that can be used to clone DNA fragments are described. Cloning genomic DNA and complementary DNA (cDNA) to generate libraries of cloned fragments remain two of the most common methods for primary library construction. Fragments may also be generated by polymerase chain reaction (PCR), or may be designed from a sequence database and synthesised in vitro. The choice of vector (plasmid, bacteriophage, virus or artificial chromosome) depends on the intended outcome, the size and origin of fragments and whether it is a primary cloning or a sub-cloning protocol. Restriction-dependent and restriction-independent methods can be used to join fragments to vectors. Techniques such as Golden Gate cloning, Gateway technology and Gibson assembly have mostly replaced earlier methods and can be used to assemble several fragments into a multi-fragment construct.

In Chapter 17, the topic of organismal cloning is described, noting the difference between reproductive and therapeutic cloning. Preformationism and epigenesis (as concepts of development), and the work in the early 1900s that led to the development of the concept of nuclear totipotency, are outlined. As the concept was refined, cells that are pluripotent, multipotent or irreversibly differentiated were described. Cloning using nuclear transfer was first proposed in 1938, and achieved in 1952. More correctly known as somatic cell nuclear transfer (SCNT), the birth of Dolly in 1996 was a milestone in that she was the first mammal to be cloned using a fully differentiated somatic cell as the source of the donor nucleus.

Chapter 3 outlines the need for a broad ethical framework in all areas of human activity, in particular the importance of ethics and bioethics in the context of genetic engineering. The basis of the ethical framework is outlined, and some key ethical issues across the eras of genetics are described.Current and emerging ethical dilemmas, and the need for active engagement with ethical issues in science, by both scientists and the public, are noted.

In Chapter 7, the host cells and vectors that are used to enable recombinant DNA to be propagated and amplified are discussed. A range of prokaryotic and eukaryotic cells can be used to propagate DNA in vectors derived from plasmids, bacteriophages and plant and animal viruses. Vectors are engineered to have particular properties such as unique cloning sites, origins of replication, selectable markers and promoters for expressing cloned genes. Vectors are designed to be compatible with one or more host cell types to enable flexibility in use. When recombinant DNA has been generated in vitro, the vector/insert combination is introduced into the target host cell by processes such as transformation and transfection. Alternatively, a mechanical process can be used, such as biolistic delivery using a ‘gene gun’ to fire DNA directly into cells.

In Chapter 10, the methods used to identify and select specific clones from clone banks (libraries) are described. The terms selection and screening are defined, and selection using selectable markers for antibiotic resistance is outlined as an example. Other genetic selection methods such as use of chromogenic substrates, insertional inactivation and complementation of defined mutations are described. Screening clone banks with nucleic acid probes and hybridisation methods is outlined to illustrate the powerful and specific nature of this approach. Radioactive and non-radioactive labelling techniques are compared, with the advantages and disadvantages of each outlined. The use of the polymerase chain reaction in screening, and immunological screening for expressed genes are described. Automation of screening is discussed to illustrate how high-throughput screening protocols enable very large numbers of clones to be screened efficiently. Analysis of cloned genes is described, covering techniques such as restriction mapping, Southern blotting and its derivative methods, sub-cloning and DNA sequencing.

The generation of transgenic plants and animals is discussed in Chapter 16. The technology is now well established, but remains a controversial area in terms of public perception and acceptance. Scientific, regulatory, ethical, political and commercial factors together present a complex framework within which the development of transgenic plants and animals is placed. As well as considering the technical procedures used to generate transgenics, these broader aspects (and their impact on the success or failure of a transgenic product) are considered. The development of Golden Rice, and the subsequent political issues around its deployment, illustrate the complexity of the topic. Paradoxically, transgenic animals often generate less negative reaction from the anti-GMO (genetically modified organism) movement than transgenic plants, with most concerns being around animal welfare issues. This is particularly true where the product is, for example, a demonstrably positive therapeutic; when transgenic animals are generated for consumption, the GMO debate tends to become polarised to the same extent as transgenic crops.

In Chapter 12, the techniques used for editing genomes are discussed, from early gene targeting approaches to highly specific CRISPR and prime editing techniques using engineered nucleases. The development of techniques through zinc-finger nucleases and TALENs is used to set the context for the CRISPR and prime editing methods that have become widely accessible and are used extensively to make changes to genomes. As an alternative to editing the genome, RNA editing offers a different approach that enables modulation of gene expression without complete inactivation or alteration of genes and may be more appropriate in some therapeutic situations. The potential for genome and RNA editing is outlined, and the ethical aspects noted.