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Table of Contents
Why Do Genetics
More Basic Concepts
Mutagenesis in vitro
Effects of Mutations
Two Factor Crosses
Other Mapping Methods
Problem Set 1
Problem Set 2
Analysis and use of clones and sequences
IX C. ANALYSIS AND USE
IX C1. ANALYSIS OF SEQUENCE
Rather than provide a description of sequencing technique, this section will touch on the requirements for sequencing and its uses. At the present time, sequencing methods are adequate to analyze 100-300 base pair stretches of DNA at a time. The rate of advances in technique has been so great, however, that this limit will probably be increased significantly in the near future. Similarly, until quite recently, any DNA to be sequenced needed to be cloned so that the region could be amplified. Now, however, the technique of PCR threatens to change the way we think about sequencing.
More important than considerations of specific methods is the question, what information is gained from sequence analysis?
The sequence does not necessarily identify functional genes, since there are a number of cases where an "open reading frame" does not correspond to the region encoding a gene product. These include "stop signals" that actually code for amino acids, cases of "natural" frameshifting, non-standard start codons, cases of ribosomes "jumping" over 2-20 codons in an mRNA, and cases of cryptic genes. Sequence analysis also does not identify biochemical functions, since the presence of "motifs" does not prove that such sites are important for the biological function of the gene product. For these reasons, while sequence provides very powerful insights into probable function, it is careless to make either biochemical or genetic conclusions based solely on sequence analysis.
IX C2. GENERATION AND ANALYSIS OF MUTATIONS IN THE CLONED REGION
There are two rather different reasons for generating mutations in your cloned region. The first is to help identify which region encodes products relevant to the phenotype under analysis and the second is to perform a precise "structure/function" analysis of the encoded products. For the former goal, loss-of-function mutations without polarity problems are best. These might include in-frame deletions are small in-frame insertions, which would be analyzed for any effect on the phenotype of interest. Structure/function analysis would almost certainly require site-directed or localized mutagenesis, coupled with a biochemical analysis of the affected protein product.
By any means of mutation generation, the resulting mutated clones will need to be analyzed for interesting phenotypes either by examining the products of the clone directly or by introducing the cloned region into the chromosome, replacing the wild-type version. The latter is clearly preferable in terms of its physiological "correctness", but is significantly more time- consuming. A good strategy would be to analyze the products of the mutated clones and then move any "interesting" alleles into the chromosome for better characterization.
IX C3. MORE COMPLICATED CONSTRUCTIONS
The knowledge of the primary structure of a nucleic acid and its encoded product, coupled with the ability to rearrange sequences in a precise way, allows the production of novel products in novel amounts. While the possibilities are vast, here are two general examples. While the fusion proteins, described in section X, are used to monitor regulation, more precise gene fusions can be constructed which produce products retaining activities encoded by both fused genes. Alternatively, constructions can be performed that place the expression of a gene under a novel promoter, for example one which can be regulated by temperature or the addition of a small molecule to the culture medium. This allows the experimenter to produce large quantities of a product toxic to the cell by growing the culture to high cell density and then triggering the expression of the gene when cell growth is no longer necessary. This can be particularly useful in the site-directed mutagenesis described immediately above, since large amounts of the altered product are available for easy purification and characterization. As a small note of caution, proteins that are dramatically overproduced are occasionally found to be "odd" in some way because of their overproduction, so that the blind analysis of their properties would yield a misleading result.
[See sample problems 23-24]
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