Assay Design Training Draft-JH
1. Copy and paste the sequence into the Assay Design Software (ADS).
2. From the Assay Type drop-down menu, select the “allele quantification”, or “SNP”.
3. Select a Target Region
Highlight a 1-100bp section of sequence containing the sites of interest. Right-click and
choose “Target Region”, then “Set Target Region”
a. It is not recommended to place primers in regions with a lot of repetitive
sequences.
b. Especially for AQ assays, try to avoid homopolymers in the target. You can try to
overcome a homopolymer by placing the sequencing primer over a few of the
bases in the stretch
4. Press “Play”
The Assay Design software will then identify the best primer sets based on your target
region selection and the Default settings that you currently have set.
5. Examine the Reports:
Double-click on the top 5 or 10 assays that appear in the primer set area, and then read
through the primer set reports. The most important penalties in this report include the
following:
a. Biotinylated PCR Primer Hairpin Analysis:
b. Duplex Formation Analysis:
c. Mispriming Analysis:
d. Template Loop Analysis:
e. Duplex Formation Analysis (Biotinylated PCR primer):
f. Mispriming Analysis (Biotinylated PCR primer): *note the location of the
mispriming event. Mispriming outside of the amplicon can be ignored.
g. Hairpin Loop Analysis (Biotinylated PCR primer):
h. Duplex Formation Analysis (Sequencing primer):
i. Hairpin Loop Analysis (Sequencing primer):
Penalties of over 50 are considered to be significant in any of these categories.
It is important to minimize these penalties by editing primer sets or redesigning
the primers altogether. If these penalties can be reduced into the 30s or even
the 20s, the design should be relatively clean.
6. Edit Primers if necessary: Please refer to section 4.5.2 in the Assay Design Software
User’s manual for an explanation of how to manually edit primers. If your design efforts
fail for a particular target region, then try another target region and repeat your primer
design efforts on the new region. If you cannot design a clean assay by editing the
primers or moving the target region, see “Tips and Tricks” below for more information on
optimizing the design.
7. Optimize your PCR
a. Biotinylated primers should be ordered with HPLC purification and a standard 5’biotin label. Non-biotin labeled primers can be standard-desalted.
b. In order to improve your success rate while keeping your assay development
costs down, it is recommended that you order the most favorable primer set,
along with a few back up sequencing primers and non-biotinylated primers.
c. Different sequencing primers and non-biotinylated primers are preffered in order
to produce a higher level of specificity. If a particular sequencing primer is giving
d.
e.
f.
g.
h.
problems, and you do not have an alternative sequencing primer, you can try the
non-biotinylated PCR primer as the sequencing primer.
You should run a pcr gradient with at least 10 to 15 different annealing
temperatures. For each temperature, it is best to run as many as three different
MgCl2 concentrations: 1.5 mM, 3.0 mM, and 4.5 mM. More information on PCR
optimization can be found in Biotage’s PCR Optimization document.
Run a gel and pick out the cycling conditions that produce strong, specific
products.
Run these products through the pyrosequencing reaction on some cell lines as
well as some samples. Pick the set of cycling conditions that give you the best
pyrograms with the most accurate measurements.
It is recommended that assay designs and cycling conditions be tested on
samples with known genotype/percentages to test for bias. There was an
excellent publication that discussed the importance of this bias testing in CpG
methylation testing.
Optimizing annealing temperature overcomes bias in bisulfite PCR
methylation analysis Lanlan Shen, Yi Guo, Xinli Chen, Saira Ahmed,
and Jean-Pierre J. Issa. BioTechniques. January 2007. Volume 42,
Number 1: pp 48When optimizing PCRs you should run the following controls to test for
background. See the document :
i. Template only control
ii. –ve PCR control (i.e. water. This control should be run on each plate to
test for nucleotide degradation)
iii. Sequencing primer
iv. Biotinylated Primer
v. Sequencing + Biotinylated Primers
Tips and Tricks
1. For quantitative apps, if possible, position your sequencing primer to avoid dealing with
“A” dispensations in the mutation site dispensations. For example, reverse an assay G/A
to C/T. In pyrosequencing, we use chemically modified Adenosine, resulting in "A peaks"
that a slightly larger than the rest of the nucleotide peaks. The bottom line is that
“reverse” methylation assays are less quantitative than forward methylation assays due
to these "A peak" artifacts. Our Pyromark and CpG software packages both have an "A
peak" correction factor, but it is always better to avoid “A” dispensations in variable
positions whenever possible. If the ADS scores are equal when comparing two similar
assay design candidates, the assay without an A is almost always a better choice. If the
ADS scores and penalties are favorable for the A assays and unfavorable for the other
assays, ignore the direction of the design and go with the design that will give you
cleanest results.
2. There are ways of correcting the penalties aside from your typical primer editing
strategies.
a. Biotinylated primer penalties are the easiest of these penalties to correct. By
using less than 0.2 micro molar of the pcr primers in the pcr setup and increasing
the cycle numbers up to 45 or 50, these biotin problems are easily corrected in
most cases.
b. Template loops structures can sometimes be destabilized by adding or removing
one or more bases to the 5’ end of the non-biotinylated pcr primer. In some
extreme cases, one might consider adding one or more “G” bases at the 5’ end
of the non-biotinylated pcr primer on forward designs, and one or more “C” bases
on 5’ non-biotinylated pcr primer for reverse assays. These “G” or “C” bases
would only be capable of annealing to methylated sequences, so the addition of
c.
these bases should, in many cases, prevent the template loop from having a
stable 3’ end. The affect of adding these extra bases should be carefully
considered using the ADS before ordering the primers with these 5’ end
sequence modifications.
Biotinylated PCR primer mispriming events on the amplicon can sometimes be
destabilized by creating a mismatch near the 5’ end of the primer. A mismatch of
this type will usually allow the primer to continue to anneal to the target, but
destabilize a non-specific priming site enough to reduce the penalty.
3. Location: The location that different non-specific annealing events occur on the amplicon
should not be ignored. The locations of these non-specific annealing events can be
found in the Primer Report, the Sequence Tab, and the Assay Overview Area.
a. All non-specific events that don’t occur on the pcr amplicon can be safely
ignored, along with any non-specific events that involve the non-biotinylated pcr
primer.
b. Non-specific annealing events that occur just downstream of your sequencing
primers can often block the “specific” polymerase activity that you are trying to
measure. Mispriming events and loops that occur further downstream of the
sequencing primer are usually more favorable.
c. The further that these mispriming events occur from your sequencing primer
annealing site, the more information you’ll get before the sequence pattern starts
to deviate from the expected sequence pattern. Even if these downstream
mispriming events block the specific polymerase activity, you might still be able
to get valuable information from the first portion of the sequence run if the
mispriming occurs far enough away from the sequencing primer.
4. Tm values should always be kept below 73 degrees. A Tm of 73 degrees would likely
require a Ta of approximately 68 degrees for the amplification. Qiagen normally
recommends a maximum Ta of 68 in their amplification protocols. Reducing the length of
a pcr primer at the 5’ end of the pcr primers will reduce the Tm of the primer, but it may
also affect the template loop penalties as well (non-biotinylated PCR primer only).
Caution should be exercised when manually editing primers to reduce high Tm values.
Please refer to section 4.5.2 in the Assay Design Software User’s manual for an
explanation of how to manually edit primers.
Controls to run for a new Pyrosequencing assay
1.
2.
3.
4.
5.
Template only control
–ve PCR control (i.e. water)
Sequencing primer
Biotinylated Primer
Sequencing + Biotinylated Primers
1. Should be a normal PCR product but in the Pyrosequencing plate just put annealing
buffer, no Sequencing Primer.
2. Should be a water PCR control which should be processed as a normal sample.
3. Should have nothing in the PCR plate and Sequencing primer in annealing buffer in the
Pyrosequencing plate
 For MA/ID 1.5uL of 10uM Sequencing primer + 38.5uL Annealing buffer.
 For HS/MD 0.5uL of 10uM Sequencing primer + 11.5uL Annealing buffer.
 For Q24 0.75uL of 10uM Sequencing primer + 24.25uL Annealing buffer.
4. Should have nothing in the PCR plate and Biotinylated PCR primer in annealing buffer in
the Pyrosequencing plate



For MA/ID 1.5uL of 10uM Biotinylated primer + 38.5uL Annealing buffer.
For HS/MD 0.5uL of 10uM Biotinylated primer + 11.5uL Annealing buffer.
For Q24 0.75uL of 10uM Biotinylated primer + 24.25uL Annealing buffer.
5. Should have nothing in the PCR plate and Sequencing primer + Biotinylated PCR primer
in annealing buffer in the Pyrosequencing plate.
 For MA/ID 1.5uL of 10uM Sequencing primer + 1.5uL of 10uM Biotinylated
primer + 37uL Annealing buffer.
 For HS/MD 0.5uL of 10uM Sequencing primer + 0.5uL of 10uM Biotinylated
primer 11uL Annealing buffer.
 For Q24 0.75uL of 10uM Sequencing primer + 0.75uL of 10uM Biotinylated
primer + 23.5uL Annealing buffer)
All of these controls should be heated to 80°C and run on the instrument as ‘normal’ samples.