Introduction

In the last 30 years or so, there has been a revolution in the field of genetics, which has had a profound effect on virtually every other area of biology. This has been due to the development of new techniques that have enabled scientists to analyse and manipulate DNA in a quite unprecedented way. Genetically modified crops, DNA 'fingerprinting' and gene therapy are just three of the many applications made possible by these advances. The subject of 'genetic engineering' is too huge to be discussed here in detail, and indeed it extends into areas far beyond the remit of this book. In this chapter, however, we shall examine some of the ways that microorganisms have contributed to the genetic revolution. As we shall see, their role in the development of new techniques of DNA manipulation since the 1970s has been just as important as their earlier contribution to the elucidation of the structure, role and replication of DNA several decades earlier.

The beginnings of genetic engineering can be said to date from the discovery, in the late 1960s, of a class of bacterial enzymes called restriction endonucleases (REs). These are enzymes that cleave DNA into pieces by making breaks in the sugar-phosphate backbone; in nature, they serve to destroy any foreign DNA that may enter the cell. They do not cut the DNA in a random fashion, however; their unique usefulness to the molecular biologist lies in the fact that they break the DNA in a precise and reproducible manner. They do this by cutting only at specific recognition sites, sequences of typically four to six nucleotides (Figure 12.1). Thus, under favourable conditions, a particular RE will digest a given piece of DNA into an identical collection of fragments, time after time. In the ensuing years, many hundreds of restriction endonucleases have been discovered, many of which recognise different specific sequences, providing biologists with a hugely versatile tool for the manipulation of DNA, often likened to a pair of molecular 'scissors'. Not long after REs were first isolated, they were used to create the first man-made recombinant DNA molecule (Figure 12.2). This involved cutting

Restriction endonucleases do not destroy the host bacterium's own DNA, because certain nucleotides in the recognition sequence are modified by methyla-tion. The REs are unable to cleave the DNA at methylated sites.

Site of cleavage

Hind III

Site of cleavage

Site of cleavage Bal I

Figure 12.1 Restriction endonucleases fragment DNA molecules by breaking the sugar-phosphate backbone within a specific sequence of nucleotides. Depending on the site of cleavage, the fragments so produced may be blunt-ended or 'sticky'-ended fragments of DNA from different sources, then using another enzyme, DNA ligase to join them together, a process facilitated by using fragments with compatible 'sticky' ends. Remember from Chapter 11 that A always pairs with T and C with G; because of this, complementary sequences that come into contact with one another will 'stick' together. DNA, it seems, is DNA, wherever it comes from; consequently DNA from plants, animals, bacteria or viruses can be joined together to create novel sequences undreamed of by Mother Nature.

Of course, a single molecule of our newly recombinant DNA is not much use to us. The important breakthrough came with the development of cloning - the ability to produce huge numbers of copies of a given molecule. To do this, two further things are needed: a carrier DNA molecule called a vector, and a host cell in which it can be replicated.

Cloning is the production of multiple copies of a specific DNA molecule. The term is also used to describe the production of genetically identical cells or even organisms.

SOURCE 1 SOURCE 2

Mix fragments and join with DNA ligase i

RECOMBINANT DNA MOLECULE

Figure 12.2 DNA from different sources can be joined together. 'Sticky'-ended restriction fragments from one DNA source have single-stranded sequences that are compatible with fragments produced from another source by the same RE. Compatible base pairing attracts the fragments together and the join is made more permanent by the action of DNA ligase

Hind ill

Figure 12.3 shows the main steps of a cloning protocol:

• 'donor' DNA and vector are digested with an RE to provide compatible sticky ends

• a fragment of donor DNA is spliced into the vector molecule

• the recombinant vector gains entry to a host cell (e.g. E. coli)

• the vector replicates inside the cell, making further copies of the inserted DNA

• host multiplication results in the formation of a clone of cells, all containing the same recombinant plasmid - we now have millions of copies of our donor DNA 'insert'. A collection of such clones is called a DNA library.

Let us look at role of vectors in a little more detail. The main features required of a cloning vector are:

• it must be capable of replicating autonomously inside a host cell- when it does so, any DNA it carries will also be replicated. Vectors make multiple copies of themselves inside the host cell.

• it must be relatively small - to facilitate manipulation and entry into a host cell, vectors must not exceed a certain size.

A vector is a self-replicating DNA molecule used in gene cloning. The sequence to be cloned is inserted into the vector, and replicated along with it.

O Donor DNA

^^ RE digestion

Recombinant vector

Clone of identical cells

Figure 12.3 Gene cloning. DNA fragments obtained by restriction digestion can be spliced into a similarly digested vector molecule and transformed into a host bacterial cell. See the text for details

Clone of identical cells

Figure 12.3 Gene cloning. DNA fragments obtained by restriction digestion can be spliced into a similarly digested vector molecule and transformed into a host bacterial cell. See the text for details

• it must carry a selectable marker - since only a proportion of host cells will take up the vector, there must be a means of differentiating them from those that do not. A common way to do this is to use a vector that carries a gene that confers resistance to an antibiotic such as ampicillin. When bacterial cells are plated out on a medium containing the antibiotic, only those that have taken up the vector will be able to form colonies. (The host strain must, of course, normally be susceptible to the antibiotic.)

A selectable marker is a gene that allows cells containing it to be identified by the expression of a recognisable characteristic.

• it must carry a single copy of RE restriction sites - in order to accommodate a piece of donor DNA, a vector must be cut by a restriction endonuclease in one place only (Figure 12.3).

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