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Rapid and multiplex preparation of engineered Mycobacterium smegmatis porin A (MspA) nanopores for single molecule sensing and sequencing

Chem Sci. 2021-06; 
Shuanghong Yan , Liying Wang , Xiaoyu Du , Shanyu Zhang , Sha Wang , Jiao Cao , Jinyue Zhang , Wendong Jia , Yuqin Wang , Panke Zhang , Hong-Yuan Chen , Shuo Huang
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Custom DNA/RNA Oligos DNA oligonucleotides (Table S1) were purchased from Genscript (New Jersey, USA). The Ni-charged magbeads (Cat. No. L00295,Genscript) were supplied in phosphate buffered saline (PBS), pH=7.4 containing 20% ethanol. Get A Quote
Proteins, Expression, Isolation and Analysis Get A Quote

摘要

Acknowledging its unique conical lumen structure, Mycobacterium smegmatis porin A (MspA) was the first type of nanopore that has successfully sequenced DNA. Recent developments of nanopore single molecule chemistry have also suggested MspA to be an optimum single molecule reactor. However, further investigations with this approach require heavy mutagenesis which is labor intensive and requires high end instruments for purifications. We here demonstrate an efficient and economic protocol which performs rapid and multiplex preparation of a variety of MspA mutants. The prepared MspA mutants were demonstrated in operations such as nanopore insertion, sequencing, optical single channel recording (oSCR), nanopore sin... More

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