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Biomolecular and Bioanalytical Techniques
book

Biomolecular and Bioanalytical Techniques

by Vasudevan Ramesh
June 2019
Beginner to intermediate content levelBeginner to intermediate
576 pages
25h 29m
English
Wiley
Content preview from Biomolecular and Bioanalytical Techniques

4 Gene Cloning for the Analysis of Gene Expression

Huw B. Thomas and Raymond T. O'Keefe

Faculty of Biology, Medicine and Health, University of Manchester, Oxford Road, Manchester, M13 9PT, UK

4.1 Identifying Target Sequence

Identifying and defining a target sequence is an important first step in expression studies since it forms the basis for all subsequent experiments. Several well‐established and fully curated databases are available online that may be used to provide the necessary nucleotide and/or protein sequence(s). These include fully comprehensive multiorganism online resources such as the National Center for Biotechnology Information (NCBI), UCSC Genome Browser and Ensembl, or more bespoke, organism‐specific databases such as those available for yeast ( Saccharomyces Genome Database [SGD], PomBase), Zebrafish (ZFIN) or Drosophila (Flybase). Links to each of these databases can be found at the end of this chapter. Multiorganism depositories are useful for identifying orthologous genes for cross‐species expression studies, whilst single‐species databases are often updated with greater regularity.

Selection of the correct sequence, and therefore the correct database source, will depend on the specifics of the intended experiment. For example, analysis of splicing consequences of a particular exon using a mini‐gene splicing assay will require not only the sequence of the exon in question but also the upstream and downstream intron sequences and thus would be ...

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