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Cell Line Optimization

Electrophoresis for DNA/RNA Analysis MCE-202 MultiNA

Checking for Genome Editing Mutations benefits

by Heteroduplex Mobility Assay Cell Line Optimization
click here • Reduces the cost and time involved in analysis
• Enables fully automatic batch analysis of up to 108 samples
Operating Principle and Features Measurement Method and Result
• Achieves high sensitivity, high resolution, and high reproducibility
MultiNA is an automatic electrophoresis system that uses a microchip After the mutation has been induced in an individual, PCR is conducted
to measure the size of DNA or RNA. It automates all steps, such as for the area in the vicinity of the deletion/insertion. The PCR product is
creating the gel for agarose gel electrophoresis, applying the sample, denatured, then reannealed to form a heteroduplex product. Then, by Culture
electrophoresing, staining, detecting, and rinsing. MultiNA uses dedicated checking the migration pattern of the sample using the MultiNA, the
reagents, fluorescent dyes, and microchips to fully automate analysis and presence of short deletions can be verified by means of the structural
achieve quick, easy, and high-sensitivity electrophoresis (Fig. 1). change, which would be difficult to determine solely by comparing
differences in chain length (Fig. 2).
Application Sample① (wild type: homo) Sample② (variant: homo) Sample③ (wild type + variant: hetero)
PCR product (no deletion) PCR product (deletion present) PCR product (no deletion)
When Transcription Activator-Like Effector Nuclease (TALEN) or a CRISPR/ PCR product (deletion present)
Cas system is used to break a genome at any particular point in a
Denatured
Denatured
Denatured
sequence, the cell will repair the double-stranded DNA break. Reannealed Reannealed Reannealed
Genome editing is a technology that uses the repair errors that occur Purification
during repairing to modify genomes by inserting or deleting code in the Sample① ⇒ No structural Sample② ⇒ No structural Sample③ ⇒ Structural change present
change
change
Reannealed product (4 fragments)
original sequence. Reannealed product Reannealed product
(deletion present)
(no deletion)
One technique used to verify whether the genome editing process
successfully introduced the intended genetic modification is the Wild type /deletion heteroduplex occurring
in addition to the original structure
heteroduplex mobility assay (HMA). It uses electrophoresis mobility to Forcing a heteroduplex structure to be taken results in a disparity
in mobility, facilitating separation.
discriminate between homoduplex and heteroduplex DNA, which have Heteroduplex separated and detected Specifications
different steric structures. using microchip electrophoresis.
Instrument MCE-202 MultiNA
Heteroduplex band
Wild type: homo band Sample rack Compatible with 96-well PCR plate (An aluminum sheet can be applied to prevent sample
evaporation.) and 12/8-strip PCR tube (Shimadzu recommended product)
Variant (deletion): homo band Characterization
Microchip Quartz, 23 mm separation channel length, on-chip electrodes (insert up to four microchips)
Example of analysis results from analysis of mobility of heteroduplex
(+): Wild type homozygote Pretreatment Automatic sample injection, automatic separation buffer replenishing, automatic chip cleaning
(-): Variant (8 bp deleted) homozygote
(±): Heterozygote (wild type + variant)
Fig. 2 HMA Principles and Analysis Procedures Electrophoresis voltage Max. rated voltage: 1.5 kV, max. current: 250 μA
Detection method LED-excited fluorescence detector (470 nm excitation wavelength) <Class 1 LED product>
Sample Well Display Loaded samples Up to 108 samples
Displays the analysis progress status in Separation size range 25 to 500 bp (DNA-500 Kit)
different colors. (reagent kits dedicated for 100 to 1000 bp (DNA-1000 Kit)
Gel Image MultiNA) 100 to 2500 bp (DNA-2500 Kit) Quality Control
Can be saved as image data ( jpg, bmp, tif ). 100 to 12000 bp (DNA-12000 Kit)
Up to 28S rRNA (5.0 knt) (RNA Kit)
Microchip rinsing Chip rinsing kit RA
Peak Table
Predicted size values and concentrations Sample volume 5 µL
can be saved to a csv file. Quantitation range DNA analysis: 0.5 to 50 ng/μL (at 10 mM Tris-HCI, containing 50 mM KCl and 1.5 mM MgCl2)
RNA analysis: 25 to 500 ng/μL (total RNA), 25 to 250 ng/μL (mRNA) (10 mM Tris-HCI buffer,
Electropherogram containing 1 mM EDTA)
Can be saved as image data ( jpg, bmp, tif ).
External dimensions W 415 mm × D 545 mm × H 508 mm
Weight 43 kg Pharmacokinetics
Power supply 100 to 120 V, 220 to 240 (CE Marking) 300 VA max.
Controller Creating analysis schedules, real-time control, automatic analysis pretreatment, automatic analysis
Fig. 1 Displaying Analysis Results in the MultiNA Viewer
post-treatment, automatic error processing, analysis log management, analysis performance checks
Conclusion Application Examples
Data processing Batch display/detailed display of gel images/pherograms, automatic quantitation and size prediction
by size markers, data searching, data import/export, manual editing and re-analysis
The MultiNA automatic analysis platform solves previous shortcomings • Verify mutations created by genome editing Changes in average size and concentration with respect to smear samples (during smear analysis)
of agarose electrophoresis. It provides an easy way to check the • Check libraries of next-generation sequencers Others
presence and size of DNA/RNA with good reproducibility. • Genotyping or detecting microorganisms or viruses Reports Multilevel data display, tree display of samples/files, RNA structural comparison, analysis performance
check results, analysis log
This article was prepared with help from Assistant Professor Masato Kinoshita of the Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University. Note: MCE-202 MutiNA is currently not available in US, EU and UK.
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