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Gene isolation

©2000 written by Gary Roberts, edited by Timothy Paustian, University of Wisconins-Madison

IX A2. GENE RECOGNITION BASED ON SIMILARITY TO GENES IN OTHER ORGANISMS

Given a cloned gene of interest from one organism, a different organism can be screened for a similar sequence as follows:

1. Perform a chromosomal Southern digest and probe with your labelled gene. If one or a small number of fragments shows hybridization, you have reason to be optimistic. If more--something's fishy. If none, try decreasing the stringency of the hybridization conditions or try another approach.

2. Use your probe to screen a library for the hybridizing fragment. Isolate and amplify a clone carrying the hybridizing region.

3. Verify the correctness of the clone by making a selectable mutation in that clone, replacing the chromosomal version with this mutated one, and checking the phenotype.

Structural similarity of gene products from different organisms can also be employed: antibody to a given protein can be used to screen for the presence of a clone synthesizing that protein or an antigenically related one. In this example, of course, the clones need to be screened in a strain incapable of making the antigen by itself, since such a background signal will probably ruin the screen.

The scheme based on nucleic acid hybridization is extremely powerful as it is becoming evident that there is significant conservation of coding sequences (and therefore detectable hybridization) for particular products among widely diverse organisms. This approach is based on the reasonable presumption that similarity at the level of DNA sequence will reflect a similarity of function between the encoded products. Potential problems include: (i). DNA similarity is too low to allow good hybridization or (ii) the two encoded proteins so detected might share a section of similarity (for example, reflecting a ATP-binding domain), but be otherwise completely different.

IX A3. GENE ISOLATION BASED ON PROTEIN SEQUENCE

One of the more elaborate examples of "inverse genetics" is the situation where the protein of interest has been purified, but the gene that encodes that protein has not been identified. In the first step, a portion of the protein is sequenced until a sequence of at least six contiguous amino acids is found for which the coding sequence is reasonably non-redundant. (Such a region would contain amino acids encoded by relatively few different codons.) All possible coding sequences of this region are then synthesized (a nucleic acid sequence of at least seventeen nucleotides is desired for specificity reasons) in a single batch. Alternatively, one either looks at codon use by the organism and generates a "best guess" oligo, or puts inosine at positions of uncertainty. This pool of sequences is labelled and used in a Southern analysis of the entire cell's restriction enzyme-treated chromosome. Typically, one or a small number of restriction fragments will show hybridization indicating similarity to one particular probe in the pool. Having shown that there is a hybridizing sequence in the chromosome, the probe pool is then used to screen a random library of cloned chromosomal fragments. Alternatively, a new library can be generated using the chromosomal fragments of the same size as the hybridizing band; this "enriched" library would then be screened with the probe pool. Any clone that shows homology to the probe would likely carry at least a portion of the gene of interest.

At this point, there are several possibilities: (i) the region may be sequenced in order to determine the open reading frames. Since you already know one end of the protein sequence, this will allow the conclusive identification of the desired gene. This sequence information can be used to clone a selectable marker (for example drug-resistance) into the coding region, for use in mutationally altering the chromosomal version. (ii) Alternatively, a restriction map of the cloned region can be produced that will indicate the relative position of the probe-hybridizing region within the clone. The clone can now be mutagenized with transposons and such mutations analyzed physically to choose a pair flanking the probe-hybridizing region (see section IX.C2). One of these must therefore be in the gene of interest.

Before serious conclusions are drawn, a mutation in the target region should be introduced into the chromosome in order to confirm that the region really does encode the product of interest.

IX A4. HYBRIDIZATION TO A TRANSPOSON INSERTION CONFERRING A DESIRED PHENOTYPE

If a mutation with an interesting phenotype is generated by transposon mutagenesis, the transposon will provide a "tag" for both genetic or physical mapping. Genetic mapping would rely on the drug resistance phenotype, but physical mapping would use a probe specific for the transposon; clones carrying the transposon will necessarily also contain the mutant version of the desired gene. This fragment can then be used as a probe for the isolation of the wild-type gene from an appropriate library.

IX A5. PCR AMPLIFICATION

The PCR (for Polymerase Chain Reaction) system employs two oligomers, homologous to regions on opposite strands, hundreds to thousands of base pairs apart, to amplify the intervening DNA. One uses the oligomers as primers for DNA synthesis in many repeated cycles of DNA denaturation, oligomer priming, and DNA synthesis to amplify a region up to 10⁵-fold without cloning. There are also a range of other technologies on the horizon, with names like "self- sustained sequence replication", "strand displacement activation", and "ligase chain reaction", that achieve similarly massive amplifications of nucleic acids, but through different enzymes and protocols.

By this method, one is able to obtain "biochemically" useful amounts of DNA without cloning. You can even amplify regions from relatively uncharacterized organisms, relying on sufficient sequence conservation that the priming oligomers still function (you are also requiring that the regions complementary to those oligomers are so positioned that reasonably sized amplified regions are obtained). Other uses include: amplification of a region from mutants for direct sequence analysis, use in both site-directed and localized mutagenesis, and the generation of specific gene (and therefore protein) fusions through choice of "hybrid primers". There is little doubt that this procedure, and its variants, will play an ever larger role in our future science.

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