Design
of
a
pair of primers
for
amplification of the coding region of mRNA
of
the SRY gene (sex determining region on the Y)
"Primer-BLAST" ("Primer3"-based) web software
Copy the complete mRNA sequence of
interest (database Gene; search for the entry of
human SRY gene; Go to "reference sequence details").
Paste it into the Primer-BLAST program and ask
the program to suggest primer pairs,
suitable for amplifying the region of interest within the mRNA.
If necessary, delimit the sequence region around
which the primers are to be placed,
by entering the start and end coordinates of the area (From...
To...) in which the program will search for a good forward primer
sequence
(repeat for the reverse primer sequence).
Click on the "Get Primers" button to obtain
the proposed primer pairs.
Modify the parameters, trying to obtain primer pairs by trial and
error , also by entering the best and most stringent parameters.
Copy and paste the sequences of the best primer pairs obtained
separately (on Mac, copy=cmd + c key, paste=cmd +v key).
Use the "Text Edit" program from the icon collection at the
bottom.
Some of the main parameters that can be
modified as desired:
"Primer size" (we
suggest 22-26 bases as the ideal length for each primer).
"Products Size Ranges":
you can modify the preferred length ranges of the amplification
product.
"Primer GC content": ideally, 50%
(45-55%).
"GC Clamp":
number of bases at the 3´ end that must be G or C.
"Max Self Complementarity":
number of self-complementary bases, ideally no more than 5 (and 0
at the 3´ end).
"Max Pair Complementarity":
number of complementary bases between the two primers, ideally
no more than 5 (and 0 at the 3´ end).
Etc.
Verify the specificity of the proposed primers by comparing
the sequence of each primer with all known sequences
(BLASTN program; restrict the
search to
Organism:
Homo sapiens).
In addition to the list of sequences complementary to the primer
reported, also observe the alignments between primers and
sequences found,
by clicking on the "Alignments" tab on the BLAST results web page.