How to sequence orthologs of my favorite gene

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p_rogers
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How to sequence orthologs of my favorite gene

I would like to obtain the sequence of as many orthologs of my favorite human gene as possible. I need help designing PCR primers for these orthologs. How can I design the best primer to ensure I get a product?

P.S. I have DNA from a few dolphin species, elephant, cow, pig, anteater and opossum.

ryan_m
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p_rogers wrote:I would like

p_rogers wrote:

I would like to obtain the sequence of as many orthologs of my favorite human gene as possible. I need help designing PCR primers for these orthologs. How can I design the best primer to ensure I get a product?

P.S. I have DNA from a few dolphin species, elephant, cow, pig, anteater and opossum.

Sounds like an interesting project. I will assume yfg (your favourite gene) has more than one exon. You will likely aim to only amplify the exons (for simplicity) unless it is the non-coding sequence that interests you? So what you want to do to start is find where all your species fit in the tree of life. You will also want to find out which species in that tree have 1) complete and 2) partial genomic sequence available. You can then take the 'nearest cousins' of each species (or check for genomic sequence from the same species in the case of cow, opossum and potentially pig).
Once you have these, you should find the genomic regions that flank each exon of your gene (5'UTR, Introns and 3'UTR). These will be where your PCR primers will go. You will have one pair of primers for each exon(or two, if using nested PCR). Desigining the primers will be tricky. There is no "best" way to do it. I would suggest that you align the genomic sequence of all the neigbouring genomes to identify regions of highest conservation. You may have to sacrifice primer Tm to get a primer that sits in a region of high conservation. You should also aim to put the 3' end of the primer in the most conserved region if possible since this is where annealing is most important. I hope this information helps you. If you want a more complicated anser involving the prediction of your genomic sequence using evolutionary models let me know.

Good luck.

Ryan

ryan_m
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I found a document online

I found a document online regarding the status of low-coverage mammalian genome sequencing. It turns out that the following genomes should be done the 2X sequencing stage by now:

elephant, armadillo, rabbit, tenrec, guinea pig, cat, shrew and hedgehog.

In addition, the following animals appear to be in progress (who knows how many reads there are at present):
Squirrel, tree shrew, bat.

If you wait another year, you can expect to see many more mammals added. The following animals are in the proposal for phase III of the low coverage genome project:

Elephant shrew (that's three shrews now!), flying lemur, dolphin, horse, llama, mole, mouse lemur, pika, kangaroo rat and tarsier.

Check out the phylogenetic tree to see where all these creatures fit in.