Stanford/BIOE44:Module 1:Day5: Difference between revisions
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===Designing Primers=== | ===Designing Primers=== | ||
You are probably familiar with PCR reactions, but may not have considered | You are probably familiar with the use of PCR reactions to selectively amplify a region of DNA, but you may not have considered what goes into designing a primer set. If you are amplifying a particular region of DNA, you may need to know the regions upstream and downstream of an open reading frame in order to design regions where your primer can "touch down." These regions are called regions of homology, and serve as the template for your primer. | ||
Let's use the arsR gene as an example. We know the sequence of this gene from either a literature search or from the registry of standard biological parts description. | ====Finding a Target Sequence==== | ||
Let's use the arsR gene as an example. We may know the sequence of this gene from either a literature search or from researching the registry of standard biological parts description. '''Note that the sequence listed for a std. biological part may be different than the original sequence''.(This may be the result of point-mutations added to eliminate a restriction site or could be the product of codon optimization). In the case of the short arsR gene (518 bp), there are no point mutations so we can use the sequence directly as we search. | |||
We can see what organisms might have this sequence or a similar one. | We can see what organisms might have this sequence or a similar one. | ||
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You can search by gene name or simply a sequence fragment to find what organisms might contain your gene of interest. | |||
'''Use the Comprehensive Microbial Resource to see if the arsR gene is present in a wide range of organism.''' | |||
There is a "great deal to explore on this site, but you can navigate from the home page, by selecting the "Searches" tab. Select find genes. In the search field type arsR and select "all genomes" | |||
You could design a primer for a wide range of microbes, but you want to make sure the primer you design will work with one of the organism you have readily available. We have been using a strand of E.coli named K12-MG1655. | |||
'''Use either the comprehensive microbial resource or a BLAST search to determine if E.coli MG1655 has the arsR gene.''' | |||
====Questions==== | |||
Revision as of 22:41, 12 April 2010
Introduction
You should define and understand the following vocabularly words.
Open Reading Frame (ORF) Primer Homology Dimer Aneal
Designing Primers
You are probably familiar with the use of PCR reactions to selectively amplify a region of DNA, but you may not have considered what goes into designing a primer set. If you are amplifying a particular region of DNA, you may need to know the regions upstream and downstream of an open reading frame in order to design regions where your primer can "touch down." These regions are called regions of homology, and serve as the template for your primer.
Finding a Target Sequence
Let's use the arsR gene as an example. We may know the sequence of this gene from either a literature search or from researching the registry of standard biological parts description. 'Note that the sequence listed for a std. biological part may be different than the original sequence.(This may be the result of point-mutations added to eliminate a restriction site or could be the product of codon optimization). In the case of the short arsR gene (518 bp), there are no point mutations so we can use the sequence directly as we search.
We can see what organisms might have this sequence or a similar one.
Two tools you may want to use are the
You can search by gene name or simply a sequence fragment to find what organisms might contain your gene of interest.
Use the Comprehensive Microbial Resource to see if the arsR gene is present in a wide range of organism.
There is a "great deal to explore on this site, but you can navigate from the home page, by selecting the "Searches" tab. Select find genes. In the search field type arsR and select "all genomes"
You could design a primer for a wide range of microbes, but you want to make sure the primer you design will work with one of the organism you have readily available. We have been using a strand of E.coli named K12-MG1655.
Use either the comprehensive microbial resource or a BLAST search to determine if E.coli MG1655 has the arsR gene.
