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FREQUENTLY ASKED
QUESTIONS
General questions - technology,
company
Does Asper provide genotyping
instrumentation or service?
What
does Asper mean by medium-sized genotyping project (in terms of technology
transfer)?
Why
should I prefer Asper's genotyping?
What
technology is used by Asper in its genotyping service?
What
are the advantages of Asper's technology?
What
are the sensitivity and the specificity of the APEX technology?
Does APEX technology enable to
identify heterozygous genotypes?
Is
the technology also suitable for VNTR (Variable
Number of Tandem Repeats) and
STR (Short Tandem Repeats) analysis?
Does
Asper provide SNP discovery service?
Where
can I get technical and scientific support (for example more detailed
interpretation of the genotyping results)?
Microarray
Slides & Spotting Solutions
What types of probes can be
immobilized onto the Genorama® Slides?
How
does Asper guarantee the quality of slides?
Do you provide bar-coded slides also?
Why should I prefer SAL (aminosilane
with linker) coated slides instead of epoxy
silane coated slides?
What is the shelf life of the Genorama®
Slides and Spotting Solutions?
How important is to use Genorama®
Spotting Solutions together with Genorama®
Slides?
Where can I get the preferred
protocols for Genorama®
Slide?
How
many spots and slides will 50 ml 2x Genorama®
Spotting Solution typically
provide?
What could be possible causes of
irregular spot morphology and how can I avoid this
problem?
What
could be possible cause of too big spots and how can I avoid the problem?
What could be possible reason for the
spots appearing to have tails (Comet shaped
spots) and how can I avoid the
problem?
What could be possible causes of ring
shaped or doughnut-like spots and how can I
avoid the problem?
What could be possible causes for a
low signal over the entire slide and how can I
avoid the problem?
What could be possible causes for low
or no signal in some areas and how can I avoid
the problem?
What could be possible causes of high
background and how can I avoid the problem?
What should I do if the
troubleshooting advice given above does not help to solve my
problem?
Where can I find a local distributor
for your slides and spotting solutions?
Chip development, Chip design Software, Bioinformatics
What kind of information should I
enclose with my inquiry regarding chip development
and routine DNA
analysis?
What kind of information and materials
does Asper need to start the chip development
process?
Do I need to know the positions of the
SNP in the genes or the chromosome regions of
my interest?
Do
you provide genotyping solution only for the human genome?
How
much does the chip development cost?
How long does the chip development
take?
Is it possible to add new
SNPs/mutation
sites to the developed chip?
Is Asper´s SNP selection and PCR
design software suitable only for APEX or can also
be applied with other
platforms?
Why should I prefer your PCR design
software?
DNA Routine analysis, genotyping software, detector,
consumables
How much does the routine analysis of
DNA samples with the developed chip cost?
Can
I submit blood samples, fixed tissue samples to Asper for analysis?
How should I send the DNA samples?
May
I amplify genomic DNA in our lab and send the PCR products to Asper?
How does Asper guarantee the quality
of the genotyping service?
How
do I receive the completed genotyping results from Asper?
Can
I use the Genorama®
QuattroImagerTM for gene expression studies?
What
is the advantage of your detector compared with other microarray scanners?
Is
the 10 micron resolution of Genorama® QuattroImagerTM
available?
Would
the 10 micron resolution option improve the APEX genotyping technology?
General
questions - technology, company
Does Asper provide genotyping
instrumentation or service?
Asper offers solutions for
both kind of genotyping needs. Customers can choose between genotyping
service and technology transfer. Technology transfer includes custom chip
design, four-colour microarray detector, genotyping software, consumables
and training.
Technology transfer - medium-sized projects
Service - large and medium-sized projects
What does Asper mean
by medium sized genotyping project (in terms of technology transfer)?
We mean by
medium-sized project 100-3000 SNP per sample/DNA.
Why
should I prefer Asper's genotyping?
- Custom
chip design – the assay is compiled exactly according to
customers’ needs.
- Flexible
solution for genotyping – Service and technology transfer available
- up to customer’s choice.
- We
provide all the necessary items for genotyping: genotyping service,
strong bioinformatics support, custom chip design software,
instruments, image analysis software, consumables, training.
- Low
initial investment compared to other solutions
- Asper
has skilled professionals who have years of experience in genotyping
- Reliable
and accurate technology – demonstrated in recent
publications in Nature, PNAS and Clinical Chemistry.
- ISO
9001:2000 quality standard.
What
technology is used by Asper in its genotyping service?
Asper's genotyping platform
is based on APEX
- Arrayed Primer Extension. This method combines the accuracy of primer
extension with the high-throughput capacity of microarray.
What
are the advantages of Asper's technology?
Simplicity
o Four color detection system; all four possible sequence variants are
detected in the same reaction;
o Low reagent complexity - oligonucleotides, dye terminators, DNA
polymerase and DNA sample.
o Robust and durable detection system
High quality and accuracy
o Excellent power of allelic discrimination
o Two- step biological quality control - hybridization and primer
extension
o Parallel SNP detection on both DNA strands – additional internal
control at negligible extra cost
o High specificity, sensitivity and reproducibility
Fast and high throughput
o time required for the complete APEX reaction is less than an hour,
several APEX reactions can be carried out in parallel with minimal effort.
o From 1 to thousands of SNPs per assay
o As the chip production is based on printing, it is highly parallel and
less time consuming than in situ synthesis.
Cost-effective
o Low initial investment
o Low running costs
o No need for expensive optically flat polished slides
o One oligo per SNP to be arrayed to call all 4 nucleotide variants
Flexibility
o Chips can be designed to meet client’s specific needs (one gene
– many genes; human DNA – other species’ DNA; disease specific –
region specific; for SNP analyzes – for mutation detection –for DNA
resequencing; small assays – large assays, etc.)
o Customer can add, remove and substitute SNPs or mutation sites from the
array easily
o Technology is suitable for both research and diagnostics.
What
are the sensitivity and the specificity of the APEX technology?
Specificity
of APEX-based b-thalassemia test=TP/(TP+FN)= 99.4%*
Sensitivity of APEX-based b-thalassemia test=TN/(TN+FP)= 99.8%*
TP - true positive TN - true negative
FP - false positive FN - false negative
* Reliable Detection of b-Thalasseemia and G6PDMutations by a DNA
Microarray.
Gemignani F. et al., Clinical Chemistry 48, No.11, 2002 2051-2054
Does APEX technology enable to
identify heterozygous genotypes?
Yes. For example from the images below you can see how the signals of
heterozygous genotypes look like.
Is
the technology also suitable for VNTR (Variable
Number of Tandem Repeats) and STR (Short Tandem Repeats) analysis?
No, the technology is
adequate only for SNP (Single Nucleotide Polymorphism) and mutation
detection analysis.
Does
Asper provide SNP discovery service?
No, we do not provide de-novo sequencing and SNP discovery. If our
customer is interested in looking for new uncharacterized SNPs, we could
provide contact details and put you in touch with our partners, who are
able to perform custom SNP discovery.
Where
can I get technical and scientific support (for example more detailed
interpretation of the genotyping results)?
Please send your e-mail to
your project manager or info@asperbio.com
. You may also call +372 7 441 772 between 9 am and 6 pm (GMT +2:00).
Microarray
Slides & Spotting Solutions
What types of probes can be
immobilized onto the Genorama®
Slides?
Genorama®
microarray slides are developed and tested primarily for DNA-based
applications. However other nucleic acids, proteins, small molecules,
cells and extracts can also be immobilized.
Our SA type slides are developed for binding unmodified DNA, long aminated
DNA and other biological material, SAL type slides are developed to bind
short aminated DNA (8-75mer oligonucleotides).
How
does Asper guarantee the quality of slides?
The production of the slides involves 3-steps of quality control:
I.
Manual selection of the raw glass material to avoid slides
with scrapes and other mechanical damage
II.
After coating several slides of every batch are scanned to
inspect the uniformity of the silane layer
III.
A number of slides of every batch are tested for their DNA
binding capacity by immobilization of fluorescent dye labeled oligos.
The
protocols of stringent production procedures have been developed in Asper
and the process is certified to be in accordance with ISO 9001 standards.
Do you provide bar-coded slides also?
Yes, upon request we do provide also bar-coded slides, please contact our customer
manager to get more information.
Why should I prefer SAL (aminosilane
with linker) coated slides instead of epoxy silane coated slides?
According to the tests (.pdf 157 kb) made at Institute of Molecular and Cell Biology, University
of Tartu, SAL slides have at least
2 times better binding capacity of aminated DNA than epoxy coated slides.
What is the shelf life of the Genorama®
Slides and Spotting Solutions?
Coated slides can be stored at least for 6 months in an unopened original
package. After opening the foil package, you should store the box of
slides at +4°C in a clean environment in the dark.
Printed slides should be stored in a closed box in the dark, preferably at
+4°C. We have noticed that printed slides have a longer self-life
compared to the ready to spot slides.
Genorama®
Spotting Solutions' shelf life is also at least 6 months and must be
stored in the dark at +4°C.
How important is to use Genorama®
Spotting Solutions together with Genorama® Slides?
The image of a SAL-slide
scanned using the PerkinElmer ScanArray 5000
The oligos were printed as follows:
Our tests have shown that the choice
of the printing buffer plays an important role in binding capacity and
spot morphology. So it is extremely important to test slides with the
appropriate buffer.
Therefore we strongly recommend the use of optimized Genorama®
Spotting Solutions together with the SAL and SA slides in order to ensure
proper quality.
Where can I get the preferred
protocols for Genorama® Slide?
We will send the protocols to you
with the ordered slides and buffers, but you can also download the manual
from our website (pdf.
252 kb).
How many spots and
slides will 50 ml 2x Genorama® Spotting Solution typically provide?
1. It depends how much spotting mix will be put into one well.
We recommend 50 μM final oligo concentration in the spotting mix.
Then the spotting mix consists of: 1 part oligos (200 μM) : 3 part
spotting solution (1×). (NB! Asper provides 2× concentrate).
If you put 50μl spotting mix into one well (37,5 μl 1× Spotting
Solution), then
50 ml of 2× spotting solution is enough for 2666 wells.
2. The number of spots
per well depends on rate of humidity in printing chamber and how long the
wells must be open for printing process.
For example 50μl spotting mix in one well is enough for 100 to 1000 spots depending
on conditions.
To summarize: 50
ml of Genorama®
Spotting Solution must be enough for 25 slides even with
quite big number of spots, in case the recommended protocol is used.
What could be possible causes of
irregular spot morphology and how can I avoid this problem?
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Cause
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Solution
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- sub-optimal washing of the
printing pins both during and after the print run
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Printing pins should be cleaned
thoroughly after each run. In addition, washing and drying steps of
pins during the printing program need to be optimized for the
application at hand.
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- differences in the volume of
printing solutions among wells
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Fill the wells with equal volume of
printing solution.
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- the inclusion of detergents (10
ppm or more) or presence of contaminants such as polysaccharides
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Check the purity of the components
of printing solution. Do not use solutions containing detergents.
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- bent or damaged printing pins
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Check
the printing pins of the microarrayer
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What could be possible cause of too
big spots and how can I avoid the problem?
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Cause
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Solution
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- damaged or dirty spotting pins
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Check
the printing pins of the microarrayer
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- excessive rehydration before
cross linking
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Optimize the rehydration time
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- excess solution on the eternal
surface of the pin
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Use blotting steps before starting
spotting
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- slide not firmly seated in
arrayer
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Control slide positioning
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What could be possible reason for the
spots appearing to have tails (Comet shaped spots) and how can I avoid the
problem?
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Cause
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Solution
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- Too high DNA concentration
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Use
a lower concentration of PCR products/oligos |
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- Insufficient blocking
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Optimize
the blocking step
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What could be possible causes of ring
shaped or doughnut-like spots and how can I avoid the problem?
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Cause
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Solution
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- sample evaporates too quickly
after spotting
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– print in humid environment, try
a different spotting solution
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What could be possible causes for a
low signal over the entire slide and how can I avoid the problem?
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Cause
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Solution
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- target concentration too low
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Increase the concentration of
target
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- poor incorporation of dye(s)
during labeling
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Use alternative enzyme
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- concentration of printed DNA
(probe) too low
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Increase the concentration of
printed DNA
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- poor quality of dyes used for
labeling
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Try a new batch or a different dye
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- poor immobilization of printed
DNA
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Optimize immobilization protocol
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- hybridization wash stringency too
high
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Decrease the stringency of
hybridization wash
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- non pure water used for
processing and making reagents (<18.2 megohms-cm resistance in
the destillator)
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Use water with better quality
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- incomplete denaturation of array
or target DNA
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Optimize your denaturation protocol
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- improper storage of substrates
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Follow the recommended storage
instructions
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- the photosensitivity fluorescent
dyes have been bleached in brightly-lit environment.
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Keep probes and hybridized arrays
protected from light. Keep the tubes wrapped in aluminum foil.
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What could be possible causes for low
or no signal in some areas and how can I avoid the problem?
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Cause
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Solution
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- area covered by bubble or
particles during hybridization
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Try to avoid bubbles and particles.
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- slide not seated properly
in scanner
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Try reversing orientation to see if
artifact remains
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- array surface subjected to
abrasion during processing
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Avoid surface abrasion
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- insufficient amount of
hybridization solution used
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Increase the volume of hybridization
solution
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- arrayer not properly calibrated
for slide thickness
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Recalibrate the arrayer
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What could be possible causes of high
background and how can I avoid the problem?
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Cause
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Solution
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- hybridization solution evaporated
during hybridization
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Increase the sample volume,
increase the humidity in the hybridization chamber
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- hybridization buffers contain too
much fluorescently labeled DNA
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The hybridization buffer should be
prepared shortly before use, using reagents of the highest quality
available.
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- deposition of volatile organics
onto the slide surface
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Try to avoid the presents of
volatile organics near the coated slides.
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- labeled target attached to
incompletely blocked slide
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Try washing again with more
stringent solutions
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- inadequate washing or slides
dried during wash steps
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Avoid slides becoming dry during
the wash steps
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- dust or other particulate
contamination introduced during processing
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Work in dust-free environment
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- unincorporated label not removed
from sample
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Remove unincorporated label
before hybridization
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- improper storage of substrates
after opening the foil
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Follow the storage instructions
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What should I do if the
troubleshooting advice given above does not help to solve my problem?
Please contact our tech. support slides@asperbio.com
of call +372 7 441 772.
Where can I find a local distributor
for your slides and spotting solutions?
The information is available on slides distributors
web page.
Chip
development, Chip design Software, Bioinformatics
What kind of information should I
enclose with my inquiry regarding chip development and routine DNA
analysis?
Regarding chip development
- number on SNPs/mutations to be screened
- do you already know all the SNPs and mutations in the genes or regions
of your interest or you need help with collecting the information?
- no of SNP/mutation to be validated
Regarding routine DNA analysis with a chip
already available in Asper
- number of DNA samples
- type of the sample (genomic DNA, PCR product)?
- number of PCR amplicons
- whether you want to order the full sample analysis service from Asper or
you prefer to do it in your own lab (technology transfer)?
What kind of information and materials
does Asper need to start the chip development process?
SNP analysis
- SNP ID numbers (rs#, http://snepper.chip.org)
- DNA concentration and method of extraction
- If the SNP is not available in public databases we need sequence of
- 400 bp flanking area (if you send us genomic DNA)
- 25 bp flanking area (if you send us a PCR product).
NB! PCR must
be performed according to our protocol in order to ensure incorporation of
a small fraction of dUTP into your PCR products for fragmentation.)
- location/sequence of the PCR primers in case you already have designed
them
- in case you order chip validation, please send us appropriate DNAs, one
per each SNP on the chip and including information about the SNP in the sample.
Mutation analysis
- location of the mutation with
- 400 bp flanking sequence, if you send us
genomic DNA
- 25 bp flanking sequence, if you send us a PCR product.
NB!
PCR must be performed according to our protocol in order to ensure
incorporation of a small fraction of dUTP into your PCR products for
fragmentation.
- DNA concentration and method of extraction
- location/sequence of the PCR primers in case you already have designed
them
- in case you order chip validation, please send us appropriate DNAs, one
per each mutation on the chip and including information about the mutation
in the sample
Do I need to know the positions of the
SNP in the genes or the chromosome regions of my interest?
No, it is not a must. Asper has developed new software to help you, to
pick SNPs from the public databases and estimate their compatibility with
APEX based genotyping.
The selection is based on the following information:
- location relative to genes (exonic SNPs, SNPs in the transcribed region
of a gene, intronic SNPs etc.)
- maximum distance from the nearest exon
(e.g. all SNPs within 3 kb from nearest exon)
- minimum distance between two consecutive SNPs
- compatibility with APEX based genotyping
- haplotype block structure (select only haplotype tag SNPs)
Our program is specially tailored for selection of a uniformly distributed
set of SNPs over large chromosomal regions or over the whole genome. This
is often required for determination of haplotype structure in certain
regions of the chromosome.
Haplotype tag SNPs can be used for SNP selection only in genomic regions,
where haplotype block structure is known.
Do you provide genotyping solution only for the human genome?
Asper can confidently genotype all the species whose genome information is
available (human, mouse, different plants, viruses, bacteria etc.)
How
much does the chip development cost?
It depends on the size
and the complexity of the chip and the rate of PCR multiplexing. Please
contact your local authorized distributor or Asper's customer services
manager info@asperbio.com
in order to get a quotation.
Before mailing your enquiry please make sure you have enclosed all the
information required to be able to give you a quotation.
How long does the chip development
take?
The duration depends on the complexity and the size of the chip. An
average time for chip development is one month. Please contact your
local authorized distributor or Asper's customer services info@asperbio.com
in order to get more detailed information.
Is it possible to add new SNPs/mutation
sites to the developed chip?
Yes, it is. One of the advantages of Asper´s genotyping platform is, that
even during the routine analysis it is very easy and cost-effective to
include, remove and replace SNPs/mutation.
Is Asper´s SNP selection and PCR
design software suitable only for APEX or can also be applied with other
platforms?
Yes, both of the software packages are available as separate products and
also be used in connection with other applications.
SNP selection software is an excellent tool, when you are launching any
kind of large-scale project.
Why should I prefer your PCR design
software?
The PCR design software includes
Genome TesterTM, a powerful module, which enables speedy
testing of PCR primers against the whole genome. The program helps to find
and eliminate primers with multiple binding sites (to avoid failed
PCR) and to choose alternative PCR products.
The programs are optimized for the human genome, but can also be used with
other completely sequenced genomes.
Routine
DNA analysis, genotyping software, detector, consumables
How much does the routine analysis of
DNA samples with the developed chip cost?
Please contact local
authorized distributor or Asper's customer services at info@asperbio.com
in order to get a quotation.
Before contacting, please read the checklist of the information required
to be able to give you a quote.
Can
I submit blood samples, fixed tissue samples to Asper for analysis?
We do not accept blood or any other tissue samples.
The preferred format of material for analysis is genomic DNA, but
we can also accept PCR products. PCR must be performed according to our protocol in order to
ensure incorporation of a small fraction of UTP into your PCR products for
fragmentation.
How should I send the DNA samples?
We prefer to get DNA in an
aqueous solution. We strongly recommend you also enclose the list
concentrations in your samples and a copy of your extraction protocol.
Before sending the DNA, please, find out the min. required DNA quantity.
May
I amplify genomic DNA in our lab and send the PCR products to Asper?
Basically yes, but due to the need for a small fraction of dUTP
incorporated in your PCR products for fragmentation purposes, please
contact us before the amplification and we will send you an appropriate
protocol.
However this might be your only option in case the law prohibits
transportation of genomic DNA out of the country.
How does Asper guarantee the quality
of the genotyping service?
1.
Asper Biotech services are certified to be in accordance with ISO
9001 quality standards by Bureau Veritas Quality International (BVQI)
auditors.
2.
The client information and the genotyping information related to
the client will be kept confidential.
3.
The genotyping process can be fully tracked and controlled from our
central electronic database.
4.
After the arrival of the DNA samples they will be labeled with
unique codes and saved in our central database.
5.
Genotyping procedure includes quality control criteria at every
step of the analysis.
How
do I receive the completed genotyping results from Asper?
We
send the analyzed genotyping data in digital format (for example in MS
Excel, but the other data formats are also available). Upon request and as
specified in the contract you can also receive some images or illustrative
material.
Can
I use the Genorama® QuattroImager for gene expression studies?
No. The detector is designed for 4-color APEX (Arrayed primer Extension)
based genotyping.
What
is the advantage of your detector compared with other microarray scanners?
It is not reasonable to compare our detector with other microarray
scanners (that are mostly designed for gene expression studies). Because
the detector has been devised for APEX based genotyping and is an
inseparable part of our genotyping platform and we do not sell it as
separate product. Therefore our competitors in this field are rather the
Pyrosecuencing' and Sequenom' instruments, than other microarray scanners.
Due to the application of TIRF (Total Internal Reflection) principle and
CCD camera it is ideal and economical tool for fast data acquisition and
gives you a smart solution for genotyping - low investment, easy to
handle, durable and high
throughput.
Is
the 10 micron resolution of Genorama® QuattroImager available?
Genorama QuattroImager has the
resolutions of 20 and 40 um, which are optimal for APEX-based genotyping.
Would
the 10 micron resolution option improve the APEX genotyping technology?
Genotyping by APEX method is
not a quantitative process, it is rather than yes or no approach.
Therefore the 10 um resolution, which is optional for quantitative
applications like gene expression profiling, is not needed for Asper´s
genotyping procedure. As here the analyzing speed is very important
and it is optimized to be fast, the pictures with 10 micron resolution are
much bigger and it makes analysis slow and heavy-handed.
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