Nucleic Acids Research, Vol. 18, No. 10 3089

.-. 1990 Oxford University Press

Error rates in oligodeoxynucleotides synthesized by the Hphosphonate method Mark Vasser, Peter G.Ng, Parkash Jhurani and Norbert Bischofberger* Department of Molecular Biology, Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, CA 94080, USA Submitted April 1, 1990

A number of reports have been published on the error rates of oligodeoxynucleotides synthesized by the standard solid support phosphoramidite procedure (1), corresponding data for the Hphosphonate method, however, are not currently available. We wish to report on the accuracy of oligodeoxynucleotides synthesized by the H-phosphonate method. A number of oligomers were synthesized using the H-phosphonate methodology (2) without a capping step on a Biosearch 8600 DNA synthesizer. The oligomers were purified by PAGE, ethanol precipitated, annealed and ligated into appropriate vectors by standard techniques. After transformation, clones containing the insert were selected and sequenced by the dideoxy method. The results are given in the table. Out of a total of 8937 bp sequenced, 37 errors were found (= 1 error/241 bp). The types of errors were mainly deletions (20) and insertions (12) with only a few substitutions (5). These results show that long oligodeoxynucleotides can be synthesized by the H-phosphonate method and be successfully used for the assembly of genes with acceptable error rates.

Although the amidite method gives lower error rates, the Hphosphonate method is superior in speed, preparation and stability

of the reagents, and cost.

ACKNOWLEDGEMENTS We wish to thank Don Dowbenko, Tony Mason, Rein Strijker, Mark Dennis and Dennis Henner for their help.

REFERENCES 1. (a) McClain,W.H., Foss,K., Mittelstadt,K.L. and Schneider,J. (1986) Nucl. Acids Res. 14, 6770. (b) Nassal,M. (1988) Gene 66, 279. (c) Bell,L.D., Smith,J.C., Derbyshire,R., Finlay,M., Johnson,I., Gibert,R., Slocombe,P., Cook,E., Richards,H., Clissold,P., Meredith,D., Powell-Jones,C.H., Dawson,K.M., Carter,B.L. and McCullagh,K.G. (1988) Gene 63, 155. (d) Hein,F., Jansen,H.W. and Uhlmann,E. (1988) Nucleosides and Nucleotides 7, 497. 2. Froehler,B.C., Ng,P.G. and Matteucci,M.D. (1986) Nucl. Acids Res. 14, 5399.

TABLE

*

Fragment Length

Number of Oligomers

Oligomer Length

Clones Sequenced

318 bp 225 bp 124 bp 220 bp 209 bp 197 bp 225 bp 190 bp 175 bp 233 bp 170 bp 90 bp 34 bp

12 6 4 8 6 6 8 6 6 8 4 2 2

45-55 65-85 40-85 35-72 56-79 59-72 41-70 51-68 40-69 33-68 71-78 88 and 93 31 and 36

4 1 1 1 1 1 1 4 4 5 8 20

To whom correspondence should be addressed

20

Del

Errors Ins

Sub

1 -

2 1

-

-

-

1

-

-

1 2 2 3 4 5 2

-

2 1 1

-

-

-

2 2

3 2

-

-

Errors/ Bp Sequenced 3/1272 1/225 0/124 1/220 0/209 0/197 1/225 4/760 3/700 6/116 6/1360 8/1800 4/680