How do I enter a primer sequence that is degenerate?
MiCA amplification algorithms recognize degenerate bases defined by
IUPAC/IUB. For example, the degenerate base W in the primer sequence
GWATTACCGCGGCKGCTG (Eub-536r) will be resolved to A and T before the
On the output pages, what do the forward and reverse numbers
Consider this example,
The number, 22, refers to the size of the fragment, from the first
nucleotide of the forward primer to the last nucleotide of the first
restriction site digested by the enzyme. The number, 310, refers the the
size of the fragment, from the reverse primer to the last restriction site
digested by the enzyme. Think of PCR experiments you will do in the tube.
If the capillary instrument had the perfect resolution, these are the
sizes the machine will report.
Is there a way to bypass the need for the reverse fragment data?
I have tRFLP data that was generated with only the forward primer being
If you only have forward fragments, use PAT+ instead. However, the reverse
primer is still required to perform in silico amplifications and
I created a database with several restriction digestions but
several organisms have the same fragment sizes. How do I resolve these
If the organisms that produce the same fragments belong to the same genus,
you should probably try different restriction enzymes. Use the enzyme
resolving power analysis tool to see which enzyme gives the most number of
unique fragments. On the other hand, if these organisms do not belong to
the same genus, you should probably try different primers. The primers you
are not sufficiently specific.
Do I have to use both labeled forward and reverse primers for
For tRFLP to resolve species adequately, it is recommended to use both
labeled forward and reverse primers. Restriction fragments generated from
a single primer typically is not sufficiently accurate for species
identification. For a really complex community such as soil, multiple
restriction enzymes are much needed.
I did not get any results from the ERPA (enzyme resolving power
analysis). What seems to be the problem?
If you could not get any results, perhaps it is the primers and number of
mismatches (search sensitivity) you chose. Try different parameters and
see if you get the results.
Is there any way I can upload my database to MiCA?
Currently, there is no way for users to upload their own databases to
MiCA. If you have a customized database you would like to use on MiCA, you
can send it to me (shyu at starlab.phys.uidaho.edu). Make sure you zip
up the file first before send it to me.
I would like to know where some of the primers listed on MiCA are
from, such as Eub-536r, ...
The primer sequences are largely collected from the literatures in AEM
(Applied and Environmental Microbiology). I do not have the complete
It is possible to use only one labeled primer for tRFLP analysis?
APLAUS+ requires both forward and reverse fragments. If you only used one
labeled primer, presumably forward primer, please use PAT+ intead.
Can MiCA compare multiple tRFLP profiles from a single sample
using different enzymes?
No, it is not possible. Multiple profiles must be handled manually.
Uploading multiple profiles to MiCA for analysis introduces huge technical
complexity. The association among different fragments becomes an
impossible for MiCA to complete in a reasonable amount of time. MiCA used
to be able to handle multiple profiles. However, most people found it
difficult to work with on the web interface.
Would it be possible for you to allow linking of your tools to
other sequence databases than those specified?
Currently, it is not possible yet. I guess the answer depends on how
exactly the links are established.
Is it possible to get a list with the accession number of all
matching strains in the PSPA?
Currently, it is not possible yet. The list of accession numbers of all
matching strains can be quite large. It might be too cumbersome to show on
the web site.
My restriction digests did not produce any results. What was the
If you can not get any results using a particular pair of primer
sequences, perhaps you should increase the number of mismatches (increase
Is it possible to perform restriction digestions on a limited
number of sequences from the RDP database?
No, currently it is not possible.
How often does the database get updated?
Well, it depends. I am quite overloaded with work. I will try to keep the
databases updated as frequent as possible. If you notice that the database
should be updated, please send me an email (shyu at