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:
YOUR ASSIGNED GENE IS EC# ______
(example given is for EC 1.17.1.2)
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.
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.
At the top of this page is a link to the
“Contig Graphical Alignment View”. Follow this link to the next page
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.
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
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,
Go to the “Primer 3” program and paste your sequence in the main window as shown:
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”
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: _____ Sequence source: ______
step 7 step 4
Paste Primer 3 program data here:
Sequences to order: (GmaxEc# _ your initials _ L (L for left, R for right) sequence