Glen Report 2-12: DNA Purification

In the most recent issues of the Glen Research Report, several techniques for the chemical modification of synthetic oligonucleotides have been discussed. Over the last few months, we have received feedback f ram researchers and we feel that some of their comments may be of general interest.

Booklet

5'- Thiol-Modifier

In response to several requests, we are appy to offer the 5'-Thiol-Modifier C6 designed to allow the production of thiol specific probes1 and attachment of proteins to oligonucleotides. We chose the 6 carbon linker arm which has proved to be most popular in the 5'-amino-modifier series. A trityl group has been used to protect the thiol group. However, this group is stable to acid deprotection, requiring oxidative cleavage with, for example, silver nitrate.

Biotinylation Kit

Labelling with biotin has become a standard technique in DNA chemistry. We are happy to introduce a kit shown to generate the most sensitive assay results. The biotin includes a 14 carbon (XX) spacer terminated in NHS ester for attachment to a primary amine at the 5'terminus. The kit includes a protocol and all reagents necessary for biotinylation at the primary amine.

5'-Amino-Modifiers

Glen Research 5'-Amino-Modifiers have proved to be quite popular and have performed admirably in a variety of situations. The six carbon linker has proved to be the most useful in a variety of applications. Consequently, we have added 0.5g packs of this product to our catalog. Several requests were obtained for a two carbon linker due to its applications in DNA sequencing. Accordingly, we have also added this item to our catalog; details are shown below.

Phosphate Modificatio 

Phosphorothioates 

Of the phosphate modifications accessible using H-phosphonate chemistry, far and away the most popular seems to be phosphorothioates with applications in anti-viral research. Oligophosphorothioates can be prepared by either phosphoramidite or H-phosphonate chemistry. Phosphoramidite chemistry is the most popular method, although the sulfurization step for converting phosphite to phosphorothioate is slow and, by necessity, must be performed once per cycle. In this regard, H-phosphonate chemistry seems more practical because the sulfurization process occurs faster and needs to be performed only once per synthesis.

The greatest advantage of the H-phosphonate method, however, may be in the preparation of oligonucleotides containing a mixture of phosphorothioate and phosphodiester linkages. Typically, two phosphorothioate linkages are prepared adjacent to the 3'-terminus to protect against exonuclease hydrolysis. These linkages are unlikely to survive a large number of iodine oxidations. Thus, with phosphoramidite chemistry, the length of sequence that can be prepared is limited. Using H-phosphonate chemistry, however, a single oxidation step converts all internucleotide H-phosphonate linkages to phosphodiester linkages. The phosphorothioate linkages, therefore, are more likely to survive when H-phosphonate chemistry is used.

Phosphoramidates 

Feedback from several researchers has indicated that phosphoramidate linkages can be conveniently and successfully prepared from internucleotide H-phosphonate linkages. Oligonucleotides containing these modifications may be an interesting alternative to those containing methyl phosphonate linkages. By varying the choice of the amine used to prepare these linkages, it is possible to modify the physical properties of the resulting oligonucleotides.

Phosphotriesters

While these linkages are prepared in a manner analogous to phosphoramidates, it is now clear that the synthesis is difficult to perform because of the necessity of maintaining absolutely anhydrous conditions. Even traces of moisture lead to the production of a mixture of phosphotriester and phosphodiester linkages. It is, therefore, doubtful that this procedure will lead to reproducible and sensible results.

RNA Supports

The main difficulty encountered by researchers using RNA supports has been the packing of synthesis columns. We now offer these supports packed in disposable columns suitable for use in ABI and Du Pont/Vega instruments as well as Biosearch instruments. Ordering information is shown below.

In our review of the use of RNA supports for the production of oligodeoxynucleotides modified at the 3'-terminus, we omitted to reference a paper2 by Zudhir Agrawal and Mike Gait. In this issue we correct our omission and thank Dr. Agrawal for bringing it to our attention.

References

1. B.A. Connolly and P. Rider, Nucleic Acids Res., 1985, 13, 4485. 2. S. Agrawal, C. Christodoulou, and M.J. Gait, Nucleic Acids Res., 1986, 14, 6227.

Product Information

H-Phosphonates
5'-Chemical Phosphorylation Reagent (10-1900)
5'-Amino-Modifier C2 (10-1902) has been discontinued.
5'-Amino-Modifier C3 (10-1903) has been discontinued. Please see:
5'-Amino-Modifier C3-TFA (10-1923)
5'-Amino-Modifier C6 (10-1906)
5'-Amino-Modifier C12 (10-1912)
5'-Thiol-Modifier C6 (10-1926)
5'-Branched Modifier C3 (10-1950) has been discontinued.
Biotin-XX-NHS Ester Labelling Kit (50-1950) has been discontinued.
Standard RNA Phosphoramidites and Supports