We synthesize "degenerated" oligonucleotides with equimolar base mixes at no additional charge. These are DNA products in which one (or more, or all) positions is (are) coded not with a single character (G or A or T or C) , but with another English (Latin) alphabet character meaning a combination of two, three or all four bases. These special characters comply with the international nomenclature. For non-equimolar mixes or equimolar manual mixes ("doped" oligonucleotides, produced by installing additional reagent bottles with manually mixed bases) additional charges apply according to the prices posted here. When the degenerated oligos are used as custom primers for DNA sequencing, the level of degeneracy should not be very high, to avoid mispriming.
Oligonucleotides with modified bases
There are numerous possibilities for modification of some or all of the bases in an oligonucleotide: bases could be coupled with a fluorescent dye (e.g. fluorescein), psoralen (for cross linking), cholesterol (for easy penetration into living cells), 5'-phosphate group (to facilitate cloning), the phosphate backbone of the oligonucleotide could be partially or completely replaced by a phosphorothioate backbone (for the "antisense" technology), etc. We do synthesize all modified oligonucleotides, for which reagents are freely available on the market, and some prices are included here . Please note that the price for most of these modifications could be substantially reduced if you have more than one modified base (or dye) from the same type to order, please inquire. We may need some additional time (usually 24 h) before we can start the synthesis, because we do not stock on some of these expensive and rare chemicals.
Oligonucleotide purification - desalting, OPC, PAGE, HPLC
Standard purity oligos are desalted at no extra cost. Optional alternative methods of purification include OPC (oligonucleotide purification cartridge, $15 per oligo), HPLC ($25 per oligo for the 100 nmole scale, $35 per oligo for the 200 nmole scale) and PAGE ($35 for the 100 nmole scale, $50 for the 200 nmole scale); prices for other scales are available on request.
Longer than 35-mer oligonucleotides. Polyacrylamide gel electrophoresis (PAGE) purification, HPLC (high performance liquid chromatography) purification
Let's assume that the efficiency of DNA synthesis is 99%. With the addition of each consecutive base, the proportion of the "aborted" oligonucleotides increases and at 40 bases the final reaction will contain 67% "true" oligos and 33% shorter products. At 100 cycles only 36% of the products will be of the correct sequence. Therefore, the synthesis of long oligos necessitates purification by PAGE, HPLC or OPC. For oligos longer than 50 bases, PAGE gives better results than HPLC.
We offer OPC, HPLC or PAGE purification for the long oligos (see the above text for prices). In addition to this fee, we require an extra 24 hours to complete the purification. Please note that even PAGE purification, although the best currently available method, does not guarantee 100% error-free oligonucleotide products. It was reported by others that a PAGE-purified 123-mer and 126-mer, when used for cloning, were proven to contain errors in about half of the clones (Hecker KH, Rill R. Error analysis of chemically synthesized polynucleotides. Biotechniques 1998 Feb;24:256-60). Even if the article was published in 1998, it is still valid, because very little has changed in the chemistry of the DNA synthesis for the last 40 years, it is still the same chemistry, called “phosphoramidite method of DNA synthesis”. This is a method that does not involve enzymes. In the biological synthesis of DNA, in the eukaryotic cells, the sequence infidelities are at the order of 1 in 1,000,000,000, because the eukaryotic cells have elaborate systems of DNA proof-reading and reparation, comprising many families of genes and proteins. In the prokaryotic cells, the biological synthesis of DNA generates mutations at the rate of approximately 1 mutation per 1,000,000 bases, which is a thousand times lower fidelity compared to the eukaryotes, because the DNA repair systems in the prokaryotic cells involve a relatively limited number of genes and corresponding proteins. In the non-cellular system of the PCR, the mutation rate during DNA synthesis is approximately 1 in 1000, thousand times lower fidelity compared to the prokaryotic cells, because it is just a one-enzyme reaction (Taq polymerase or similar thermostable enzymes), no DNA repair at all. Finally, the lowest fidelity of all is during the chemical synthesis of DNA (the oligonucleotide synthesis), approximately 1 mutation per 100 bases, because not only there are no proof-reading and DNA repair systems, but there is not even a single enzyme (unlike the PCR), therefore, the non-enzymatic reactions are never 100% efficient. The coupling step (the reaction of adding the next DNA base) is approximately 99% efficient, the capping step is 97% efficient, and the deblocking step is 99% efficient. The inefficiencies in these three chemical steps/reactions generate sequence infidelities, cycle after cycle, therefore, the longer the oligo, the higher the percentage of molecules with sequence infidelities.
We strongly encourage you to order OPC, HPLC or PAGE purification for your long oligos. However, if you prefer that we supply you the basic product without purification, we could still accept your order if you explicitly confirm that you want your oligos non-purified. Generally, our replacement warranty does not cover these non-purified long oligos. You are advised that you would have to purify them, preferably by polyacrylamide gel electrophoresis (PAGE).
Oligonucleotides for cloning purposes
Regardless of the length of the oligos, if they are intended for cloning purposes (usually for PCR, followed by cloning), it would be a much better choice to order these oligos additionally purified, at least by OPC. However, even when the oligos are additionally purified, the customer may wish to consider the following dangers:
1) Internally deleted (n-1, n-2 etc.) products. This is the most common and upfront limitation of the chemical DNA synthesis, which cannot be completely overcome by additional purification. The chemical synthesis of DNA is not as exact as the DNA synthesis in the living cell, whereby numerous "proofreading" and reparation systems exist to reduce sequence infidelities down to one mutated base per million bases or even one in billion. Even the PCR reaction has a better fidelity, such as one in thousand or one in ten thousand, because the fidelity of the synthesis is additionally enhanced by its enzymatic nature. Unlike the DNA synthesis in the living cell, or the DNA synthesis in the enzyme-based PCR reactions, the currently used protocol for chemical synthesis of DNA has the upfront limitation of generating sequence infidelities at a rate of approximately one in one hundred for each cycle of the synthesis. Unfortunately, the CapA and CapB reagents, used to block the aborted products from being extended, are not 100% efficient. Another cause of internal deletions is the incomplete deblocking. A third problem is the hindrance of the DNA synthesis caused by interference of the growing DNA chains with each other and with the free flow of synthesis reagents within the pores of the solid support. There are no perfect solutions for these problems, because even very long capping times will not guarantee 100% efficiency; as for the deblocking step, increasing the deblocking solution's strength, or reaction step length, will lead to unwanted oligo depurination, so a balanced compromise must be reached, at which some low-level incomplete deblocking and depurination will simultaneously occur. The internally deleted products cannot be removed easily; in fact they can never be removed 100%. The OPC or RP-HPLC will not remove them, the best method to decrease their amount is purification by PAGE. The OPC and HPLC are just different types of column chromatography, while PAGE is just another electrophoresis; it is not easy to separate by chromatography or electrophoresis a full-size (n) oligo from (n-1), or (n-2) and similar products. This may be easier for analytical purposes, when very small amounts of DNA are loaded on the gel (or column), but for preparative purposes, the method of separation is less powerful. Therefore, a certain amount of shorter oligo molecules will always "contaminate" the OPC, HPLC or PAGE-purified final product (the term "contaminate" is not precise, because the "mutant" molecules are not a contamination from an exogenous source, they are inherently generated by the normal chemical synthesis of DNA). Because of the random nature of these infidelities (different molecules have different mutations, and some molecules do not have any), the sequence infidelities usually do not present a problem for regular PCR, sequencing or hybridization, especially for oligos that are not very long. However, the cloning process by its very nature is a selection and amplification of a single oligo molecule, and if that single parental molecule had a sequence infidelity, all molecules in the clone would display the same sequence infidelity.
2) Insertion of one nucleotide, for example G-duplication, is another frequent sequence infidelity in some molecules of the final product. When a G-duplication or another one-base insertion is combined with a (n-1) deletion in the same DNA molecule, the total length will be equal to that of the wild-type (desired) DNA molecules, and thus impossible to separate by OPC, HPLC or PAGE.
3) In addition, the OPC, HPLC and especially the PAGE are "lossy" methods, resulting in low yields (30% to 50% for OPC and HPLC, 5% to 30% for PAGE), because a large number of "good" DNA molecules (of full length and correct sequence) will be removed together with the undesired DNA molecules.
These drawbacks of the oligonucleotide synthesis are explained in the above-mentioned article of Hecker KH, and Rill R. (Error analysis of chemically synthesized polynucleotides. Biotechniques 1998 Feb;24:256-60). In that article, the authors synthesized and PAGE purified long oligos, used them for cloning, and sequence verified 10 clones. They found 7 single base pair deletions, one 4-base deletion, and one G-C transversion. In addition to these infidelities, others have described G-duplications, branching and other n+x products.
There are some methods to fight infidelities: a) additional purification is recommended; in that case not only are the oligos purified, but the synthesis method is modified to enhance the purity; b) when used for cloning, the OPC-, PAGE- or HPLC-purified oligos (and the PCR products obtained with them) should be expected to give some "mutant" clones (clones with sequence infidelities, originating in the oligonucleotides), therefore selection and sequence analysis of several truly independent clones is advised (not just one or two clones). Generally speaking, the chances of finding a clone with the wild-type sequence are very good, as long as truly independent clones are selected. For this purpose, c) we suggest shorter pre-incubation times without antibiotic immediately after transformation (shorter than 15-20 minutes), because after 15-20 minutes, some bacterial cells will divide, and during subsequent spread onto the Petri dish, will give falsely-independent clones; d) the bacterial colonies should be picked up when not very large, and preferably located away from each other on the dish; e) where possible, shorter oligos should be used instead of longer ones.
In conclusion, any oligo, synthesized by any company or core facility, on any DNA synthesizer, is always a mix of "good" and "bad" ("mutant") DNA molecules. However, the customer should not feel intimidated by the above warnings; the existence of molecules with sequence infidelities in all oligo products does not mean that these products are unusable for cloning, quite the opposite; usually, there are very good chances to find a clone with the desired sequence, especially if the oligonucleotides are less than 100 bases long, and are ordered with additional purification. In addition, most oligos (all short ones, and the additionally purified long ones) are covered by unconditional 90-day replacement warranty ("free replacement, no questions asked"). The above text is intended mainly to facilitate customer's understanding of the potential problems, and provide some help during the ordering and use of custom DNA oligonucleotides.
