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We have previously described a protocol for the' chemical sequencing of 3'-labelled DNA (1), further developed for onelane fluorescent DNA analysis (2). The method can be coupled to an automatized system (laser scan) and to a quantitative software that allows a rapid determination of the sequence of cloned, dideoxy fluorescently terminally labelled DNA. The resulting procedure (2) is based on the treatment of DNA with piperidine and formamide, in this order, and is particularly useful for the identification of mutations. This method is not suited for the sequencing of 5 '-labelled DNA because the reaction with formamide (alone or used after piperidine treatment) results in smeared sequence profiles (1), due to the preferential cleavage of the phosphodiester bond at the 3' side and to the concurrent, less efficient cleavage at the 5' side (4). It is known that piperidine efficiently cleaves phosphodiester bonds at both the 3' and 5' sides of removed or damaged bases (3), in addition to a base-selective cleavage (2, 4). Therefore the following protocol based essentially on the inversion of the 'piperidine followed by formamide' treatments was developed, yielding base-selective and non-smeared cleavages. This protocol allows the sequence determination of 5'-labelled DNA fragments, obtained by amplification from radioactive or fluorescent oligos (Figures 1 and 3) or by kinase end-labeling (Figure 2). 1 5 ng ( .5-2x 10 cpm) of 5'-labelled DNA were dissolved in 80% formamide, and heated 10 min. at 110°C. After cooling on ice, piperidine was added to 10% final concentration and the sample was heated 30 min. at 95°C. After 5-fold dilution with water and evaporation to the original volume (to eliminate piperidine) the sample was denatured (2 min., 90°C) and loaded on a sequencing gel. Alternatively, piperidine can be eliminated by ethanol precipitation. One practical important feature of the protocol used is that piperidine is added directly to the DNA-containing formamide hot aqueous solution, thus making the whole procedure simple and rapid. The intensity of the bands produced i s G > A > O T . This method can be used to sequence a DNA fragment starting directly from genomic DNA, amplifying it with two oligos, one of which is 5'-labelled. After amplification, the DNA product is ethanol-precipitated or purified on a gel filtration column and sequenced (Figure la). The method is compatible with fluorescent labelling, thus allowing direct and accurate quantitative evaluation (Figure lb). In the case of ambiguity, an easy solution is provided Figure 1. Sequencing of PCR-amplified DNA samples by fonnamide-piperidine treatment, a. A 170 bp portion of the S.cerevisiae 5S RNA repeat gene (1) was PCR-amplified using as a template 5 ng of pBBl 11R piasmid (3125 bp) containing the entire gene. Two internal 20 bp-long oligonucleotides were used as primers, one of which had been terminally labelled at the 5'-end by polynucleotide kinase. The amplification mixture (20 cycles) was ethanol !"•*• i|iimn-H, treated as described in the text and loaded on a 6% sequencing gel. Scanning denshometry (coding strand from residue +57 to +89) is shown, b. Densitogram obtained by the 373A automatic sequencer (ABI) software (2) on a DNA sample amplified using an ABI fluorescently labelled primer and treated as in Figure la.

Chemical method for DNA sequence determination from the 5′-extremity on PCR amplified fragments