Assay Design: SNP
1. Identify mutation of interest and it’s position within the genome
2. Find sequence:
a.
b.
c.
d.
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 (ie: chr17:7577121)
Hit search to go to the location, and verify that the nucleotide at that position is
correct
e. 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. Constrain probe to land over the SNP: On Internal Oligo tab, set Hyb Oligo Excluded
Region
i. Target the probe to be between 15 bases downstream and 15 bases
upstream of the SNP position
ii. Example: Entering “1,85 116,85” would exclude 85 bases starting from
position 1 and 85 bases starting from position 116. 1-85 and 116-201 will be
excluded
e. Paste your sequence from Step 2 into the Main tab.
f. Change your mutation to lowercase so you can see where it is after primer selection
g. Click "Pick Primers"
h. Confirm that the probe falls on top of the mutation of interest and that the mutation is
roughly centered within the probe. Specia case: for MGB probes it's best to have the
mutation in the 3' half of the probe, but not in the last couple bases.
i. To get the mutant design, change the target base to the mutant and repeat this
process. This should yield the same primers. One exception is where a primer and
probe are directly adjacent. If one probe design is longer, this will cause a
discrepancy in primer design.
4. Adjust design parameters if needed
a. If SNP is not approximately in the center of the probe (+/- 4 bases) can change the
"Hyb Oligo Excluded Region" to force SNP to the center
b. If you get a 5' G on the probe, shift probe or primers to remove
c. 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
6. Confirm strand choice
a. Go to http://biophysics.idtdna.com/
b. Enter probe sequence and select box marked “mismatch, dangling ends”
c. Enter the reverse complement of the mutation at the SNP site as the mismatch
i. Example for an A  G mutation (A is WT, G is MT) for the WT probe, C
would be introduced as the mismatch (reverse complement of G). It would
replace a ‘T’ (reverse complement of A)
ii. Calculate the delta Tm (Tm of the perfect match vs. Tm of the mismatch)
d. Repeat the process with the reverse complement of the probes
e. Choose the strand with the largest delta Tm and redesign from step 3 with that strand
(see 4c)
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