Haybrant

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Hello Scientists,

Can someone tell me what it means to clone a gene? I know it used to be a big thing back in the day but the technology is such that it is now easy; why is it easier now, and what does cloning a gene tell you? How would you know where to start? Overview of the procedure? Thanks!
 

Molecule

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Haybrant said:
Hello Scientists,

Can someone tell me what it means to clone a gene? I know it used to be a big thing back in the day but the technology is such that it is now easy; why is it easier now, and what does cloning a gene tell you? How would you know where to start? Overview of the procedure? Thanks!
How about: to incorporate a segment of DNA corresponding to a gene in a vector that can then replicate in another organism.

This can be a genomic clone (introns and gene regulatory elements included)...or it can be a cDNA clone (coding sequence only).

It's easier now because of the completion of various genome sequencing projects--and gene annotation (preliminary identification of what a gene encodes), which is becoming more and more complete everyday.


People can start by one of many routes. But two of the most common are by:

(1) a genetic screen - Mutagenizing many individuals and then searching for the one mutant that has a biochemical, developmental, or behavioral phenotype of interest. This phenotype is then followed over a series of genetic crosses with marker strains (phenotypic or genotypic "signposts" at different chromosomal sites) that allow determination of a gene's coordinates.

(2) screening by homology - Taking a known gene and looking for relatives (which should have similar sequence motifs) by using, for instance, bacterial clones with diced-up DNA fragments from the organism of interest. By Watson-Crick basepairing between a labeled probe and your bacterial clones (under conditions which are permissive of less than perfect basepair matching), you could pull-up gene cousins, this way.


In Case #1, isolating the clone--and finding that overexpression (or, conversely, knocking-out) a gene either rescues or reproduces a phenotype helps prove your argument that gene X is responsible for the mutant phenotype you isolated. This can then be used to help you construct gene pathways and gain traction into a process whose molecular underpinnings are not yet fully understood.

In Case #2, isolating related genes can help you determine what function(s) similar gene products (to the one you started with) mediate. Sometimes, you can gain a fuller understanding of your starting gene, this way, by seeing what a cousin does in a different context. For instance, if the starting gene is neuronally-expressed--and behavior is too difficult a thing for you to study--perhaps by understanding what a related gene does in development will give you biological metaphors with which to understand your starting gene.
 

PostalWookie

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It's definitely easier to clone a gene now than in the days before PCR and the genome projects, but it's still a bitch if you're working with a species that hasn't been lucky enough to have its genome sequenced. I'm working with Aplysia californica, and all we have to go by is a crappy, poorly annotated EST database, so cloning a gene for us takes a lot of time and effort.