scholarly journals Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format

10.3791/55586 ◽  
2017 ◽  
Author(s):  
Muhammad Khairul Ramlee ◽  
Jing Wang ◽  
Alice M. S. Cheung ◽  
Shang Li
Keyword(s):  
High Throughput ◽  
Gel Electrophoresis ◽  
Capillary Gel Electrophoresis ◽  
Knockout Mutants ◽  
Fluorescent Pcr ◽  
Electrophoresis Technique

scholarly journals High-throughput genotyping of CRISPR/Cas9-mediated mutants using fluorescent PCR-capillary gel electrophoresis

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Muhammad Khairul Ramlee ◽  
Tingdong Yan ◽  
Alice M. S. Cheung ◽  
Charles T. H. Chuah ◽  
Shang Li
Keyword(s):  
High Throughput ◽  
Gel Electrophoresis ◽  
Capillary Gel Electrophoresis ◽  
Fluorescent Pcr

scholarly journals Tracing CRISPR/Cas12a Mediated Genome Editing Events in Apple Using High-Throughput Genotyping by PCR Capillary Gel Electrophoresis

2021 ◽  
Vol 22 (22) ◽  
pp. 12611
Author(s):  
Susan Schröpfer ◽  
Henryk Flachowsky
Keyword(s):  
Genome Editing ◽  
High Throughput ◽  
Gel Electrophoresis ◽  
Cost Effective ◽  
Capillary Gel Electrophoresis ◽  
Guide Rnas ◽  
Large Deletions ◽  
Multiple Loci ◽  
Fluorescent Pcr

The use of the novel CRISPR/Cas12a system is advantageous, as it expands the possibilities for genome editing (GE) applications due to its different features compared to the commonly used CRISPR/Cas9 system. In this work, the CRISPR/Cas12a system was applied for the first time to apple to investigate its general usability for GE applications. Efficient guide RNAs targeting different exons of the endogenous reporter gene MdPDS, whose disruption leads to the albino phenotype, were pre-selected by in vitro cleavage assays. A construct was transferred to apple encoding for a CRISPR/Cas12a system that simultaneously targets two loci in MdPDS. Using fluorescent PCR capillary electrophoresis and amplicon deep sequencing, all identified GE events of regenerated albino shoots were characterized as deletions. Large deletions between the two neighboring target sites were not observed. Furthermore, a chimeric composition of regenerates and shoots that exhibited multiple GE events was observed frequently. By comparing both analytical methods, it was shown that fluorescent PCR capillary gel electrophoresis is a sensitive high-throughput genotyping method that allows accurate predictions of the size and proportion of indel mutations for multiple loci simultaneously. Especially for species exhibiting high frequencies of chimerism, it can be recommended as a cost-effective method for efficient selection of homohistont GE lines.

scholarly journals High-throughput glycosylation analysis of therapeutic immunoglobulin G by capillary gel electrophoresis using a DNA analyzer

mAbs ◽  
2013 ◽  
Vol 6 (1) ◽  
pp. 185-196 ◽  
Author(s):  
Dietmar Reusch ◽  
Markus Haberger ◽  
Tobias Kailich ◽  
Anna-Katharina Heidenreich ◽  
Michael Kampe ◽  
...  
Keyword(s):  
High Throughput ◽  
Immunoglobulin G ◽  
Gel Electrophoresis ◽  
Capillary Gel Electrophoresis
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