Evaluation of sample preparation techniques for mass measurements of PCR products using ESI-FT-ICR mass spectrometry.
Elimination of PCR buffer components and alkali metal cations (i.e., Na+, K+) is of critical importance to allow for accurate mass measurements of PCR products for genotyping and sequencing applications. Ethanol precipitation followed by microdialysis has been repeatedly shown to efficiently desalt PCR products for analysis by mass spectrometry and is considered the gold standard. Alternative cleanup techniques that are compatible with automation are explored here with the intent of expanding the bottleneck that exists between the production of PCR products and analysis by electrospray ionization mass spectrometry (ESI-MS). Numerous combinations of approaches were evaluated that included PCR purification kits and alcohol precipitations. The data shown here support alternative approaches to an ethanol precipitation followed by microdialysis that have comparable desalting efficiency and can be utilized for cleanup of PCR products generated from single reactions.
Null
Allison P
AP
Department of Chemistry, Virginia Commonwealth University, Richmond 23284, USA.
George
Laura T
LT
Muddiman
David C
DC
D019840:2-Propanol / Q000737:chemistry / Q000302:isolation & purification; D004247:DNA / Q000737:chemistry*; D000431:Ethanol / Q000737:chemistry / Q000302:isolation & purification; D007202:Indicators and Reagents; D017551:Microdialysis; D016133:Polymerase Chain Reaction / Q000379:methods*; D012997:Solvents; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D007202:Indicators and Reagents; D012997:Solvents; D000431:Ethanol; D004247:DNA; D019840:2-Propanol