Identifying Polymorphisms in the Steroid Biosynthesis Pathway of
Soybean
The Steroid Biosynthesis pathway currently has 40 genes matching soybean EST
sequences. This pathway is located at:
http://www.genome.jp/dbget-bin/www_bget?path:egma00100
YOUR ASSIGNED GENE IS EC#
_________
(example given is for EC 1.17.1.2)
STEP 1
Click on the hyperlink above to go
to this pathway. Find your assigned
gene in the pathway (look for green
highlighted boxes) and click on it to
view the sequences identified in
soybean.
This representation of the steroid
biosynthesis pathway is from the
KEGG database. Green highlighted
sequences are from soybean, while
un-highlighted genes are found in
organisms other than soybean. All
of the enzymes listed in this
pathway are sequenced from
transcribed genomic DNA.
STEP 2
Look for “Other DBs” and select the
“alignment” link (shown in red at the
right).
Count the number of records ______
(17)
Transcribed genes are usually
sequenced randomly, thus the number
of genes is an excellent indicator of
the level of transcription of a particular
gene – the more records the higher the
transcription rate. If you have only one
sequence the alignment option will not
be available, go to STEP 5.
STEP 3
At the top of this page is a link to the
“Contig Graphical Alignment View”.
Follow this link to the next page
STEP 4
Select the left-most (top) sequence
in the Contig Graphical Alignment
View and follow the link to NCBI.
Example Sequence ID: 58020548
Your Sequence ID:_______________
This alignment shows all of the
sequences from soybean identified for
this gene. By selecting the left-most
sequence you get the mRNA with the
most 5’ transcribed sequence.
STEP 5
Find the sequence length
(circled in red) and subtract 100
from it below. Enter this value after
the “50” below..
Length of sequence 933 -100 = 833
50, 833
Enter for YOUR EC #: ___________
Length of sequence ___ -100 = ____
50, ______
You are selecting the region that will be amplified during PCR. The "50" entered above is
the first 50 base pairs of the gene. You will use this information to design a forward
primer located in the first 50 bp and a reverse primer located in the last 100 bp of your
sequence. This gives you the greatest chance of detecting a size polymorphism using
PCR
STEP 6
Change the display type to FASTA
(the display menu is circled in red the page will automatically
change). Copy the sequence in
FASTA form by selecting the text
on the page as shown.
This is the nucleotide sequence of
your gene. The (un-transcribed and
un-modified) DNA located on a
chromosome in soybean will be the
target of your PCR reaction,
STEP 7
Go to the “Primer 3” program and paste
your sequence in the main window as
shown:
http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi
Exclude the middle of the sequence by
typing 50,833 (in this case the sequence
is 933 bp long, so 50, 833 is appropriate).
Use the value you computed for your
sequence in step 5.
Select “PICK PRIMERS”
STEP 8
If the program was able to
design primers, simply cut and
paste them into the report page
at the end of this document.
If the program could not
design primers, select a
smaller target (and thus a larger region for primer design) by increasing the left side
exclude value and decreasing the right side exclude value in step 7 by 25 bp increments.
Pathway:
Steroid Biosynthesis
Enzyme EC number:
Students Name:
Date:
___________
___________
___________
# of records _____
step 2
Product Size: _____
step 7
Sequence source: ________________
step 4
Paste Primer 3 program data here:
Sequences to order: (GmaxEc# _ your initials _ L (L for left, R for right)
Example:
GmaxEC1.17.1.2_JS_L
GmaxEC1.17.1.2_JS_R
GATACTGCAACTGGCAACATTT
CATGGGTGCAAAGGTATGAA
sequence