FREQUENTLY ASKED QUESTIONS :
I am interested in the promoter of gene X. What do I do now?
Regardless of whether you have a
great deal of information about the promoter of gene X or not, your
first stop should be SwitchDB.
You will need to create a free user account which will allow you to
save your searches. You can begin a query using gene Ids, symbols,
aliases or accession numbers as well as other types of identifying
information.
What is SwitchDB?
SwitchDB
is a freely available, open source database for studying human gene
regulatory sequences. It is a very powerful tool that permits users to
identify transcription start site (TSSs) corresponding to a single gene
or a large collection of genes. For detailed information on how to use
SwitchDB, click on the help link at SwitchDB.
I performed a query using my gene identifier, what do I do now?
If your input information was
correctly recognized, a green window should appear indicating the
number of gene models and promoters found. If you submitted a long
list, you may choose to filter the results using additional criteria.
(For details on how to filter, go to the help link.)
If you choose to browse the gene models, you can view details about
each gene model identified for your initial query including coordinates
of the gene model, annotation information as well as the coordinates of
the transcription start site(s) and their corresponding scores. You may
also extract the data in tabular format or save the query for later.
Why does my gene have more than one transcription start site?
It has been estimated that
greater than 25% of all human genes have more than one transcription
start site. A given transcript may initiate from different start sites
in different cell types or under different cellular conditions or
stimuli. Additionally, several transcription start sites may be
utilized in a single cell type. SwitchDB catalogs all potential
alternative transcription start sites for genes and ranks them based on
experimental evidence.
Which transcription start site should I choose?
Choosing the correct
transcription start site is important to obtaining relevant results
from a promoter assay. For best results that reproduce previous
findings, we strongly recommend identifying the coordinates of the
tested promoter fragments. The genome browser at UCSC
is an invaluable tool in this process. If you have sequence information
from a previous promoter construct, you can use the BLAT tool at the
top of the home page to identify the coordinates of that sequence in
the human genome. Make sure you select the May 2004 assembly, as all
SwitchDB coordinates are from that freeze. Click on the browser link to
view the search results and coordinates in the genome browser. If
you only have primer information, you can use the PCR link from the
genome browser web page to obtain the coordinates and amplified
sequence from a pair of primers. Click on the coordinates to view the
amplified fragment in the genome browser.
Once you have identified the
coordinates of a previously-tested fragment, you will want to see which
transcription start site is contained within that fragment.
SwitchGear's genome-wide transcription start site predictions can be
viewed on the genome browser using the SwitchGear TSS annotation track.
If you scroll down on the genome browser page, you will see a number of
pull down boxes grouped into different categories. You will want to
scroll down to the Expression and Regulation category and select
“full” from the SwitchGear TSS pull down box. You will then
scroll back up the page and click on the refresh button. The genome
browser will now contain the SwitchGear Genomics Transcription Start
Sites track. If any transcription start sites are present within the
region you are interested in, they will show up as a vertical tick
mark. You can click on any tick marks to view more detailed
information.
What if there is no information for the promoter I am interested in?
For many genes, it may not be
possible to identify a region that has demonstrated promoter activity.
In some cases, you may still be able to choose between different start
sites using transcript information. Many transcripts are alternatively
initiated or alternatively spliced. In those cases, the start site most
proximal to the 5' end of the transcript you are interested in should
be selected.
In the absence of any additional
information about your gene, the start site with the most experimental
evidence would be the most reasonable to begin testing. In SwitchDB,
start sites are ranked according to their experimental support
(indicated as their “Score”), with the highest scoring
start site designated with the suffix R1.
I've chosen a transcription start site, but what about the promoter?
To capture most proximal
regulatory elements, both positive and negative, we have targeted a 1
kb size for the majority of our promoter constructs, encompassing the
region approximately 900 bp upstream and 100 bp downstream of a
particular transcription start site. If you would like to know
the exact size or sequence for a particular promoter, please contact us.
Where can I find more information on the vector and reporter gene?
SwitchGear uses a destabilized form of luciferase called luc2P
developed by Promega for it's reporter gene. This optimized luciferase
protein has a half-life of ~1 hour enabling detailed analysis of
kinetic responses yet still provides a very robust absolute signal.
Vector maps for all of SwitchGear's reporter vectors can be found
at the Vector Map link on our Technical Resources page.
How do I place an order?
On the Ordering page, simply paste the transcription start site IDs for the promoters you are interested in and we will contact you.