Polylysine-coated diamond nanocrystals for MALDI-TOF mass analysis of DNA oligonucleotides.
A protocol based on aminated diamond nanocrystals has been developed to isolate, concentrate, purify, and digest DNA oligonucleotides in one microcentrifuge tube for matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry. It is shown that use of diamond nanocrystals as a solid-phase extraction support not only permits concentration of oligonucleotides in highly diluted solutions but also facilitates separation of oligonucleotides from proteins in heavily contaminated solutions. Enzymatic digestions can be conducted on particle, and additionally, the digests can be easily recovered from the solution for base sequencing. In this method, the aminated diamond nanocrystals ( approximately 100 nm in diameter) were prepared by noncovalent coating of carboxylated/oxidized diamonds with poly(L-lysines) (PL), which form stable complexes with DNA oligonucleotides. While the complexes are sufficiently stable to sustain repeated washing with deionized water, the DNA molecules can be readily eluted after incubation of the diamond adducts in aqueous ammonium hydroxide at elevated temperatures. No preseparation of PL or diamond nanocrystals is required for subsequent MALDI-TOF mass analysis.
Kong
Xianglei
X
Institute of Atomic and Molecular Sciences, Academia Sinica, P.O. Box 23-166, Taipei, Taiwan 106.
Huang
L C Lora
LC
Liau
S-C Vivian
SC
Han
Chau-Chung
CC
Chang
Huan-Cheng
HC
D000327:Adsorption; D019921:Biotinylation; D004247:DNA / Q000032:analysis* / Q000737:chemistry; D018130:Diamond / Q000737:chemistry*; D053758:Nanoparticles / Q000737:chemistry*; D009841:Oligonucleotides / Q000032:analysis* / Q000737:chemistry; D011107:Polylysine / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation* / Q000379:methods*
D009841:Oligonucleotides; D011107:Polylysine; D018130:Diamond; D004247:DNA