An Introduction to Genetic Engineering by Dr Desmond S. T. Nicholl

By Dr Desmond S. T. Nicholl

During this 3rd version of his renowned undergraduate-level textbook, Des Nicholl recognises sound snatch of uncomplicated ideas is essential in any creation to genetic engineering. consequently, in addition to being completely up-to-date, the ebook additionally keeps its concentrate on the basic rules utilized in gene manipulation. The textual content is split into 3 sections: half I presents an creation to the correct simple molecular biology; half II, the equipment used to govern genes; and half III, functions of the expertise. there's a new bankruptcy dedicated to the rising significance of bioinformatics as a special self-discipline. different extra gains contain textual content containers, which spotlight very important features of issues mentioned, and bankruptcy summaries, which come with goals and studying results. those, besides key observe listings, notion maps and a thesaurus, will allow scholars to tailor their research to fit their very own studying types and eventually achieve a company grab of an issue that scholars ordinarily locate tough.

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DNA polymerase I catalyses a strand-replacement reaction that incorporates new dNTPs into the DNA chain. If one of the dNTPs supplied is radioactive, the result is a highly labelled DNA molecule (Fig. 3). Fig. 3 Labelling DNA by nick translation. (a) A single-strand nick is introduced into the phosphodiester backbone of a DNA fragment using DNase I. (b) DNA polymerase I then synthesises a copy of the template strand, degrading the non-template strand with its 5 →3 exonuclease activity. If [α-32 P]dNTP is supplied this will be incorporated into the newly synthesised strand (solid circles).

In this way nanogram or picogram amounts may be dispensed with reasonable accuracy. Precipitation of nucleic acids is an essential technique that is used in a variety of applications. The two most commonly used precipitants are isopropanol and ethanol, ethanol being the preferred choice for most applications. 2 M salt, ethanol causes the nucleic acids to come out of solution. Although it used to be thought that low temperatures (−20◦ C or −70◦ C) were necessary, this is not an 13:16 P1: SBT 09780521850063c03 CUUS128/Nicholl 978 0 521 85006 3 March 11, 2008 WORKING WITH NUCLEIC ACIDS absolute requirement, and 0◦ C appears to be adequate.

If mRNA is needed for cDNA synthesis, a further purification can be performed by affinity chromatography using oligo(dT)-cellulose to bind the poly(A) tails of eukaryotic mRNAs (Fig. 1). This gives substantial enrichment for mRNA and enables most of the contaminating DNA, rRNA, and tRNA to be removed. The technique of gradient centrifugation is often used to prepare DNA, particularly plasmid DNA (pDNA). In this technique a caesium chloride (CsCl) solution containing the DNA preparation is spun at high speed in an ultracentrifuge.

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