Mar 17, 2014  The implications of oligonucleotide synthesis include implications to both RNA and DNA. Find out about the implications of oligonucleotide synthesis. Oligonucleotide sequences combinatorial synthesis and assembly fabrication of oligonucleotide array. Oligonucleotides are synthesized only from four different nucleotides (A, G, C, T), and, therefore, a lot of different oligonucleotide sequences can be synthesized by combinatorial chemistry at the same time.

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1. Unicorn For Oligonucleotide Synthesis User Manual 18-1134-71 5
2. Custom Oligonucleotide Synthesis

The DNA Synthesis Service uses the MerMade-192 synthesizer from BioAutomation. The MerMade has the capability to synthesize up to 192 oligos in a single run. A full run of 20-mers can be completed overnight. These oligos are suitable for a wide range of applications including PCR, dye terminator sequencing, cloning, microarray experiments, hybridization, reverse transcriptase PCR, and custom gene synthesis. Oligos up to 120 bases in length can been made with a coupling efficiency in excess of 98%. Oligos are synthesized in individual columns in a 96-well format. Cleavage and deprotection are done off instrument. Purification and other post processing steps are done robotically to ensure rapid, accurate and consistent processing of oligo orders. Most orders placed for 50-nmol oligos (up to 70 bases in length) will be available for pickup the next working day. Orders for 200-nmol scale, and for very long oligos, may take additional time.

Scale

Two synthesis scales are available, 50-nmol and 200-nmol. The 50-nmol scale is usually sufficient for clients using the oligos for sequencing or PCR. A single OD of an oligo is typically good for thousands of sequencing or PCR reactions (see expected yield information below). The 200-nmol scale is available for clients who need to run the same assay or reaction very frequently.

Please Note: Due to the small number of 200-nmol scale oligo orders, we will be processing all 200-nmol scale orders on Wednesdays.

Purification

All oligos synthesized at the facility are desalted with a Sephadex column to remove ammonium ions and deprotection by-products at no extra charge. Additional purification is available using Varian's TOP: Trityl On Oligonucleotide Purification System at an additional charge. This is sometimes referred to as cartridge, OPC or reverse phase purification. Oligos to be purified via the TOP method are synthesized with the 5' terminus protecting group (trityl or dimethoxyltrityl (DMT)) left on. The hydrophobic nature of the trityl group permits tighter retention of the desired full-length oligo than the truncated failure sequences that do not contain a trityl group. Failure sequences are washed from the tube with a low percent acetonitrile rinse. Retained oligos are detritylated on-column with trifluoroacetic acid to remove the acid-labile trityl group. Residual acid is rinsed from the column and the full-length oligo is recovered with an acetonitrile/water solution. Oligos purified in this way are greater than 90% pure full-length oligo.

Yield

Yield is affected by oligo base composition and length. In general you can expect the following yields for an average 20-mer:

Please note that for purified oligos, yield will decrease with increased oligo length. Oligos that do not meet minimum yield standards will be resynthesized at no additional charge. For particularly difficult oligos you may receive the product of several syntheses to achieve an acceptable final total yield.

Specialty Reagents

The facility provides inosine at no additional cost. Clients desiring fluorescent tags or other modifications should contact the facility to determine availability and cost. Most modifications that use standard phosphoramidite chemistry may be made on the MerMade. Please be aware that these reagents are usually packaged in such a way that a single bottle provides enough for several modifications. Once a bottle of specialty reagent is opened, it has a limited stability. Due to this, we must charge for an entire bottle of reagent no matter how many oligos are actually modified. For this reason, it may not be cost effective to label only a small number of oligos. It may be better to pool orders with other researchers or save up oligos until the bottle can be used efficiently. The client will be charged for any specialty reagents plus the usual per base charge.

Ordering Instructions

• All orders are placed through our OnCore synthesis system.
• Orders should be placed using all caps without spaces.
• Please use the following codes to order your oligos:

Standard Bases

Mixed Bases

Specialty Bases / Modifications ¹

^{(Others available on request. Please contact the facility)}

_{1 - The MerMade is limited to a total of 6 different specialty or different
mixed base positions in a single oligo or run
2 - Additional Reagent Charges
3 - TET is not supported for Genotyping on our newer Applied Biosystems instruments.}

Sequencing Primers

If you need custom primers synthesized for your sequencing job, the facility can make it fast and simple to coordinate your order and eliminate the need for multiple or delayed shipments of samples. When ordering custom primers for sequencing at our facility, you must place your sequencing order prior to submitting your primer synthesis order.

Please follow these steps when ordering custom primers for sequencing:

• Place your sequencing order using the Sequencing OnCore system prior to placing your primer synthesis order.
  • In the 'Comments' section of the Sequencing OnCore system, enter the phrase 'Primer Ordered' and note the primer annealing temperature if it is outside the range of 54-58°C.
  • Note the sequencing tracking number assigned by the system, you will need this tracking number when submitting your synthesis order.
• Place your primer synthesis order using the Synthesis OnCore system.
  • Check the 'Use for sequencing' box
  • Enter your sequencing tracking number in the box provided.
• Send your samples with your name and OnCore tracking number to the facility.
  • Please include a copy of your order form, or your name and OnCore tracking number, with all orders sent to the facility. The OnCore tracking number information is displayed on the last screen during the ordering process and can be printed and sent with your sample(s).
  • See our shipping page for suggestions on how to ship your sequencing samples to our facility.

Once your primer synthesis order is complete, our facility will automatically make a primer dilution and start your sequencing order. Your primer stock(s) will be available for pickup once your sequencing order is complete. The primer tracking number(s) are required for pickup your custom primer(s). Please pickup your custom primer stock(s) as soon as possible once they are done. If you do not pick up your primer stock(s), they will be kept at the facility for a limited period of time and then we will dry them down and mail them to the address on file in your profile.

To reconstitute a primer stock(s) that has been dried down after being used for sequencing:

1. Look up the synthesis reports in the OnCore Synthesis system (Track Oligo Synthesis) using the tracking numbers sent to you by email during the project. If you don't have the tracking numbers, leave the field blank and you will be presented with a list of jobs to choose from. You can also find the yield information printed on the stock tubes.
2. We used approximately 1% of then initial yield to make the dilution for your sequencing order.
3. Please use this information to dilute your dry primer stock to 5 pmol/µl (5 µM) for use as sequencing primer at the facility or any other concentration you wish for your own purposes.

For help with primer design, please see our 'Designing Custom Primers page. The facility also provides a primer design and primer walking service. Additional information can be found on our Primer Walking service page.

An Overview of Chemical DNA Synthesis

Unicorn For Oligonucleotide Synthesis User Manual 18-1134-71 5

DNA synthesis is a cyclical process that assembles a chain of nucleotides from the 3'-end to the 5'-end. The 3' nucleoside is covalently attached to a solid support and successive nucleotide monomers are added one by one through a cycle of four chemical reactions. In the first step, detritylation, the dimethoxytrityl (DMT) group is removed with trichloroacetic acid to free the 5' hydroxyl for the coupling reaction. The second step is coupling, in which the 5'-OH of the nucleotide reacts with an activated monomer created by simultaneously adding the phosphoramidite nucleoside monomer and tetrazole. The tetrazole protonates the nitrogen of the phosphoramidite, making it susceptible to nucleophilic attack. The third step, capping, terminates any chains that did not undergo coupling. Unreacted chains have a free 5'-OH and are capped by acetylation with acetic anhydride and 1-methylimidazole. Capping minimizes the length of impurities, making it easy to do post-synthesis trityl-selective purification of the final product. Finally, the internucleotide linkage is converted from a phosphite to the more stable phosphotriester by oxidation. After oxidation the DMT group is removed with trichloroacetic acid and the cycle is repeated until the chain elongation is complete. The protected oligo is cleaved from the support with concentrated ammonium hydroxide and methylamine and is ready for post-synthesis deprotection and purification.

Custom Oligonucleotide Synthesis

MerMade-192 Oligonucleotide Synthesizer

There are three main hardware components in the MerMade-192, the inner reaction chamber, the outer chamber, and the motion/injection system. The two 96-well plates are placed inside two separate vacuum chucks which are then placed into the inner chamber. This outer chamber is of sufficient size to accommodate the full motion of the X-Y table. The injection head is mounted on the top of the outer chamber lid. The X-Y table is controlled by the computer and moves the plates around under the injection head, positioning the wells to receive the reagents. The two chambers ensure the process takes place in a moisture-free and chemically-inert environment. During the course of synthesis, argon is continually introduced into the inner chamber and the small gap at the top of the chamber guarantees a constant flow of gas through the chamber.

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