Modified oligonucleotides containing 5-methylcytidine and/or 2-aminoadenosine form tighter hybrids with DNA and are, therefore, more efficient primers for DNA sequencing as compared to their natural counterparts. Strings of contiguous modified pentanucleotides can be used for DNA sequencing by primer walking.One of the promising approaches to speeding up large-scale DNA sequencing is a recently described "primer walking" technique (1). In this technique, the first primer complementary to a known portion of DNA under study is used for sequencing the adjacent region. The new sequence information obtained is then used to prompt a new primer for further sequencing beyond the known regions. By repeatedly performing this procedure, the sequence of long DNA stretches can be established. However, the approach includes expensive and time-consuming synthesis of new and rather long primers after each sequencing step. This shortcoming was elegantly circumvented by Studier's group (2). Instead of synthesizing long sequencing primers, they proposed to use strings of short contiguous oligonucleotides constituting together the sequence of a desirable primer. Oligonucleotide constituents of such composite primers could be simply chosen from a premade oligonucleotide library. The authors (2) have demonstrated that strings of three contiguous hexamers can serve as specific sequencing primers in the presence of single-stranded DNA-binding protein (SSB). The full library of all possible hexanucleotides should contain as many as 46 = 4096 individual components. Still this figure seems too high for routine sequencing, and a further decrease in size of the library is highly desirable.In principle, the decrease could be achieved by shortening oligomers that form composite primers. However, it was convincingly demonstrated (2) that six is the lowest limit of the length of natural oligomers capable of functioning as components of composite primers, whereas pentamer combinations provide only "weak and ambiguous" priming.We have previously described the use of modified oligonucleotides containing 5-methylcytidine (m5C) and 2-aminoadenosine (n2A) instead of their natural counterparts as primers for sequencing (3). Oligonucleotides with m5C (4) or n2A (see the discussion of the problem in ref. MATERIALS AND METHODS Phosphoramidites. 5-Methyl-2'-deoxycytidine and 2-amino-2'-deoxyadenosine were prepared according to refs. 6 and 7; protected using the 4,4'-dimethoxytrityl group for the 5' position, the benzoyl group for the N4 in 5-methyl-2'-deoxycytidine, and the dimethylacetamidine group for N2 and N6 in 2-amino-2'-deoxyadenosine (8); and converted to the corresponding 3 '-cyanoethyldiisopropyl phosphoramidites (9). Other phosphoramidites were synthesized by a standard procedure.Oligonucleotide Synthesis. Oligonucleotides were synthesized on a controlled porous glass support with a Milligene 7500 DNA synthesizer. After deprotection by aqueous ammonia at 60°C for 20 h, the oligonucleotides were purified by denaturing polyacrylamide gel ...

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