Nucleotide Database

[elynn]

I am trying to design a good primer for isolating my target genes..unfortunately the sequence references are very limited..most of the database lead me to arabidopsis..n i dont want to only use arabidopsis..i wanna isolate apetala genes but due to the limited sequences, i cant design a good primer..
What is other database i can use apart from NCBI and TAIR?

[JackBean]

which gene from what organism? Do you know the gene sequence? If not, you will have to try either degenerated primers or screening genomic/cDNA library or something like that.

[elynn]

APETALA gene from hibiscus sabdariffa. and i dont know the gene sequence..thats why i need to design a pair of primer to use it for isolating full length cDNA

[Cat]

Aren't you putting a cart before a horse? Do you have an apetala mutant of hibiscus to work with?

You might get lucky if the hibiscus apetala gene is similar in sequence to that of arabidopsis, so you can design a bunch of primers to that and hope to get something. However, unless you have a mutant that you can use to prove your gene IS apetala, your work would be insufficient as everything would be "putative".

[JackBean]

PubMed Contains only about 500 nucleotide sequences related to Hibiscus
http://www.ncbi.nlm.nih.gov/nuccore?term=%22Hibiscus%22[Organism]&cmd=DetailsSearch
You may try to look for higher taxa to see, whether you will be able to find some apetala gene sequence.

However, what you will probably end up with will be DNA library and degenerated probe, which will lead you to putative apetala gene from Hibiscus. After sequencing and further characterization you should be able to say how related it is to known apetala genes and whether could it be really apetala gene.
(mainly homology and spatial expression should be enough to say)

[Cat]

After sequencing and further characterization you should be able to say how related it is to known apetala genes and whether could it be really apetala gene.
(mainly homology and spatial expression should be enough to say)

Not in plants. Here is an alignment of a couple of genes to help visualize the problem:

BWB16106 VRN1 Wheat
BWB16185 VRN1 Arabidopsis


BWB16185 MPRPFFHKLIFSSTIQEKRLRVPDKFVSKFKDELSVAVALTVPDGHVWRVGLRKADNKIW
BWB16106 MGRGKVQLKRIENKIN-RQVTFSKRRSGLLKKAHEISVLCDAEVG---------------
* * .: :...*: ::: ...: . :*. .::* . *

BWB16185 FQDGWQEFVDRYSIRIGYLLIFRYEGNSAFSVYIFNLSHSEINYHSTGLMDSAHNHFKRA
BWB16106 ------------------LIIFSTKG----KLYEFSTES-----CMDKILERYERYSYAE
*:** :* .:* *. . ::: ..:

BWB16185 RLFEDLEDEDAEVIFPSSVYPSPLPESTVPANKGYASSAIQTLFTGPVKAEEPTPTPKIP
BWB16106 KVLVSSESEIQGNWCHEYRKLKAKVETIQKCQKHLMGEDLESLNLKELQQLEQQLESSLK
::: . *.* . .. *: .:* .. :::* :: * ..:

BWB16185 KKRGRKKKNADPEEINSSAPRDDDPENRSKFYESASARKRTVTAEERERAINAAKTFEPT
BWB16106 HIRSRKNQ-LMHESISELQKKERSLQEENKVLQKELVEKQKAHAAQQDQTQPQTSSSSSS
: *.**:: *.*.. :: . ::..*. :. ..*:.. * ::::: :.: ..:

BWB16185 NPFFRVVLRPSYLYRGCIMYLPSGFAEKYLSGISGFIKVQLAEKQWPVRCLYKAGRAKFS
BWB16106 -----FMLRDAPPAANTSIHPAAAGERAEDAAVQPQAPPRTGLPPWMVSHING-------
.:** : . :: .:. . :.:. : . * * :

BWB16185 QGWYEFTLENNLGEGDVCVFELLRTRDFVLKVTAFRVNEYV
BWB16106 -----------------------------------------

As you can see, there is very little sequence similarity...

[JackBean]

So? I'm sure you could find such examples also in animals.

[Cat]

So? I'm sure you could find such examples also in animals.

Probably. My point is APETALA is only known in Arabidopsis and thus it's probably totally different from Hibiscus.

[elynn]

APETALA is not only known in Arabidopsis.. it is a flowering gene..so it should be presented in angiosperm.. and i hv few sequence of apetala from other species..unfortunately its not enough to design a good primer

[JackBean]

elynn: in such case it would be probably best to prepare slightly degenerated probes for whole length and do some screening of libraries.