Retrieval of DNA using soft-landing after mass analysis by ESI-FTICR for enzymatic manipulation [11]

Journal of the American Chemical Society

Volumn 121, Issue 38, 1999, Pages 8961-8962

  Feng, Bingbing ^a   Wunschel, David S ^a   Masselon, Christophe D ^a   Pasa Tolic, Ljiljana ^a   Smith, Richard D ^a  

^a PACIFIC NORTHWEST NATIONAL LABORATORY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

BIOPOLYMER; DOUBLE STRANDED DNA;

CYCLOTRON; DNA DETERMINATION; DNA HYBRIDIZATION; DNA MODIFICATION; DNA TEMPLATE; GENE AMPLIFICATION; INFRARED SPECTROSCOPY; LETTER; MASS SPECTROMETRY; POLYMERASE CHAIN REACTION;

EID: 0033615345     PISSN: 00027863     EISSN: None     Source Type: Journal    
DOI: 10.1021/ja9923888     Document Type: Letter

References (35)

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32. 15 were soft-landed at low energy on the membrane. An electrically biased metal grid in front of the membrane prevented unselected species from reaching the surface during ion injection and trapping.
33. The sequence of the soft-landed single strand DNA is: 5′-ACATCT-TACACATCACCAC TTAAACTGGAATCTTCCCATACATTCAATCC-3′ while the other synthetic DNA in the ESI solution has a G instead of C (at the underlined position). Both DNA and related primers were synthesized in the University of South Carolina oligonucleotide synthesis facility. PCR were carried out using synthetic and commercial reagents (95 °C, 30 s; 46 °C, 30 s; 72 °C, 30 s for 10 cycles, followed by 95 °C, 30 s; 48 °C, 30 s; 72 °C, 30 s for 30 cycles). PCR products were purified and desalted by ethanol precipitation, microdialyzed, and concentrated before analysis by ESI-FTICR.
34. The amount of one attomole DNA soft-landed was estimated based on the visual relative "spot" intensity on gel electrophoresis (compared to controlled amplification, e.g. Figure 1B). The FTICR cell typically has a charge capacity of 1 million. After 300 cycles of ion injection, manipulation, and soft-landing, tens of millions of ions may be ejected onto the membrane. The cell charge capacity sets an upper limit for the number of soft-landed ions (tens of attomoles) and the experimental results from gel electrophoresis after PCR amplification indicated a maximum of a few percent of ions survived the entire process.
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