Assay Design: General
1. Identify region of interest and it’s position within the genome
2. Find sequence:
a.
b.
c.
d.
e.
Go to http://www.genome.ucsc.edu and click on Genome Browser.
Select Human GRCh37
Paste the position into the "Search Term" box with a "chr" in first
Hit search to go to the location, and verify that to nucleotide at that position is correct
Click on View in the blue bar at the top of the window and select “DNA” from the drop
down menu.
f. In the "Get DNA" page that comes up, add 100 bases upstream and 100 bases
downstream to the position in the “Sequence Retrieval Options” section
g. Change the “Sequence Formatting Options” to “Mask Repeats to lower case”.
This gives you the sequence you will use in step 3 below
3.
Select Primers and Probes via Primer3Plus
a. Go to http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi
b. Load settings file: On the "General Settings" tab of Primer3Plus, under the "Load and
Save" section, select “Choose File”
c. Upload settings file (see attached) and select "Activate Settings”
d. Paste your sequence from Step 2 into the Main tab.
e. Change your mutation to lowercase so you can see where it is after primer selection
f. Click "Pick Primers"
4. Adjust design parameters if needed
a. If you get a 5' G on the probe, shift probe or primers to remove
b. If you are having trouble, considering design primers and probe against the reverse
complement (select “reverse complement sequence” in Step 2f).
5. Confirm specificity and amplicon in PrimerBlast and In-Silico PCR
a. Go to http://www.ncbi.nlm.nih.gov/tools/primer-blast/ and enter primer sequences
b. This allows you to check for mispriming
c. Go to In-Silico PCR to check amplicon specificity and Tm http://genome.ucsc.edu/cgibin/hgPcr
Tips about Tm:
For standard probes: target probes to ~3 deg above the primer Tm
Examples: Primer Tm = 60, probe Tm = 63; Primer Tm = 55, probe Tm = 58
For MGB probes, aim for a Tm that’s 5-7 degrees lower
For Primer3Plus:
Save the following parameters as a text file (.txt) and upload the file for later use. The feature is
available from the "General Settings" tab of Primer3Plus, in the "Load and Save" part of the
screen (at the bottom of the screen).
> PRIMER_MISPRIMING_LIBRARY=HUMAN
> PRIMER_PRODUCT_SIZE_RANGE=40-100 100-150 150-200
> PRIMER_DIVALENT_CONC=3.8
> PRIMER_DNTP_CONC=0.8
> PRIMER_SALT_CORRECTIONS=1
> PRIMER_TM_SANTALUCIA=1
> PRIMER_INTERNAL_OLIGO_OPT_TM=58
> PRIMER_INTERNAL_OLIGO_MAX_TM=70
> PRIMER_INTERNAL_OLIGO_DIVALENT_CONC=3.8
> PRIMER_INTERNAL_OLIGO_DNTP_CONC=0.8
> PRIMER_INTERNAL_OLIGO_MISHYB_LIBRARY=HUMAN
> PRIMER_IO_WT_SIZE_GT=3
> SCRIPT_DETECTION_PICK_HYB_PROBE=1