A nucleoside analogue, 1-(2’-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole (4) was designed to function as a universal replacement for any of the natural nucleosides in DNA sequences. Compound 4 was synthesized by the reaction of 3-nitropyrrole with sodium hydride and 1-chloro-2-deoxy-3,5-di-O-toluoyl-D-erythropentofuranose, and the structure was confirmed by X-ray diffraction. Nucleoside 4 was transformed to 1-(2’-deoxyl-5’-dimethoxytrityl-beta-D-ribofuranosyl)-3-nitropyrrole-3’-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite) (6) for incorporation into oligonucleotides by conventional synthesis protocols. Analogues of the oligonucleotide, 5’-d(CGT AAT CAG AAA ACA AT)-3’ with nucleoside 4 replacing the natural nucleosides in UP to 9 positions were constructed and tested as primers for dideoxy sequencing. Sequencing studies show that a substantial number of nucleotides can be replaced by 4 without loss of primer specificity. Sequencing primer 4 with substitutions of 4 at the third position in each of four codons gave a sequencing ladder comparable to primer 1, the exact match, while a 256-fold degenerate oligonucleotide mixture (primer 2) gave an unreadable sequencing ladder. Primers containing two or more mismatches gave indecipherable results.