Oligonucleotide synthesis is a complex process that requires more than one hundred sequential chemical reactions to make a single, 25-base sequence. Contemporary synthesis chemistry is robust and modern synthesis platforms are reliable and highly automated. Still, each oligonucleotide synthesized at IDT is evaluated for quality before shipping to ensure that the correct sequence was made. The best method available to assess compound identity in a high throughput environment is mass spectrometry (MS).
|IDT Oligo MALDI-TOF Analysis
|Figure 1. Routine MALDI-TOF mass spectrogram of a 17mer oligonucleotide.
What is mass spectrometry?
In MS analysis, a small amount of a synthesized oligonucleotide is ionized and the ions are propelled into a mass detector/analyzer where molecular weight is measured. The analysis compares the calculated molecular weight of the given sequence to the measured molecular weight. Two methods of mass spectrometry are routinely used. These are MALDI−TOF (matrix-assisted laser desorption ionization−time of flight) and ESI (electrospray ionization) MS. (Read more about these MS methods in the Technical report, Mass spectrometry analysis of oligonucleotide syntheses).
Interpreting MS results
MALDI-TOF (Figure 1) and ESI (Figure 2) MS results both have a main peak representing the synthesized oligonucleotide. MS analysis at IDT can detect deletions, additions, or substitutions. IDT will remake oligonucleotides with a significant amount of these products, or with significant secondary peaks. However, there can be, and often are, additional peaks present in the final traces. During MALDI-TOF analysis, depurination of the oligonucleotide can occur as a result of heating (laser ionization) in an acidic environment (the matrix). Depurination also can occur during ESI analysis because of heating in the transport region of the ESI instrument.
|IDT Oligo ESI Analysis
|Figure 2. Routine ESI mass spectrogram of a 20mer oligonucleotide.
Depurination can create secondary peaks having approximately 135 (dA) or 151 (dG) mass units less than the major peak. These species are created by the process as the sample is measured, but are not present in the product itself.
In addition, synthetic oligonucleotides made using phosphoramidite chemistries employ protecting groups on the primary amines in dA, dC, and dG phosphoramidites in order to prevent branching and other undesired side reactions during chain elongation. Protecting groups are cleaved off post-synthetically during the final steps. Incomplete removal of these side groups results in additional masses that are easily detected by MS.
Finally, oligonucleotide modifications add mass to the product. Modifications commonly used in oligonucleotides are well characterized and their masses are taken into account in the final mass spectrograms produced. A list of mass contributions for the most often requested modifications of DNA and RNA oligonucleotides are listed on the IDT Modifications page. Further, the anhydrous molecular weight of both unmodified and modified oligonucleotides can be calculated using OligoAnalyzer Tool.
MS data available for IDT orders
IDT offers ESI MS QC free of charge on all standard oligonucleotides. This QC data is available online for oligonucleotides that were ordered on the website once they have shipped.
To view the data, go to My Account > Order History and find the order for which you would like to see the data. Click on the zip file located under the QC/COA heading for the order you want to see. This file will contain all of the trace files for your order.
Product focus—Oligos, modifications, dsDNA fragments
Custom oligonucleotides and primers
You can order up to 1 µmol desalted, custom synthesized DNA oligonucleotides and they will be shipped to you the next business day (larger scales are shipped within 5 business days). You can also specify whether to receive them dried down or hydrated, and whether you want them already annealed. Every IDT oligonucleotide you order is deprotected and desalted to remove small molecule impurities. Your oligos are quantified twice by UV spectrophotometry to provide an accurate measure of yield. Standard oligos are also assessed by mass spectrometry for quality you can count on.
Learn more or order now.
Review a list of the common modifications IDT can add to oligonucleotides here. Not finding a modification you need on the IDT website? IDT will consider any modification you need. Just send your request to firstname.lastname@example.org.
Custom dsDNA Fragments
Rather than annealing oligonucleotides to obtain dsDNA fragments, when your fragment size is 125 bp or longer, it might make more sense to order gBlocks® Gene Fragments. gBlocks Gene Fragments are double-stranded, sequence-verified, DNA genomic blocks, 125–3000 bp in length, that can be shipped in 2–5 working days for affordable and easy gene construction or modification. These dsDNA fragments have been used in a wide range of applications including CRISPR-mediated genome editing, antibody research, codon optimization, mutagenesis, and aptamer expression. They can also be used for generating qPCR standards.
Learn more about gBlocks Gene Fragments at www.idtdna.com/gblocks.
ESI mass spectrometry—why we use it for oligonucleotide quality control—IDT was, and still is, a pioneer in using mass spectrometry for quality control in oligonucleotide synthesis. Learn about why a particular method, electrospray ionization (ESI), is used ubiquitously in our manufacturing processes.
Determining the physical characteristics of your oligos—the OligoAnalyzer Tool—Use this free web tool to determine many of the physical characteristics of your oligonucleotides. By simply inputting your sequence, you can find out its length, GC content, melting temperature range, molecular weight, extinction coefficient, and optical density.
Oligo synthesis: Why IDT leads the oligo industry—Read about the phosphoramidite method of oligonucleotide synthesis that IDT uses in its manufacturing processes. We also highlight the additional measures we take to ensure our customers receive the highest quality oligos and nucleic acid products in the shortest time possible.
Understanding melting temperature (Tm)—Read this advice from our own thermodynamics specialist, Dr Richard Owczarzy, on the effects of melting temperature (Tm) on hybridization. He provides considerations for better oligo and PCR/qPCR assay design, including oligo concentration, salt, and base pairing mismatch positioning.
Review other DECODED Online newsletter articles on oligo handling and analysis, and oligo modifications.
You can also browse our DECODED Online newsletter for additional application reviews, lab tips, and citation summaries to facilitate your research.
Author: Jaime Sabel is a scientific writer at IDT.
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