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*****Glen Research Glen Report*****
The design of oligonucleotide primers and probes is frequently
made more complicated by the degeneracy of the genetic code or
incomplete peptide sequence data. To confront this problem, two
strategies are popular but both have significant drawbacks. The use
of a mixed base addition (N) at specific points is a logical approach
and is achieved either by delivery of equal amounts of monomer on the
synthesizer or using the monomers premixed in equivalent amounts.
This strategy is more successful the fewer the number of degenerate
sites, probably simply due to the smaller number of oligonucleotides
formed. (Remember that x degenerate sites lead to 4x
oligonucleotides, e.g., 4 fully degenerate sites lead to 256
oligonucleotides of which only one is the desired sequence.) An
alternative approach is to substitute 2'-deoxyInosine (dI) at the
degenerate sites. Hydrogen bonding of dI to each of the other bases
is low but, unfortunately, is not completely equivalent and this
factor can cause problems in PCR and sequencing experiments.
The CE phosphoramidite (2) of 3-nitropyrrole 2'-deoxynucleoside was used to prepare oligonucleotides with several unknown sites substituted with M. The correct sequencing primer in a dideoxy sequencing experiment was compared with the equivalent primers modified at the third position in 4 codons with N (a 256-fold degenerate primer), with dI, and with M. The sequencing ladder obtained using N was unreadable, using dI was only partially readable, but using M was an exact match of the correct oligonucleotide primer. Even more impressively, 17 base oligonucleotide primers with three, six and nine contiguous substitutions of M also generated accurate sequencing data. Both double and single stranded templates were successfully sequenced.
Promising results were also demonstrated in initial PCR experiments amplifying from total RNA using primers containing three M substitutions. Amplification of the correct product resulted.
The ability of an oligonucleotide containing M to function as a sequencing primer would indicate that a duplex is being successfully formed with the complementary strand. Indeed, a normal pattern was observed from the DNA double strand to single strand transition. The Tm values of oligonucleotides containing M-X base pairs (where X=A,C,G or T) all fell within a 3¡ range. In contrast, duplexes containing dI opposite the other bases vary in Tm by as much as 15¡.
Other Potential Applications
As usual, alert and ingenious researchers have already suggested
other applications for the use of M: So far, experiments involving
ligase chain reaction, in situ hybridization, mutagenesis, motif
cloning, and even in restriction fragment length polymorphism (RFLP)
have been proposed. We await feedback with interest.
(1) R. Nichols, P.C. Andrews, P. Zhang, and D.E. Bergstrom, Nature, 1994, 369, 492-493.
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