Design considerations for qPCR assays

Design considerations for qPCR assays


Hello, and welcome to this
integrated DNA technologies webinar. Design considerations
for qPCR assays. My name is Sean McCall, and
I’ll be serving as moderator for today’s presentation. Before we begin, we wanted to
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your bandwidth. A copy of today’s slide deck
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the resource list. We encourage you to download
any resources or links that you may find useful. Today’s presentation will be
given by Dr. Erik Wendlandt. Erik is a senior scientific
application specialist at IDT. Erik obtained his PhD in
molecular and cellular biology from the University of Iowa,
where he studied disregulation of gene expression
in human macrophages upon parasitic infection. As a postdoctoral fellow
at the University of Iowa, he investigated how changes
in chromosomal structure resulted in drug resistance
and disease progression in multiple myeloma. At IDT, Erik specializes
in applications of functional genomics
and gene expression. And on a daily basis,
helps scientists design and troubleshoot
their qPCR experiments. The question and
answer session will be conducted by Brendan
Owen’s, assistant manager of scientific application
support at IDT. Erik’s presentation should
last about 30 minutes. And following the
presentation, he will answer as many questions
as possible from attendees. In case you need to
leave early or want to revisit this webinar, we
are recording the presentation and will make the
link to the recording available on our website a few
days after the presentation. We will also post the recorded
presentation on our YouTube and Vimeo channels. You will receive links to
these in a follow-up email. Also, an on-demand
version of the webcast will be available approximately
one day after the webcast and can be accessed using
the same audience link that was sent to you earlier. So now, let me hand it over
to Erik for his presentation. Thank you for the introduction. As Sean mentioned, today’s talk
is titled design considerations for qPCR assays. The objective of today’s
talk is to give you insight into qPCR design and
instill confidence in you to begin designing
your own qPCR assays. Today’s talk will begin
with basic considerations for quantitative
PCR or qPCR assays. We will first look at the
different qPCR chemistries followed by useful tools for
basic qPCR design and oligos characteristics necessary
for a robust assay design. Next, we will look at
the pre-designed assays available in the IDT
pre-designed library. The talk will conclude with a
look at tools and strategies for designs requiring
more customization. qPCR assay design
covers a lot of topics, including gene expression,
copy number variation, SNP genotyping,
multi-species analysis, splice variant-specific or
variant-common expression, and quantification
of rare targets. For the scope of
today’s talk, we will focus on gene
expression assay design. In general, qPCR experiments
are composed of six steps– assay design,
experimental setup– and this includes
sample treatment– RNA or DNA collection,
and if the sample is RNA, for gene expression
analysis, conversion of that RNA to a cDNA library through a
reverse transcription reaction. Next is running of the
qPCR reaction and finally, analysis of the data. Let’s begin with some basic
qPCR design considerations. Before starting an
assay design, it’s important to consider
the different chemistries available for qPCR analysis. qPCR asses are
broadly categorized into two assay types– primer only assays and
probe-based assays. As the name suggests,
primer only assays will only have two oligos– a forward and a reverse primer– as shown in step 1. Step 2, the reaction proceeds,
and more double-stranded DNA is generated. The increasing double-stranded
DNA is detected in step 3 by interpolating agents,
such as SYBR Green. Like primer only assays,
probe-based assays also rely on amplification from
a forward and reverse primer, shown here by the arrowheads. However, the probe-based
assay also has a third oligo– the probe– as shown in step 1. The probe is labeled with a
fluorophore or dye at one end and a quencher at the other. Prior to amplification,
the probe remains intact and is
undetected by the instrument. In step 2, the polymerase
extends off the primers and encounters the probe. In step 3, the 5-prime to
3-prime exonuclease activity of the polymerase
hydrolyzes the probe, freeing the fluorophore
from the quencher. It is in step 4 that the
increase in fluorescence is detected by the instrument
and related to gene expression. To determine the assay type
best suited for your experiment, let’s take a look at a few pros
and cons of each assay type. Some of the pros of
primer only assays include the low cost of
primers, also, the longer the amplicon, the
stronger the signal, which can be advantageous
for low complexity sequences. Some drawbacks to
primer only assays include the inability
to multiplex assays within a single well,
non-specific detection, and signal generation
due to primer dimers. Because of the concern for
non-specific amplification and primer dimers, a
post-run melt curve analysis is recommended to assess
for assay specificity. Some strengths to
probe-based assays include increased specificity
as a result of the addition of a third oligo. The probe makes pathogen and
rare detection, SNP genotyping, and multiplexing possible. The increased
specificity alleviates the need for a post-run
melt curve analysis. The major drawback
to probe-based assays is the added costs associated
with the fluorophore modified probe. Now that we have reviewed
the pros and cons of the different
types of qPCR assays, let’s focus on the
steps and oligo characteristics necessary
for a good qPCR assay design. Outlined here are
some considerations for generating a new design. Know your gene. Are there multiple transcripts
for a given target? Depending on your target,
are there common or unique exons between the
different transcripts? NCBI is a great resource for
pulling sequence information, comparing splice variants
of a single target, and identifying
exon-exon junctions. Once sequences are
captured from NCBI, alignments can be
run to identify common or unique regions to
target for primary and probe design. Finally, once
designs are complete, blast the primers and probes
to look for specificity of the targets of interest. Much of this initial research
can be done through NCBI, such as obtaining
sequence information, identifying variant information,
performing BLAST analysis, and looking for SNP information. The NCBI homepage is shown
here for your reference. Just as important as
the characteristics of your target of interest,
oligo characteristics are also important to consider
when designing a robust assay. For primers, I recommend
that the melt temperature of the primers differ by less
than 2 degrees centigrade and that the primer Tms fall
between 60 and 62 degrees. Aiming for a Tm of
60 to 62 degrees, primers will fall between
18 and 30 bases in length and should also have a GC
content between 35% and 65%. Poly sequences
generally are OK, but I recommend avoiding
runs of four or more G bases, as these
sequences can result in the formation of strong
secondary structures. The probe within a reaction will
have similar characteristics in regards to GC content and
avoidance of poly G runs. Probes will often be
longer than primers, and this is the result of the
higher preferred oligo Tm. The pro Tm should be four to six
degrees higher than the primer Tms. This will mean that
the probe Tm will fall between 66 and 68 degrees. Additional
characteristics to aim for include probes less
than 30 bases in length, and no G bases at
the 5-prime end, as this can result in a loss
of fluorescence intensity. Longer probes are
OK, but addition of an internal quencher, like
zen or tau, is recommended. Finally, remember it is OK to
target the sense or antisense strand of the target. Generally, qPCR assays vary in
length between 70 and 150 basis and can vary depending
on the assay used. Longer amplicons tend
to be advantageous for primer only assays
to help discriminate between amplicon conformation
and primer dimers. A little later in the talk,
we will look at an amplication where designing to shorter
amplicons is preferred. Much of the analysis of primer
and probe characteristics can be performed using
the OligoAnalyzer tool through the IDT website. In addition to the
OligoAnalyzer tool, IDT offers a variety of tools
for designing custom assays and selection of pre-designed
assays for human, mouse, and rat targets. These tools can be found within
the tools dropdown on the IDT website. Many researchers will
have a target in mind and are looking for a quick
identification of an assay. To assist with assay
identification, IDT has compiled the library
of pre-designed to primer only and probe-based assays
for human, mouse, and rat transcripts. The focus of this
portion of the talk will show how to access,
customize, and order target-specific and
housekeeping assays. The pre-designed library can
be accessed through the tools drop down menu under the
qPCR assay design header as shown here. The assays are developed
to avoid cross-reactivity with other transcripts. The human assays
are also designed to avoid high
frequency SNPs, as SNPs within the oligo binding sites
may result in decreased assay performance. Here’s a screenshot of the
pre-designed library look-up tool. Not only will the tool
provide pre-designed assays for your target of
interest, the tool will also list a variety
of housekeeping assays. The housekeeping assays
can be found here by selecting your
species of interest. To search for an assay
using the gene name, enter the gene name here. Please, note that the tool only
recognizes official NCBI gene symbols. You can also search for
assays via RefSeq IDs or via an IDT assay ID,
allowing for easy reordering of an assay. Once you have entered
a gene ID or RefSeq ID, select your species
of interest here as well as the assay style
we offer designs for primer only or probe-based assays. Here are the
available probe-based assays when searching
for human beta actin. Recommended assays
for each target are annotated with a check
mark next to the assay ID. We also offer additional
assays to account for specific applications. To avoid confusion
related to gene aliases, I recommend checking to ensure
that the gene ID matches the gene query, and this
information is displayed here The tool also
provides information on transcript variance
each assay will target and whether this assay will
target all annotated variants for the gene of interest. Finally, the tool provides the
exon location of each assay. The probe-based assays default
to FAM-modified probes. But you can customize
the assay with a variety of different fluorophore and
quencher combinations in three different sizes. If a configuration is not
available via the tool, additional combinations
are available as custom configurations. Ideally, an assay will
not amplify genomic DNA. However, due to complexities
within a target, an assay may amplify
a genomic DNA. And such assays are denoted
with a dot G annotation at the end of the
assay ID as shown here. One instance when an assay
will identify genomic DNA is when the assay
targets a single exon, like the assay shown here. However, it is also
possible for an assay to amplify genomic
DNA, even if the assay targets multiple exons. This assay spans multiple exons,
but intron 3 between exons 3 and 4 is sufficiently
short, but an amplicon can form under standard
reaction conditions. In cases when genomic DNA
amplification is a risk, additional precautions
should be taken to prevent against
non-specific amplification. The remainder of the talk
will be spent looking closer at custom qPCR assay designs. To begin with a custom
qPCR assay design, we will go back to the qPCR
tools within the tool dropdown. The PrimerQuest tool allows
for custom qPCR assay design, regardless of your
target species. From the dropdown menu, select
the PrimerQuest design tool shown here. The PrimerQuest tool
will assist in designing primer only and
probe-based assays for qPCR and droplet digital PCR. You can enter sequences
individually or batch up to 50 sequences at a time. When entering
sequences individually, custom parameters
within the tool can be configured to
target exon-exon junctions, create custom oligo
Tms, and partially input sequences
for assay design. An example of when
you would partially input design
parameters is when you have a primer for a
primer only assay, but would like to add a
probe to convert the assay to a probe-based assay. To begin, consider
in the transcript where you would like to target. It is important to
target multiple exons to avoid concerns over
amplification of genomic DNA contamination within a sample. Here is a cartoon of a gene with
four exons and three entrons before and after splicing. I like to look at the whole gene
when deciding where to target, as I prefer to target exons
separated by the largest entron my design will allow. In this example, my target
would be exons 2 and 3, as entron 2 is the largest
of the three entrons and the resulting
genomic DNA amplicon would exceed 1,000 bases. At this length, genomic
DNA amplification is unlikely under normal
qPCR reaction conditions. If you find yourself
forced into targeting a single exon or multiple exons
separated by a small entron, you can alleviate
genomic DNA concerns by treating your RNA
samples with an enzyme to remove trace amounts
of DNA, like DNase 1. Many RNA extraction
protocols will have guidance for this additional step. To begin an individual custom
assay design, pace your target fast in the sequence
entry box shown here. Or you can also enter
it in accession ID here, and the PrimerQuest tool
will download the sequence from NCBI. You can also batch up to
50 sequences using an Excel template. In batch mode, assays
will be designed using the default parameters,
and no customization is allowed. For assistance
uploading sequences from the batch function,
an Excel template can be found here. Here, I’ve added the FASTA
sequence for human BRCA1. Once the sequence
is entered, you can select design between
probe-based assays by selecting here, or primer
only assays by selecting here. The primer plus
tool will also allow you to create an assay using
defined custom parameters here. For this example,
I will demonstrate how to design a probe-based
assay using the default parameters. The tool will default
to providing up to five assay designs
for a given target. But if you are looking for
additional assay options, the custom design parameters
can be overridden to provide up to 50 assay designs. To view the details of a design,
click on the View Assay Details link here. The tool will display the
probe and primer sequences, as well as oligo characteristics
in the table shown here. The output will also annotate
each oligo within the sequence. The green sequence represents
the forward primer. The orange sequence
is the probe. And the red sequence
is the reverse primer. Please, note that
the reverse primer is annotated as the
reverse complement, as the reverse
primer is designed against the anti-sense sequence. When ordering, it is important
that the sequence given in the table view
is used and not the sequence annotated here. Now, I will demonstrate
how to generate a custom design using the
custom design parameters here. In this design, I
will tell the tool where my exon-exon
junction is located, so that the tool will design
and assay with an oligo spanning this location. Within the custom
design parameters, ensure that you are designing
to the correct assay format. The custom design feature
within the PrimerQuest tool will allow for PCR, qPCR, and
sequencing primer designs. For this example, I will
design a probe-based assay. Once the desired assay
type is selected, the custom design
parameters will allow for adjusting primer
and probe characteristics, like Tm, GC content, as
well as amplicon length. The tool will also
allow for selection of target regions to
focus designs around or regions to exclude. You can also perform
partial design inputs like we talked about previously
if you would like to design a probe for existing primers. Here, I defined position 703
as the exon-m exon junction to direct our design around. And here’s the output
for the custom design in which I provided the input
of the exon-exon boundary. We defined the exon-exon
boundary as position 703, and the PrimerQuest tool
designed the reverse primer to span across this location. Now that the PrimerQuest tool
has provided us with the custom design, targeting an
exon-exon junction, it is important to do some
backend checks to ensure that there are no concerning
dimers, the essay is specific for BRCA1, and that
we are not targeting regions with high frequency SNPs. To check for
concerning dimers, we can use the OligoAnalyzer tool. The OligoAnalyzer
analyzer tool is located in the Tools dropdown
menu on the IDT homepage. Unlike with assay design,
the OligoAnalyzer tool is found under the oligo
design and handling header here and not under the qPCR
assay design header, where we found the PrimerQuest design
tool and the pre-designed assay library. The OligoAnalyzer
tool will allow us to assess a variety of
oligo characteristics, dimer analysis, and even
blast a primer. To get started, enter
your primer sequence here. Today, we will focus
on self-dimer analysis. But you will also want to check
for header dimer formations, for all of the oligos
within an assay, as well as potential
hairpin formations. Once a sequence
has been entered, select the Self-dimer tab here. This is an example output
of a self-dimer check from our primer of interest. The tool I’ll put dimers as
number of base in the dimer and the strength of the dimer
in kilocals per mole or delta G. The more negative the
delta G value, the more stable and concerning the dimer is. A general guideline
for dimer strength is to avoid dimers more stable
than negative 9 kilocals per mole. Here, our dimer has a delta G
value of negative 3.6 kilocals per mole. So we should not be worried,
as this dimer is not a stable dimer. Here’s another example
of a potential dimer. This dimer is much more stable
than the previous example and should be avoided,
as it can affect the efficiency of our assay. While it is recommended
to avoid this dimer, if this were the only option,
it would be an OK choice, as it is not an
extendable dimer. I will discuss what I
mean by extendable dimers in the next few slides. In this next example,
we see a dimer that looks more stable
than we would prefer. But since it is
an internal dimer, this would be OK if it
were our only option, as it is more stable than
the negative 9 kilocals per mole value I mentioned. We should also consider
all possible dimers for a given sequence. The next most stable dimer
is much more concerning. The dimer’s configured
in such a way that the 3-prime hydroxyl
group of one oligo is free and bound to the sequence
of the next dimer. And this can allow for
polymerase extension. The other oligo in this
dimer acts as a template. And this can lead
to primer extension and greatly hinder the
performance of an assay. Because of this concern, I
would not recommend an assay with this type of a dimer. For extendable
dimers like this, I would recommend
avoiding dimers that are more stable than negative
6.5 kilocals per mole. Once you have checked all
potential dimers for the oligos within an assay, I
recommend you blast the oligos to ensure they are
specific for your intended target and do not target regions
with high frequency SNPs. If you are unfamiliar
with BLAST in NCBI, I would recommend
viewing the webinar– Tips for Effective Use
of BLAST and other NCBI Tools by Dr. Matthew McNeil. A link for this
webinar is shown here. For those interested in
designing assays for droplet digital, or ddPCR,
many of the design parameters remain the same,
as they offer standard qPCR assays. The most dramatic deviation
between ddPCR assays and standard qPCR assays
is amplicon length. For ddPCR assays,
I recommend going with shorter amplicon
lengths, generally between 70 and 125 bases. For lower quality samples,
like FFPE and circulating DNA, try and stay between 70 and
100 bases for amplicon length. Other characteristics
are similar, such as GC content, oligo
Tm, and dimer concerns. For rare target detection
and SNP analysis, please contact the scientific
application support group for design assistance,
as other considerations may be needed, including
the incorporation of Tm-enhancement modifications. As I mentioned earlier, an
advantage to probe-based assays is the ability to multiplex
assays in a single reaction. This is done by adding different
fluorophores to the probes, so the instrument
can distinguish between each assay type. IDT offers a variety of
fluorophores and quencher combinations to fit all
commercial instruments. Here are just a few of
the available fluorophores and recommended
quenchers offered. With the last few
minutes, I would like to take a look
at a few possible run failures you may encounter– non-specific amplification. This may be the result
of changes to databases or the presence of pseudogenes
within your sample. In the case of pseudogenes,
moving the assay either upstream
or downstream may be sufficient to
avoid the pseudogene. It is not uncommon for a
pseudogene only replicate a portion of the transcript. And moving the assay may
be sufficient to improve specificity. Late amplification– this may be
the result of extendable primer dimers, like the example we
discussed a few slides ago. Redesigning one or both
primers will generally resolve this concern. False positives– often,
this is the result of genomic DNA contamination
within the RNA sample. This can be alleviated
by performing a DNA SWAN digestion to remove genomic
DNA prior to cDNA library generation. Alternatively,
redesigning the assay to span across an exon-exon
junction is an option. Poor assay efficiency–
this may be the result of incompatible
primer and probe Tms. This could be from
primary Tms varying by more than a few
degrees, or a probe Tm similar to the primary Tms. Redesigning one or more
oligos within the assay may be necessary to improve
the assay efficiency. Your synthetic template
amplifies, but not the sample. Seeing amplification
in a synthetic template but not in a sample may be
the result of incorrect target annotations or targeting regions
with high frequency SNPs. If the target is a
human transcript, NCBI offers a comprehensive
map of annotated SNPs. And redesigning to a
region with less SNPs may resolve the issue. For other species, ensure
that the correct annotations are used. This is especially important
for viral and bacterial samples, as subtypes can have
divergent sequences. And your samples may have a
different sequence than the one used for the design. Here are some of the
links and the tools we discussed in today’s talk. Please, recall the OligoAnalyzer
and PrimerQuest tools can also be accessed through the Tools
menu on the IDT home page. Thank you for joining
me today, and I hope today’s talk
instills confidence in you to try designing
your own qPCR assays. I would be happy to answer
any questions you may have. Thanks very much, Erik, for
that informative presentation. Audience, if you
have any questions and have not done so already,
please go and type them into the Q&A widget right now. We can start with
a few questions. Erik, here’s the first one. Can you repeat the
delta G cutoffs, please? Sure. For non-extendable dimers,
like the internal dimer that we discussed,
the general cutoff is negative 9 kilocals per mole. As we become more
negative, those dimers become more stable. So we should try to
avoid dimers that are more stable than
negative 9 kilocals per mole. In the case of the
extendable dimers, that value is decreased. And I recommend avoiding dimers
that are extendable and have a delta G value of negative
6.5 kilocals per mole or more negative. OK, thanks. And how does
annealing temperature differ from melting temperature? Oh, that’s a great question. The annealing temperature
is the temperature used in the PCR
reaction to anneal the primers to a template. Generally, this temperature
is lower than what the Tm of the oligos are. And for most standard qPCR
reactions, this is 60 degrees. The melt temp, or
melting temperature, is the temperature in which
one half of the primers are bound to the target,
and one half of the primers are free in solution. OK, great. What master mix can researchers
use with probe-based assays? They can use any
master mix that is compatible with
probe-based assays. IDT offers the prime time
gene expression master mix. That will work great with all
of our probe-based assays, as well as other commercially
available probe-based master mixes. Erik, here’s a good question. It comes in pretty frequently. How to freeze and thaw cycles
affect probe degradation? Absolutely is a concern. IDT has performed
stability studies on qPCR assays looking at
freeze-thaw cycles for primer and probe assays. What we found is that, even
after 30 freeze-thaw cycles, we don’t see a
significant decrease in performance of an assay. That being said, I do recommend
that researchers allocate out their assays to avoid
freeze-thaw cycles. Less is always better. OK, great. And here’s a question. How can I determine
what fluorophores are compatible for my instrument? Sure. There’s two ways of doing this. Your user manual that
came with the instrument will have this
information in the index. We also offer a multiplex
dye selection tool under that qPCR
assay design header that we looked at
for the PrimerQuest in the pre-design lookup tool. While this tool is
designed for really helping select fluorophores
for multiplexing, it also will give
you information about what channels are
available for your instrument and what dyes will fit
into those channels. OK, great. An attendee is asking, can I
use my SYBR green master mix with a probe-based assay? Generally, the answer is, no. I wouldn’t recommend doing this. The SYBR green dye fluoresces
in a very similar wavelength to fan fluorophores. And this can lead
to interference, so no, I wouldn’t recommend
using a SYBR-based master mix for probe-based assays. All right, Erik, we
have a couple questions asking how to determine
Tm of an oligo or what the best program
is to determine that. Yeah, absolutely. So we can go back to
the OligoAnalyzer tool that we used to
check for dimers. By entering in
your sequence, you can select the
Analyze tab that’s just above the self-dimer tab. And that will provide you
characteristics on the oligo, including Tm, GC content,
molecular weight, extinction coefficients. I do want to note one thing
on OligoAnalyzer tool. It defaults to a
spec sheet setting. So this is if the oligo
were re-suspended in water. If you’re using the
oligo for a qPCR assay, under the parameters that’s
next to the sequence entry box, select the qPCR
assay parameters set. This will also take into account
magnesium, dNTPs, and also the primers that
would be in the assay. So the Tm is going to vary. It’ll be a little
bit different than it would be if you
were to calculate it based off spec sheet values. Great, thanks. And so you had mentioned
synthetic templates. What if a researcher doesn’t
have a synthetic template to use? Is there a way to obtain one? Yeah, absolutely. So if you know the
amplicon sequence, it can be as easy as
ordering a DNA oligo, like our [INAUDIBLE] oligos. Or if it’s a little bit
of a longer amplicon, one of our G-blocks
gene fragments. If you don’t know
what your amplicon is, you can still find that. By using BLAST, you
can blast your primers to locate where in the
transcript they target. And from that information, you
can pull the amplicon sequence for ordering that
synthetic template. OK, great. We’ll go with a few
more questions, Erik. First, how long after
hydrolysis of the probe does the fluorophore emit light? It’s done through the
same step in the reaction. It’s nearly instantaneous. Once that fluorophore is
liberated from the quencher, it does disperse
throughout the liquid. It doesn’t need much separation
from the quencher to actually be detected by the instrument. So it’s very quick. OK, great. And there’s been a
couple of questions about detecting methylation. Could you speak to any
general considerations, or whether these tools can be
used in those types of assay? Absolutely, yeah. So methylation is
definitely something that researchers
are looking at more. And the PrimerQuest
tool can be used to design to methylated
bi-sulfide converted sequences. The one thing that’s really
important when designing assays for methylation-specific
studies is to know all of the methylation
sites and their status within a sequence. What will happen when you
convert those sequences, the sequence becomes
very AT-rich. So what’s going to
happen is you’re going to have to
change the parameters within the PrimerQuest tool
to allow for lower GC content primers and also for longer
primer lengths and probe lengths, if you’re going
with a probe-based assay. But if you don’t know
what the methylation status and the
methylation patterns are within your sequence,
this could be difficult. If you are assuming that a
sequence is converted from a C to a T, but indeed, it’s
not, your primer sequences aren’t going to match
your template sequence. OK, great. As questions come in here, let’s
go back to a couple of things that you covered. Could you speak a little
bit to the reasoning behind why amplicon sizes
are as they’re recommended? Yeah, it’s basically just
the efficiency of the assay. The shorter the amplicon,
the more efficient a PCR reaction is. So the general guideline
of that 70 to 150 basis will allow us to have an
assay efficiency between 90% and 110%. And really, that’s
what we’re targeting. You can go longer if need be. But as you start to exceed 200
bases in length or even longer, the efficiency of the
assay is going to decrease. OK, and could you really
reiterate again the recommended temperature difference
between melting temperature and annealing temperature and
the reason for that as well? Sure, absolutely. So with qPCR assays, the general
rule of thumb is two degrees. The reason that we go lower– so two degrees lower– is that we want more primers
bound to the template to start the reaction. So as I mentioned,
the Tm of an oligo is when half of the primers
are bound to the sequence, and half of them
are in solution. If we have a primer
Tm that’s 62 degrees, and we anneal that oligo at
60 degrees, more than 50% of our primers will
be bound to a target. And this is going to help
with the assay efficiency. OK, great. And for multiplex
assays, is there a maximum number of targets
that you generally recommend? Generally, this is more
defined by the instrument. As far as I’m
aware, the most you can multiplex using
different dyes for each assay is five on some of the
commercially-available assays. You can always put in
more, but they’re going to have duplicate fluorophores. And you wouldn’t necessarily
be able to distinguish between those assays. If you are multiplexing, it is
important to not only consider homo and heterodimers
within a single assay, but you also need to consider
what those heterodimers are between primers and probes
of one assay with the primers and probes of another assay. So the potential
for dimer formation is significantly greater
when multiplexing. Great. Thanks very much, Erik. This will be your last question. And there are some remaining
questions coming in. Thank you very much
for those questions. We will get back to any
questions not answered here via email. Thanks again for those. Erik, for one final question,
what if the PrimerQuest design tool can not design to
a researcher’s target? That’s a great question. So the PrimerQuest
tool is really designed to output the
best possible assay within a set parameter. If you’re not getting
an output, this could be due to poor GC
content, low complexity, or incompatible oligo Tms. So you can override this by
going back to the custom design parameters and adjusting GC
contents, primer lengths, probe lengths, or even amplicon length
to maybe target a larger region to design an assay to. And this would be something
you would absolutely see in that previous
question that we talked about with methylation-specific qPCR. Those sequences tend
to be very AT-rich, and it wouldn’t be surprising
if the default parameters within the PrimerQuest
tool would not design to that sequence. By changing those primer lengths
and lowering the allowed GC content, you can
change the parameters, and PrimerQuest will
design for those sequences. Thank you, Erik. OK, that is all the time
we have for questions. I want to thank all of you for
attending today’s presentation. I also would like to thank
Erik for his informative presentation, as well as Brendan
for conducting the question and answer session. This is one of a
series of webinars we will be presenting on
qPCR, as well as other topics. We will email you about
these future webinars as they are scheduled. Also, as a reminder, a recording
of this webinar will be posted shortly on our website and
at YouTube.com/IDTDNAbio. There, you will find several
other educational webinars on such topics as next
generation sequencing, genotyping, CRISPR, and
general molecular biology. Thank you again for attending,
and we wish you the best of success in your research.

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