rhAmp™ SNP Genotyping: A novel approach for improving PCR-based SNP genotyping

rhAmp™ SNP Genotyping: A novel approach for improving PCR-based SNP genotyping


Hello everyone and welcome
to this integrated DNA technologies webinar on a
new PCR genotyping system. My name is Dr. Hans
Packer and I’ll be serving as the moderator
for today’s presentation. The presentation today will
be given by Doctor Scott Rose. Dr. Rose is director
of enzyme development here at integrated
DNA technologies where his team develops
tools for functional genomics research. They’re responsible for
creating, purifying, and characterizing novel enzymes
and developing complex reaction mixes that produce
consistent results. Also, today, joining us
will be Dr. Rita Menezis. And she is the product
manager for QPCR. And she’ll be joining
us more towards the end of the presentation. But she’s been heavily
involved in the development of this new technology. So she’ll be joining
us for a little bit and helping to answer
some of the questions. The presentation today
should last about 30 minutes. And following the
presentation we’ll have a question
and answer session. You can stick around
and ask your questions. You ask questions at
any time during or after the presentation by typing
them into the GoToWebinar to our control panel. And you’ll find that at the
right hand side of your screen. And if you click on that little
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window actually pops out. Because it’s a
little larger it’s a little easier to
type of question in. So like I said,
you can type those in at any time during
the presentation. And at the end I will
present as many of those to Dr. Rose and Dr.
Menezis for their answers. Also, in case you need
to leave early today or you want to revisit this
webinar we are recording the presentation and
we will be posting it on our Vimeo channel
and our YouTube channel. And those are shown
on the screen here. And you don’t need
to remember this. We also be sending you
links to this by email after the presentation. So don’t worry about
writing this down. If you did go to
those channels you would also find a wealth
of other materials on QPC, our next generation
sequencing, crisper genome editing. We have a lot of great
content on those sites. Also, we get asked for
slides quite a bit, so we have a slideshare.net
site that’s shown on the screen. And the slide deck for this
is actually already there. So you could go grab
those slides any time. And, like I said, you don’t need
to remember any of these links. We’ll send you these
links in a follow up email in a couple of days. So with that being said, I’m
going to hand the controls over to Dr. Rose, so he can get
started on the presentation. Well, thank you, Hans,
for an introduction. I’m really excited today to be
able to tell you about a brand new product that we
just launched that we called rhAmp SNP Genotyping. And it’s one of our
answers to the growing need for a very accurate,
high level, great resolution genotyping. And what I’d like to do is, talk
you through this presentation, explain the basis for
this technology, how the technology
works, and then we’ll show you some data that
goes along with it. So, the agenda of
this talk is, first, I want to give you an
introduction to our rhPCR technology and that it stands
for arness h mediated PCR. And then after that, I’m
going to actually talk about the application that we’re
discussing today, the rhAmp SNP Genotyping, and tell you
what it is and how it works. We’ll give you some of
the technical highlights. In the end, we’ll talk
about the design tool that we have available. And then later we’ll be
discussing the product description features. So at the core of this rhPCR
technology that IDT has, is an enzyme called RNase H2. And I’m going to give you a
little bit of a background about the enzyme. The enzyme was first
purified from human cells by Dr. Joseph Walder,
who is our CEO, back when he was a professor at
the University of Iowa. And he studied this
enzyme for quite a while. And he discovered some very
interesting properties. There’s multiple members
of this enzyme family. There’s an RNase H1, RNase H2,
and, in some species, RNase H3. But we’re going to
focus on RNase H2. We had been working
with the version that we’ve gone ahead and cloned
from the hyperthermophilic archaeon Pyrococcus abyssi. We really like this
enzyme from this organism. And also our work is
being done with this. Now, RNase H2 is
an endoribonuclease and it binds two
RNA-DNA duplexes. And that’s a very
important point. This enzyme does not recognize
single stranded material, but it’s substrate for
activity is a duplex– a heteroduplex. And it will cleave as
little as a single RNA base that’s been embedded
within the heteroduplex. So you don’t need a lot of RNA
residues bound to DNA residues. A very simple single
RNA base and they’re sufficient for to the
enzyme to be active. One of the very interesting
properties of this ribonucleus that other enzymes
don’t have is, when it cleaves the RNA
containing strand it leaves a 5′-phosphate on the RNA
base and the DNA base has a 3′-hydroxyl group. So that’s going to be really
important in just a moment. It does not clean
single stranded RNA. Once again we’re talking about
the substrate for this enzyme being heteroduplex. And one of the
really ideal points about this enzyme from
a Pyrococcus abyssi is not only does it have very
high optimal for activity, but it’s monvalent
and divalent cations that are required for activity
are very similar to those used with DNA polymerases. And so that has allowed
us to combine everything into a single tube format. Let me talk about this
RNase H2 cleavage mechanism that I just mentioned. So once again it has to
recognize the heteroduplex. What I’m going to
focus on here, is just the one strand
containing the RNA base and RNA base is shown in blue. Now, this is a 5′ end and this
is the 3′ end of that strand. The enzyme will come in
and cleave right here. And what you generate
is once again a DNA base with the hydroxyl. The phosphate group
stays with the RNA base. That 3′-hydroxyl group that’s
now left on the DNA is going to be critical for
future functions. One of the other things
about this RNase H2 is that it really does not like
to cleave mismatches at the RNC base or even mismatches upstream
and downstream immediately of the RNase H2. That doesn’t mean they won’t
cleave it with the kinetics are much greatly reduced. So they become very
mismatches, they become we come very poor substrates. So rhPCR, the RNase H activated
PCR, the basic mechanism here is very simple. We have a DNA primer that
has a single RNA base in it, couple more bases
down, DNA bases downstream with an RNA base
and then a blocking group. So that primer is not
competent for any kind of extension by a DNA polymerase
until the blocking group is removed. So in the presence of a
nice, perfect heteroduplex the enzyme comes along, does
the cleavage step right here, the small fragment of DNA
with a single RNA based floats off at an
elevated temperature– basically you have
regular DNA primer ready to be extended
by DNA polymerase. And we can do this with both
the primers being blocked or one primer being blocked. The important thing
here is, the primer needs to sit down and have a
good match well within the area that the RNA base is in
or the enzyme doesn’t want to cleave it, and then
you will get extension. So one of the real strengths
of the rhPCR technology is that we can use it to
greatly reduce primer-dimers and other spurious
amplification that comes from a primer hybridizing
to a sequence partially, but enough that the DNA
polymerase extends off of it. So here’s an example of
a 96-plex PCR reaction. The correct product
is right here. What you can see is in the
presence of 10 anagrams of junk DNA with standard DNA primer–
so the non-block primers– you see a little
bit of the product, but you see a tremendous
amount of other material which turns out to
be primer-dimers. And even in the absence
of any template DNA, sure enough, you
can actually see a fair number of the primers
interacting with themselves. Now, I don’t care how good
your algorithm is for design, a certain amount of this
is going to be a Problem. Now, if we use the
rhPCR technology, in the presence of the
same amount of DNA, what you see is a very
large intense band here that constitutes that 96
different amplicons and just a teeny, tiny
amount primer-dimer. And in the absence of
the template once again you see nice clean
lane here, but just a small amount of primer-dimer. So this system by itself
really proves itself and has the ability to really
cut down on your noise. So that is the rhPCR
technology that’s the basis for everything. Now, I’m going to tell you
about the rhAmp SNP Genotyping System which works off of
the Rh PCR based technology with some minor changes in them. So what we have here is a
genotyping system that we feel is very simple, it’s
accurate, it’s cost effective, and it generates
fluorescent signal much like you would
get in other platforms, using either real-time
instrumentation or endpoint mode analysis. The reason that it works so
well for allelic discrimination, is because it’s a mixture
of two different enzymes that IDT owns. The first is that RNase
H2, which as I mentioned, provides a sequence
specific cleavage event. And the other is a novel DNA
polymerase Taq, DNA polymerase, that has superior
allelic discrimination. Both of these are enzymes
that are property of IDT. So we have a cleavable
block primers that are generated also
through a very advanced design pipeline. Pipelines are very important. And they can really
help the quality of the data you generate. So when we combine
the master mix which has two different enzymes, as
well as our advanced pipeline, we can We can generate
some very strong signals. Once again the signal generation
is fluorescent in properties. The nice thing is,
it also follows very much the standard
3-step thermal cycling profile that you would find
in most qPCR instruments if you were doing
genotyping with say Taq man assays are
you doing genotyping with LNA-based probes. Our system is platform
agnostic and it is compatible with automation. So here’s the key slide
in the whole toxo. I’m going to go a little
bit slow through, this but it’s really
important to understand how the rhAmp SNP works. We have two different
allelic specific primers. We have them labeled
here as orange and blue. Within these primers we
have the blocking group that I’ve been talking about. The triangle
represents an RNA base. The blue or the red is
actually the position of where the SNP would be,
and the template strand, so just upstream of
the RNA base and this is all a little bit
different than what I was talking about in rhPCR. Notice that this actual position
of the SNP on the primer, is going to be the free
prime end in the primer once it’s been cleaved. Then we also have a
local specific primer that is of course
blocked and has RNA base. So what happens is, when we
have a mixture in one two, we have a mixture of two
different allelic specific probes. Each one at the
end of the day will generate its own specific
fluorescent signature. When the appropriate primer
binds to the template, we have a perfect match. The RNA stage two
enzyme, which is present, then go ahead and
clips it right there, this small DNA RNA
fragment floats off. We now have a primer
that’s perfectly capable of extra extension. As I said we generated 3′
hydroxyl and an extension that can occur. Now if inadvertently this primer
sat down over this space right here– so then we would
have an AC mismatch– not only the RNase H2 not want
to cleave this substrate– It would very, very
slowly, but it would– but that mutant DNA
polymerase and I was talking about
that has approved allelic discrimination
won’t extend off of this very efficiently. So then we have two levels of
specificity there that we get. The two dyes that are used,
since I keep mentioning, is fluorescent based, is
FAM and Yakima Yellow. Yakima Yellow, for those of
you who done a lot of qPCR, is very, very, very
similar to the dye Bick. In fact, it is so similar
in terms its excitation and emission wavelengths
that you do not have to recalibrate your instrument. If you don’t have Yakima Yellow
indicated as one of the dyes you can simply use
the Bick channel. It makes things very easy. So I mentioned this
mutant Taq DNA polymerase and how important it is, because
I want to show you an example, during development where we
started with using the RNase H2 enzyme in conjunction with
wild-type Taq DNA polymerases. And what you’re looking at is
an allelic discrimination plot, where the allele A is
the FAM signal shown on the x-axis allele
G in this instance is shown on the y-axis that’s
coming from Yakima Yellow. Ideally you should
see with no template– you should see no
signal down here– and then each homozygous
material should either be over here on this
axis, or over here on this axis, and
the heterozygous it should be in the middle. What you really see on these
allelic discrimination plots, is that you can see three
clusters corresponding to the two homozygous in
the one heterozygous sample, but they’re not
very well resolved. And that’s because there’s a
lot of cross reaction going on. So it’s destroying
the specificity. If we take the same
experiment, the same genotypes, and we use the master mix now
that has the IDT mutant Taq DNA polymerase alongside
RNase H2 enzyme. Now, you see the
classic textbook example of ultimately allelic
discrimination plot. No template controls
are down here at zero. You can see that
the one homozygous sample is right here,
right along the x-axis, the other homozygous
sample is on the y-axis, and clear smack in the
middle is your heterozygotes. This is the classic pattern. This is a very good example
of a really clean, positive genotyping assay. And that’s what we’re
striving to generate. And it all really
requires this combination of the two different
enzymes, before we could get to this case. We’re very proud of
this work, because we have some very excellent
scientists here at IDT We have two world class
scientists who understand the ins and outs of RNase H2. They just know so much about it. That’s Dr. Mark Belke, he’s
our chief scientific officer, and Dr. Joseph Dobese, who’s
a senior staff scientist here at IDT. They spent many, many years
studying this enzyme– not only RNase H2, but then
this mutant Taq DNA polymerase. Are Another characteristic
of these plots is that, what we’re
finding is, we get very strong,
robust signal when we compare our assays
to a standard 5′ nucleus genotyping assay that you
get from another vendor. Here’s two different examples. Once again, wild-type allele
will be on the x-axis, the alternative allele
will be on the y-axis. The blue dots are
coming from samples that have been run with
IDT rhAmp SNP assays. The red dots are coming from
the other commercial vendor, who sells them using the
commercial vendors master mix versus our IDT master mix. We’ve gone ahead and
run these reactions under conditions that are
optimal for both technologies. And what you see is a
fairly large difference between the signal
between the 5′ nucleus genotyping assay
and the rhAmp SNP assay. This particular SNP is
another good example. Once again you see
a lot less signal coming off of the commercial
version versus the IDT’s version. And that makes a big difference
when the input gets low. As you start to get lower
and lower amounts of DNA and input– right here this
example is only three nanograms of DNA– what you’ll see is, as the
input gets lower and lower the signal is going to
get weaker and weaker. And these different clusters are
eventually going to collapse. And you’re not going to be
able to tell what they are. So having that improved signal
noise is very important. So one of the things that this
sort of assay also shows us is that, the signal is well
balanced between the two different reporter dyes. In this case we have an
example of the rhAmp SNP assay. And you can see that the
signal from both the FAM, which is in red, and the signal
from the VIC probe, which is in Yakima
Yellow, is in blue here. And you can see that the
fluorescence at 40 cycles is very well matched. Down here at the bottom is
just the ROX for normalization. These are one on the
thermal fissure platform. Now, if you take that other
5′ nucleus assay we were just talking about, what
you notice is– here’s the FAM channel,
here is the signal coming off of the VIC channel– even at 40 cycles
or even 50 cycles they’re still widely separated. That leads to an
imbalance on the plots and the angles look bizarre. One of the things
we’ve done is to look at the ability of
these assays to be run on different platforms. So we have an example of an
assay run on the QuantStudio, the more official platform. The same assay is run, now,
on the CFX from Bio-Rad. And we also have an example of
the same assay run on Biomark. Once again there’s about
three nanograms of input DNA. There is 46 different samples. This is Coriell gDNA. The reaction volumes actually
range from nanoliters to microliters. And once again that
Yakima Yellow signal is detected using
the VIC channel without any calibration. Now the technology
works very well not only with purified DNA, but DNA
coming from cruder samples. The first example
that is shown here, is where we’re showing you some
genotyping with genomic DNA that’s been isolated
from whole blood. This material was isolated from
whole blood using in the QIAgen QIAamp DNA Blood Mini Kit. So it’s partially purified. In the same set of
plots here we also have some carload of gBlocks
that uses controls as well. What you can see is, the samples
regardless of what they come from– whether they’re
purified gBlocks, DNA, semi-crude DNA that’s come
through from whole blood– we still get the same basic
pattern and the same signals for both the heterozygous
and the homozygous sample. What’s really
impressive to me is that we can also do this
rhAmp SNP genotyping with really crude lysates. And this is an example
coming from [INAUDIBLE].. We’re taking crude maize
lysates in a low-volume PCR. So we’re looking at
96 different samples from maize that
are crude lysates. They are put directly
into the reaction. And the volume of the reaction
is less than two microliters. And we’re still getting
some very good signal– excellent separation–
even though we’re using unpurified DNA. You take the same
material and put it in the standard 5′ nucleus
assay, once again you see this horrendous
change in fluorescence. So the signals are really weak. As well as you notice the
clusters start to spread out. And even then no template
controls are starting to drift. So it’s pretty clear that this
system works extremely well with crude lysates. And we’ve done this with
other lysates, not just maize. We’re working on
several others– wheat, soy, others like that. I mentioned this earlier and
I want to bring up a point. Sometimes, when you’re doing
some developmental work or initially studying
something, you may not have samples
where you know all three types are available. And if you try to run
genotyping with samples without the controls,
the automatic software can’t call whether it’s
homozygous or heterozygous. You can’t make a call with
auto calling software. So what we offer also in
IDT are these gBlocks. And the gBlocks are just
double stranded DNA sequences where they have the particular
genotype that you want. You have a homozygous,
another homozygous, and of course you
mix the two together and you get the heterozygous. With this particular
assay, what you see is, auto calling can
now say, OK this is really homozygous
allele A1, based on these reference inputs. And so once again, it’s very
easy to order these gBlocks, if you need them, as parts
of control references. Now, the last part about
this is the software. And we have this rhAmp
genotyping design tool that’s available on our web site. IDT has predesigned about 10
million assays for human SNPs. There’s plenty more than that,
but we already have 10 million predesigned there. Some of those we also
split out are ADME assays– are the types of
assays that might be interesting to
pharmacogenetics folks. ADME stands for absorption,
distribution, metabolism, excretion. These have been prevalidated
in the wet bench. The input that you can
select from as humid is where we have
all the predesigns. You can use other species
those aren’t predesigned, but we have the
ability to design for these assays on the fly. The type of input information
can vary everything from a SNP ID with the
gene symbol reference, rough seeking information,
or even FASTA sequence. So basically you
select your assay type, you select your
species, and then you have your input
DNA format, and you can cut and paste in
any [INAUDIBLE] you want or the FASTA sequence. This is the next step
in the design tool. If you are going to
use FASTA sequence, it would look like this. Or as I mentioned you cut
and paste in your sequence– right here you use
brackets to designate the genotype slash in between– enter in your email address,
and you will get a reply. The output of this and
what we’ll send to you is the actual sequence
of the DNA primers. So you will get
that information. It’s not a black box. Alright at this point I am
going to turn this over to Rita and let her tell you a little
bit more about the product here. Thanks Scott. As Scott said, we
are really very excited to offer this product,
the complete rhAmp Genotyping System, to researchers. What we’re excited
about is, we’ve been able to predesign for 10
million human SNPs We believe that’s like the
largest collection of predesigned assays currently
available on the market. However, we also
do still realize that there are a lot more
newly discovered SNPs. So there are going
to be situations where you may have an
rs number that we do not have a predefined assay for. For those situations
as well as if you’re dealing with any other
species besides human, we have a high performing
custom assay design tool. Though, thing to keep in mind,
if you use our custom assay design tool and you enter
a human SNP rs number, we are going to
design it live for you and do all the QC checks
that we would have done as if it were predesigned. So that is really the
added benefit you get, if you’re using the custom assay
design tools with the human rs number. However, for all
other species that you us label to design
for SNPs of interest, as Scott just showed you, it
be more apparently that it’s a FASTA sequence. Pay you back your SNP. We have some other
criteria in helping you put the sequence in correctly. You give us an email address
and once the custom design run is finished, we
will get back to you with the the code sequences. For those that are submitted
with a factor sequence, we are going to
give you 10 assays. That is intentional
so that you all have the ability to
blast and pick the assays that best suits your needs. We do rank our assays
even on the custom design tool based on the
thermodynamic parameters. But we do recommend
that you also blast to do any additional QC. As Scott mentioned this is a
design tool where we really put a lot of thought into– not just the current
dynamics, but also the sequence information. And because we are designing
with the current information that is present in
all the databases, we are definitely more
up to date in our design and the crosschecking
of information that we can provide. In addition, we
have a small subset of the assays that have
been vetted or validated for the [INAUDIBLE]
gene targeted for the pharmacogenomics market. We are working towards
completing our [INAUDIBLE] assay, though that assay set. One thing to keep in mind is,
that the cycling conditions that these ADME assays are on at
are the same as IDT SNP assay. So then IDT, whether you
chose regular SNP assay or you chose an ADME assay,
the cycling parameters are the same. And that’s very important
so that you can then run on one plate
on IDT SNP assay as well as an IDT ADME assay. This is a sophisticated system,
as he just described to you, it has two enzymes. So the master mix
that we provide is a one mix solution, which
contains both the enzymes. So keep in mind it’s not
two separate vials of two different cubes. It’s a one optimized mix. In addition, we have the
universal reporter system. This really enables us to keep
our costs of this product low. With some other 5′
nucleus genotyping assays you would have to design
a new probe for every SNP that you would
want to integrate. And this is really
a benefit, when you have the universal
reporter system that you can buy the enzyme mix
and the universal reporter mix at one time, and just have
to change your assays, which are a set of preprimer
based on the SNP of interest. The reporter system
is also designed such that you can choose
between a vial that contains the reference dial
not based on your platform. Something that has also
added value to this system is that we are making available
the sequence with which the target SNP sequence or it
does turn them on the flanking bases around the SNP. That if researchers so desire,
they can use that sequence and go to our gBlocks
gene fragments page, where you can order these
double stranded DNA fragments. So this is just an added value. It’s not automatically
provided with the kits, but it is something that, if you
choose to, you can go and get. And most importantly,
what I would like everyone to keep in mind is, although
this is a dual enzyme mix, it really fits the
current PCR cycling method of doing genotyping. Things So it’s very simple. You just add your DNA samples
to your assay and reporter and master mix combination. You set it up, run it
on a thermal cycler, qPCR Peter cycler and
you do data analysis. So although it is a
sophisticated system, it’s very simple to use. And really in terms of
the normal protocols– that you are all already
used to in doing genotyping– this fits right in. There no added steps and
there is no added complexity of multiple tubes, of
multiple cycles in terms of trying to accommodate
for the RNase H2 and our Taq polymerase. It’s all pretty
straight forward. The cycling time is between
90 minutes to 2 hours. So that’s how most of genotyping
assays have that runtime. Scott, the next slide please. So just going a little
bit into product details. You can get the product in tubes
or in a 96-well matrix rack. The minimum is 24
assays if you want to order a 96-well matrix rack. However, if you have
the need for less, we can work with you, there
maybe some added fees, but we can make it happen. What is key is, we have
40 action scales to choose from for the assays– the 100, 750, 2,500 and 6,000. The 100 reaction scale is
110 microliter to the action. If you need to, the
human SNP market– We believe this is
really empowering the customer, because there
are many SNPs that you are just curious to know
if this is valid, is that something
of interest, and you don’t have to invest the cost
of a higher scale when you just need a few reactions. So the 100 reactions
criticizes men to [INAUDIBLE].. However, we’re also
very conscious of making a very value pricing
of all unit sizes. So, you can put
validate the [INAUDIBLE] with the 100
reactions booth size, but then move on
without increasing the cost of your whole project. We have very affordable prices
on all the other sales as well. In addition of course we also
offer multiple configurations of the master mix
and reporter mix. To make it easy,
we have matched it. But really you can pick
any size that you need based on your project size. Another key
differentiator is, we are going to release the
core primer sequences. So even if you ordered a
predesigned assay, on the specs sheet you are going to see
the core primer sequence that was part of your SNP assay. And of course when you do it
with our sequence or FASTA sequence we’re able to get you
multiple assays of that time. But the sequence is going to be
awkward on on the specs sheet. And as I just
mentioned we also offer the gBlock sequences, so that
you could go ahead and order our template, if needed. And finally, but
most importantly, it was very important for us
to be able to develop a product that we could ship
out very quickly. It’s IDT’s benchmark
value that we don’t like to keep our customers waiting. So with this product
we have defined what we want to ship this
within 7 business days and are actually getting a
much shorter turn around time shipping out the assays. Of course the master
mix and reporter mixes will come out with even
shorter turn around time. So these are some of the
products we deal with. Thank you for listening. And we are really excited about
the performance of the product and we hope you
will give it a try. Hans, Scott– So we’ve got a few questions
already in the questions box. If you haven’t
already done so now is a great time to
enter your questions into the questions box in the
GoToWebinar control panel. And I’m just going to
jump right into this. I do want to mention
one quick thing, which is what I’d mentioned earlier
that we have the slide deck on slideshare
and so I’m just going to let everybody
have the link to that. Don’t get distracted, but
you can go look at slidedeck, if you want. And let’s get started with
some of these questions. So, Rita– I get a couple
of configuration things out of the way here– is this available
worldwide or is it just in the United States? No it’s available worldwide. OK so it’s available, now. And then the next question– I’m not sure that we
can answer this one, but I’m just going to
ask it, because at least get something out there– which is what is the
genotyping cost per sample? Yeah, so why don’t I
follow this follow-up with a more detailed
conversation on the cost per sample. I can take this
offline and follow up. Sure. There’s just a lot of
variables involved there. So it’s hard to give a complete
answer to that for this format. Just to be clear, I really need
to know where the researcher is based, in what location
and then really do a more tactful
calculation on the cost. Sure. So, Scott, I guess this
will come back to you. So this person is asking
about quantification there they’re interested in
quantifying the presence of one allele versus the other. So efficiency is
important to them. And they’re wondering if the
additional locus specific primers would compete with
the allele specific ones and affect the efficiency. Do you want to just comment
on efficiency in general? Yes, so the initial
release of this product is really more for genotyping
on germline mutations. Then one of the
applications, that we’re working on very
hard, is actually in the direction of
what you’re mentioning, and that is to be able to
look at rare allele detection. We have done some work already. We’re very heavy into the
development right now. We think this will
be an excellent way to do rare allele detection,
but it is not ready for release just yet. So I would not recommend using
this particular application for rare allele detection if
that’s where you’re going. The next question
that I have is– this is an application
specific thing– so how well does
this work in bacteria and have we tried it on
bacterial colony suspensions? Good question. We The design and everything
is the same for bacteria– I mean, just obviously
using the genome. The master mixes that
we have are very clean. So there’s no background
signal from contaminating DNA, with DNA in the mixes. And we’ve never tried it
with really crude lysates, but we have used this enzyme
in another slightly different, very lifestyle formulation
with actual crude lysates. So I know that the
RNase H2 enzyme itself works very well in
crude bacterial lysates. We just haven’t formally
done those experiments, but I would not anticipate
any difficulty at all. There’s a couple
of questions here– some of this is going to come
back to just the design tool– the first one is,
with the design tool, is there any advice
we can give people who are trying to design for
polyploid species, in which you maybe need to know
subgenome specific SNPs, in order to know
that you’re looking at the correct chromosome. That of course, is a
problem in the egg world. What I would recommend
there, is actually speak to our director of
bioinformatics, Dr. Huang, he is the one who would
be able to help you. And he could give
you some advice on how to use the tool and ways
to get around those issues. I think, that’s
beyond the scope, here, that I could
really talk about. So if somebody does want
to get a hold of us. They want to send an email. We have a short URL, which is
just www.idtdna.com/contactus. And if you just reach
out through that and ask your
question it will get directed to somebody who
can help you with an answer, if we can’t help you here. So that will be a good
place to reach out. And related to that, Scott,
then was the other question, which is, is this good for
plants and things which are– they mentioned wheat
which is hexaploid. Yes, so the next
version that we’re going to launch
with this product will actually focus
around, not the biallelic assays that we’ve
been talking about, but using indels as well as
working in egg bio samples. We have done successfully a
series of different tissue types, including,
as I mentioned, maize, and soy, canola wheat,
and a few others, that, I guess, I can’t
really say just yet. But the design becomes
critical of course and we have a lot of
sophistication and that. And we have some additional
workarounds in the pipeline. So the next generation
of this assay will definitely be targeted
towards plants in general– as well as, I mentioned other
types of mutations, not just single nucleotide
polymorphisms, but indels. The current formulation,
as we have existing now, actually does work with
biallelic, triallelic, and tetraallelic SNPs. The next question, Scott– i’m not sure if this is
something that we disclosed or not, so I’ll ask
this carefully– but can we tell them what the
distance is between RNase H2 cleavage site and
the SNP itself? Sure. If you look at the
allele specific primers, here, the RNA base is actually
the very next base away from the SNP site. So this is where the
SNP is going to lie, or be the perfect match. The very next base
is the RNA base. So the cleavage is in between. In this case it
would be in between g in the very next place,
which would be the RNA base. So the cleavage is
here, 3’hydroxl here, this little 5′ phosphate, and
the RNA little stub floats off. And this is where
I was mentioning that, if it’s a mismatch
there, the DNA polymerase– this mutant polymerase, that
we had– wouldn’t extend off of it. And the signal generation part–
and I didn’t go over this very well– is really coming from
this tail, which is not complimentary to the template. And it has a primer
floor for there. The next question is– something that probably
a lot of people would like to know
the answer to– is it possible to multiplex
assays within a while and if so how many? So we’re working
on that right now. All that is part of development. We have done some
preliminary work. We can indeed have four
colors available– so in other words, two
independent biallelics. And down the road of
course, as I mentioned, we’ve done some triallelics. We want to get to
that point where we can have four colors at
a particular given reaction. Most of the equipment
that’s out there right now doesn’t handle
five dyes overly well. So four right now is pretty
much based on the equipment. So the next question,
Scott, is something that people are
interested in, which is, can the assay have
more than one mismatch or does it support the indels? So the first point
is, yes, there can be. We’ve been working on that. The RNase H2 enzyme
itself, which is going to remove
the blocker, is really only sensitive to three spots. Where the RNA base
is a mismatch, there it’s very sensitive, too– and then one base up
and one base down. After that it doesn’t affect
the RNase H2 cleavage pattern. So there will
always be bases that will have an impact on
how well it cleaves. If there is a mismatch
elsewhere, for instance– certainly could be over
here beyond the RNase base. Remember this is a little spacer
sequence– its RNA is actually several DNA bases
and then a blocker. That has a very minimal impact. As well as more
mismatches, let’s say, upstream of where the
real mismatch that you’re interested in interrogating
is located at. Those can be tolerated for. The next question is
about reaction conditions. So will the presence
of phenol salts or other imperial impurities
from phenol chloroform extraction cause problems
with that genotyping accuracy? So that’s a good question. And with any system, if there
is a tremendous amount of it there then, yes, of
course, it would. But with what I’m familiar
with, most of those samples get diluted after you
done the extraction. You have so much
DNA that you have to dilute it down to the
appropriate concentration anyhow. So that takes some of it
out or out of the play. We have not quantitated
the exact levels, but we will tolerate some
phenols as well as some sugars. This is a good question. I’m wondering if this
is related to something we’ve talked about in a
different presentation, as well. Is there a preamplification
set up available? This particular
system really doesn’t have a preamplification. But the rhPCR
technology in itself can be used for preamp, as well. So it can be used. We showed data for
the Fluodigm biomarker at some point and
that that’s where we’ve discussed this previously,
that pre-amplification stuff. Do they have to do
anything different if they want to
use the Fluodigm? We have some recommendations
how to set up and execute the reactions on the Fluodigm. Is it compatible with
digital droplet PCR? That’s an excellent question. We had the same
question earlier. Let me say that
this version is not. We’ve been doing and are
continuing to do a lot of work in that in place. Being able to use this rhAmp
SNP technology in emulsion PCR– what it comes down to is, is
the wild type RNase H2 enzyme compatible? However, we do have
some mutants in hand right now that are compatible. We were working on
developing that system. So we fully expect
down the road to have a version of this
that is directly compatible with emulsion PCR. This next question–
this is a good question. So there’s a lot of
interest in FFPE samples where you get low DNA yield. Do we have any
recommendations on how much template DNA should be used
for analyzing FFPE samples? I can tell you that we have
not spent a lot of time on FFPE samples. We have done titrations of
how low the amount of input material we can get to. Certainly we can get away
with around 30 copies of DNA. The amplicons for this
are typically very short. We could design as little
as 40 base amplicons. And that would, of course, be
compatible with a very small amount of very short fragments
of DNA that come from FFPE. We just simply
haven’t had a chance to explore that in
too much detail, but we believe it
will be compatible. But we’ll see. Now we looked quite a
bit at the NGF stuff. Will be in an interesting
set of experiments for us to do with the rhAmp SNP tools. The next question– and
it’s the other end of that– so how large can
the amplicons be? Gosh, you know I
don’t think we’ve ever tried to make very,
very long amplicons. A couple of hundred bases is
probably the outside length that we’ve done. The technology could work
with much longer amplicons. What would be required
then of course is, the reaction conditions
would have to alter a bit. The next question is
how long is the tail that is 3′ to the RNA base? The tale that is, you mean the– I’m guessing they
mean everything that’s left after you cleaved
the base, so even the sequence specific stuff and
then the tail itself. What’s the rest of the
primer that’s left? So I’m not going to
talk about the length of the orange and the
purple regions, if you will. I can’t really give
you that length. But once the DNA
[? allele, ?] once it’s cleaved and RNA
blocking groups gone, then that length is very much
dictated by what the TM is. So it can vary. This next question is great. It’s basically just
asking if we can use this technology on the
QuantStudio Real Time machine? Absolutely. We’ve done a lot of stuff
on the QuantStudio platform. Apologies here, I’m just
kind of reading through some of these longer questions. There are quite a few questions
here about pricing or whatnot and we will just follow up
with people about the pricing issues. That’s going to
be region specific and you know even specific
to the kinds of work that they’re doing. That’s correct. When we run these
reactions, as I mentioned, the reaction size can be in the
nanometers to the microliters. So it very much depends
on your platform. We’ve actually gone and
used really small volumes with an intellycube. So we know that the
platform is compatible with different reaction volume
sizes which are dictated of course by the platform. So the overall cost
in the end of the day is going to reflect the
platform that you’re using, the number of samples that
are you are using, obviously, and other parameters. Here’s a question for you
which is are the rhAmp SNP genotyping assays
only compatible with biallelic analysis? So no, as I believe,
I just mentioned this extremely briefly. We actually do have triallelic
and tetraallelic work being done. We have some information
on our user guide on how to set up and run
triallelic– and tetraallelic would be the same thing. And there are some in
our ADME assay library that are triallelic. And if you want to
make them you can just follow what’s in that
user guide and figure out how you would design those
using the custom design portion of our design tool. So it’s possible. This is interesting– I’m not familiar
with this platform, but I’m sure that
you and Rita are– which is, have we tried this on
the applied bio qPCR platform– maybe that’s an applied
biosystems qPCR platform. Yeah but there are
many [INAUDIBLE] you need a little bit
more specifics right? Yeah, I mean are the
dyes different for different platforms on there? I mean these are pretty– Any platform that can handle
at least two dyes i.e. the FAM and Yakima Yellow slash VICs. Any of those platforms that can
at least detect those two dyes are going to work with this. And they clarified
this that they meant they applied biosystem. So Yeah, I mean, regular
thermal cycling parameters, which is just the nicety of this
system, they don’t have to do– As we mentioned this can be
used both, in a real time format with pretty much all
of the existing platforms out there as long
as they had to dyes. Also it can be grow as a– you can burn it in
a thermocycler then transfer it over
to a plate reader and do an endpoint
read if you want. That would be possible, too. Hey, Hans– Yeah. In relation to many of
the pricing questions, we could encourage our
customers to go on our web site, because all the local
regions [INAUDIBLE] pricing is pretty
easy displayed. All the different products–
there is a whole step. And then they can just figure
out the size of the reactions that they’re planning,
work backwards from there pretty easily. I’m happy to follow up at
answering those specifically and I will. But if they are curious in
terms of knowing the pricing. They can see how competitively
priced especially the 100 reactions sizes
are in US dollars. It’s well below
$100 [INAUDIBLE].. Another question about the
functioning of the design tool itself which is, does the
design tool calculate things like delta G and does it also
look for structural problems with the primers and
look for primary dimer issues and whatnot? It does a lot of that. You’re not going
to see the output. But as part of the pipeline
it takes into consideration many of those things– is when it generates a ranking
of the possible outputs, and what we think is probably
the best, all of those factors get looked at. IDT has done a lot of
research in that area. Our primary quest are– all of our analyzer tools
have been really popular for many years. I mean, I used him in
grad school some years ago and it’s built in to
all of our design tools that relate to DNA and RNA
design so, yeah, it’s baked in. So we’re pretty much
out of time here and we’ve gotten through
all of the questions that we can do here. So with all that, I would
like to thank everybody for the great questions
that you’ve asked and for participating
in this discussion. And I want to thank
Rita and Scott for helping out with answers. And, Scott, thanks for
the great presentation. This is wonderful. So anything else
anybody wants to add. No I’m good. Thank you for listening. All right, thank you, guys.

One thought on “rhAmp™ SNP Genotyping: A novel approach for improving PCR-based SNP genotyping

  1. hi, great video. I liked much.
    I am a student and am doing a review against the Taqman.

    I have a question for you, what is the rol of the probe?
    I think the probe stays at sequence of the primer, therefore the Taq Polymerase doesnt hidrolize it until after 2cicles. the probe contains the fluorescence which will allow to study th concentration of a SNP.
    i believe that i understand it but i would thank you much if yo remove my doubt.
    Thank you very much

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