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Identify single-cell mutationsand determine mutation
frequency.
CRISPR/Cas9 Gene EditingFragment Analyzer™ Automated CE
System
Mutationanalysisbycapillaryelectrophoresisprovidessignificantbenefitsoverslabgelmethods.TheFragment
Analyzer detects mutations generated from Non Homologous End
Joining (NHEJ) and
HomologyDirectedRepair(HDR)modificationstotargetedgenesequences.Asamultichannelcapillary
electrophoresis instrument that uses performance tested
non-denaturing Gel Kits coupled with a sensitive intercalating dye,
the Fragment Analyzerautomatesworkflow,reduceshandsontime and
decreases time to results while providing high detection
sensitivity and separation resolution. PROSize® integrated
analytical software package accelerates detection of mutation
eventsbyefficientlydisplayingresults.
Forgeneeditingexperiments,theCRISPR-specificGelKitwillshowthemutationfrequencyofapooledcell
population, determine the presence/absence of a mutation from a
single cell compared to its wild type complement, and under certain
circumstances, assist in calling the zygosity of alleles.
Additionally,
researcherscandeterminethequalityandquantityofisolatedgenomicDNApriortoamplification.
They can measure amplicon post-PCR to assist in combining
equimolar amounts of pre-heteroduplexed wild type and experimental
amplicon.
The Fragment Analyzer can be used in multiple ways related to
CRISPR/Cas9 mutagenesis including single guide RNA (sgRNA)
forcleavageefficiency,analyzingmutationeventsincelllinesorpools of
individual cells, plant cells and microbes. Furthermore, time
course studies or changes to cell lines during cell selective
passage can be monitored.
ADVANCED ANALYTICAL > WWW.AATI-US.COM FRAGMENT ANALYZER™
Automated CE System
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Instrument Capabilities
ADVANCED ANALYTICAL > WWW.AATI-US.COM FRAGMENT ANALYZER™
Automated CE System
FRAGMENT ANALYZER WORKFLOW BENEFITS
Fragment Analyzer™ Automated CE System
Eliminate Slab Gel Usage
With its ease of use, ability to hold multiple 96 well sample
plates, fast electrophoresis times, sensitivity and versatility,
the Fragment Analyzer is the ideal instrument to analyze
CRISPR/Cas9 mutated cells and cell lines.
Ease of Use
Simply “load and go”. With a long reagent shelf-life and room
temperature stable reagents, prepared gel can be held worry-free on
the instrument and pumped into the capillaries as needed. The long
lasting capillary array does NOT require daily handling or
maintenance and can be used for thousands of injections. Variable
capillary array length (22cm, 33cm and 55cm) and number of
capillaries (12, 48 or 96) provide the
ultimateflexibilityinexperimentdesign.
Sample LoadingHolding up to 3 x 96 well plates, sample plates
can be queued to run while new plates are loaded into the
instrument as soon as samples have been injected. And with a single
addition of a diluent post enzyme cleavage of the heteroduplexed
amplicon, sample processing requires minimal effort and can be
automated if desired.
Duetotheminimumuptakeofsample,thesamesamplecanbeinjectedmultipletimesifwarrantedforconfirmationor
reproducibility studies.
Time to Results
Withanautomatedworkflow,loadedsampleswillrununattendeduntilcomplete.Availableelectrophoresismethodsare
designed for optimum resolution of fragments with some runs as
short as 20 minutes. Electrophoresis voltage can be adjusted by the
user to decrease total electrophoresis time as conditions and
resolution requirements demand.
Sensitivity
Cleaved fragments can be detected down to 5pg/µL under the right
experimental conditions. High sensitivity allows
forthepotentialtoreducethenumberofamplificationcyclesand/orreducetheamountofcleavageenzymeused.
Versatility
WithmanyGelKitstochoosefrom,userscanqualifyorquantifyalltypesofDNAfragments.Specifically,GelKitsareavailabletoquantifyandqualifygenomicDNApriortoamplification.Postamplification,ampliconcanbesizedandthe
concentration determined for correct pooling prior to heteroduplex
formation and enzyme cleavage.
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ADVANCED ANALYTICAL > WWW.AATI-US.COM FRAGMENT ANALYZER™
Automated CE System
DeterminingwhichcellshavebeenmodifiedbytheCRISPR/Cas9machineryisacriticalfirststeppostmutagenesis.Slabgelbased
methods, which can lack the sensitivity and resolution needed to
see all mutation types as well as automation for large scale
screening and analysis, also typically lack an integrated software
analysis package.
PROSize®, the analytical software package within the Fragment
Analyzer system seamlessly calculates fragment sizes, fragment
concentration and fragment molarity. An additional software plugin
specific for CRISPR/Cas9 mutation analysis shows the mutation
frequency of pooled samples and zygosity of individual samples
(diploid analysis).
DATA ANALYSIS SOFTWARE
Mutation Frequency Calculator
Users can input the expected uncut amplicon size and
endonuclease cleaved fragment sizes either manually for each
capillary or quickly through a CSV upload. The expected fragments
are automatically compared to the observed fragment sizes and
thencolorcodedforeasyidentification.Thepercentcleavageiscalculatedfromthemolarityofidentifiedfragmentsbasedonthe
following formulation:
% cleavage =
CSV Upload of expected uncut and cut fragment sizes. The % error
can be adjusted as needed to aid in fragment identification.
Average Molarity (fragment #1 and fragment #2)
Average Molarity (fragment #1 and fragment #2) + Molarity
fragment uncut
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DATA OUTPUT
Fragment Analyzer™ Automated CE System
ADVANCED ANALYTICAL > WWW.AATI-US.COM FRAGMENT ANALYZER™
Automated CE System
Result data can be quickly exported into either PDF or CSV
format. The CSV output, as shown below, provides immediate access
to the pertinent information regarding the samples and can be used
for further analysis when necessary. The PDF output provides a page
by page report for each sample. All or some of the sample data can
be exported in either report.
Data output showing the input expected sizes and the observed
sizes. Full length and cut fragments are color coded for easy
identification. The % cleavage (highlighted in yellow) is shown for
each sample.
WellA1A2A3A4A5A6A7A8A9
A10A11A12
Name420054200642007420084200942010420114201242013420144201542016
(bp)125012501250125012501250125012501250125012501250
(bp)230230230
000
349350350437440441
(bp)100010001000
000
842837844715708712
(bp)127812881278125912781278127812781269124012591249
(bp)230230231
000
349350351437438437
(bp)994994994
000
842845845715719719
nmole/L27.6127.6231.9
64.7852.4650.5842.8450.4653.1417.0714.5617.89
nmole/L27.8525.2132.45
000
7.9510.5111.1350.3
41.1244.24
nmole/L24.7323.4729.21
000
8.5710.6210.8539.1533.3435.96
% Cleavage48.7846.8549.15NaNNaNNaN
16.1717.3117.1472.3871.8969.15
Estimated FullLength
EstimatedFragment 1
A B C D E F G H I J K L
123456789
10111213
Sample
EstimatedFragment 2
Measured Full Length
Measured Fragment 1
Measured Fragment 2
Full Length
Fragment 1
Fragment 2
CSV output of data. Expected and measured fragment size is shown
as well as the molarity of the fragments. The % cleavage
(highlighted in yellow) is calculated as described above.
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CRISPR / MUTATION DISCOVERY KITS
ADVANCED ANALYTICAL > WWW.AATI-US.COM FRAGMENT ANALYZER™
Automated CE System
The Gel Kit listed below has been optimized for detection of
point mutations and other small mutations post heteroduplexed
amplicon cleavage. The Gel Kit is available in a 1,000 sample kit
size.
CRISPR Discovery Gel Kit, 1 – 6,000 bp, DNF-910-1000CP
a. Markers at 1bp and 6,000 bp; the fast running lower marker
eliminates problems with primer dimers b. Input concentration range
5 ng/µL – 100 ng/µL c. Gel formulation suited for a wide range of
fragments
OTHER AVAILABLE GEL KITS
Genomic DNA Analysis
Standard Sensitivity Genomic DNA Analysis Kit, DNF-487
a. Sizing range: 50 bp – 20,000 bp b. Input concentration range
5 ng/µL – 200 ng/µL
High Sensitivity Genomic DNA Analysis Kit, DNF-488
a. Sizing range: 50 bp – 40,000 bp b. Input concentration range
50 pg/µL – 5000 pg/µL
DNA / NGS Fragment Analysis
Standard Sensitivity Genomic NGS Fragment Analysis Kit,
DNF-473
a. Sizing range: 25 bp – 5,000 bp b. Input concentration range 5
ng/µL – 100 ng/µL
High Sensitivity Genomic NGS Fragment Analysis Kit, DNF-474
a. Sizing range: 25 bp – 5,000 bp b. Input concentration range
50 pg/µL – 5000 pg/µL
Fragment Analyzer™ Automated CE System
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Maximum Sample Throughput: Array dependent, either 12, 48 or 96
samples
Maximum Unattended Sample Capacity: Up to 288 samples
Minimum Sample Volume: 20 µL of liquid for injection; as little
as 1 µL of actual sample required
Qualitative Kits: 35-500 bp; 35-1,500 bp; 35-5,000 bp; 75-15,000
bp; 75-20,000 bp
Quantitative Kits: NGS Fragment Analysis, Genomic DNA, Total
RNA, mRNA, microRNA and TILLiNG
Resolution: Gel dependent; to as low as 2 bp
Sizing Accuracy: Typically 5% or better
Detection Sensitivity: As low as 5 pg/µL for fragments and 50
pg/µL for a smear
Light Source: 700 mA, 10 W LED, 470 nm excitation wavelength
Detector: High sensitivity CCD; 500-600 nm emission
wavelength
Software: Fragment Analyzer instrument control, PROSize® 2.0
data analysis
Data Export Format:
CSV,PDF,flexiblenumericalorbinaryoutputoptions
Environmental Conditions: Indoor use, normal laboratory
environment 20-23°C
Relative Humidity Range: < 80% (non-condensing)
Electrical:
100-200VAC;50-60Hz;15A(alternateconfigurationsavailable)
Instrument Dimensions:
Fullyconfigured61cmHx38cmWx51cmD(24x14x20in)
Instrument Weight: 38.5 Kg (85 lbs)
Automating genomic discovery
Advanced Analytical Technologies, Inc.
2450 SE Oak Tree Ct.Ankeny, IA 50021 USAPhone:
+1-515-964-8500Fax: +1-515-964-7377E-mail:
[email protected]
Advanced Analytical Technologies, GmbH.
Im Neuenheimer Feld 583D-69120 HeidelbergPhone: ++49 6221
868058-20Fax: ++49 6221 868058-99E-mail:
[email protected]
Copyright © 2015 Advanced Analytical Technologies, Inc. All
Rights Reserved. Reproduction, adaptation or translation without
prior written permission is prohibited, except as allowed under
copyright laws. Fragment Analyzer™ Automated CE System is a
trademark of Advanced Analytical Technologies, Inc. PROSize® is a
registered trademark of Advanced Analytical Technologies, Inc.
ADVANCED ANALYTICAL > WWW.AATI-US.COM FRAGMENT ANALYZER™
Automated CE System
Fragment Analyzer™ Automated CE System
NUCLEIC ACIDS DETECTED BY THE FRAGMENT ANALYZER
PCR amplicons, restriction enzyme digests, SSR/microsatellite
fragments, NGS library preparations from all platforms, total RNA,
messenger RNA (mRNA), microRNA, small RNAs, high molecular weight
DNA, genomic DNA, plasmids (supercoiled & linear), point
mutations and indels (TILLiNG)
SPECIFICATIONS
Identify single-cell mutationsand determine mutation
frequency.