Custom DNA Oligos – QC Analysis by Mass Spectrometry

Mass Spectrometry is the technology of choice for analyzing oligonucleotide synthesis. It enables the most sensitive detection of low levels of by-products, which can affect performance such as:

  • Incomplete coupling (N-1)
  • Depurination
  • Side-chain protecting groups
  • Acrylonitrile adducts
  • High salt content identification

Every oligonucleotide is thoroughly characterized by mass spectrometry, ensuring absolute quality.

We employ two different types of Mass Spectrometry (MS) instruments to accomplish our 100% commitment of quality control. Each instrument type produces a determination of the oligonucleotide's composition through direct molecular weight measurement. While MALDI-TOF instruments typically have a higher throughput, the length limitation and lower performance for modified oligonucleotides limits the type of oligonucleotide which can be analyzed using this technique. As a natural complement to MALDI-TOF, Electrospray Ionization (ESI) MS provides the ability to analyze longer oligonucleotides as well as those oligonucleotides containing photo-labile modifications which can be problematic for MALDI-TOF analysis.

MALDI-TOF (Matrix-Assisted Laser Desorption Ionization, Time-of-Flight)
MALDI-TOF MS uses laser light in conjunction with a chemical matrix to impart a charge to the sample (ionization) in question and then accelerates the charged ions through a flight tube to the detector, which measures particle counts as a function of time. The time-of-flight (TOF) is directly proportional to the mass of the molecule and has mass resolution of approximately 0.03%, i.e., resolution of +/- 3 Da on a 10 kDa oligonucleotide.

MALDI-TOF MS is ideally suited for primers and high throughput requirements, when speed is essential. Though a powerful and efficient technology to characterize oligonucleotides of lengths below 50 bases, MALDI-TOF MS has the disadvantage that the ionization efficiency, and therefore the resolution of the procedure, decreases rapidly for samples greater than 50 bases (>13 kDa). Furthermore, the laser source used to generate the charge could be detrimental to the analysis of modified oligonucleotides that are photosensitive. ESI-MS can be utilized to bypass these limitations.

ESI (Electrospray Ionization)
Applications requiring long oligonucleotides (>50 bases) e.g., microarray applications, cloning and/or gene synthesis, have increased the need for instrumentation that can accurately characterize these molecules. The method of choice for these oligonucleotides is ESI-MS. The target molecules are ionized into multiple charge states producing a waveform that can be de-convoluted into parent peaks. As only the charge state will vary for the ions, oligonucleotides with high molecular weights can be analyzed using this method. Additionally, the inherently milder ionization conditions make this analytical technique a great tool for the analysis of labile compounds such as common quenchers, e.g., dabcyl, BHQ's, used in dual-labeled fluorogenic probes. The ESI-MS systems have mass resolution of approximately 0.03%, i.e., resolution of +/- 3 Da on a 10 kDa oligonucleotide.



 

Benefit Summary of MALDI-TOF vs. ESI

 

Criteria MALDI-TOF ESI
<50 bases + +
>50 bases - ++
Photosensitive Modified Oligonucleotides - +
Degenerate (wobble) Oligonucleotides - +
Throughput +++ +
N-1 Detection + +
Incomplete deprotection + +
Depurination + +
Mass accuracy + ++