Nucleotide sequence-based multitarget identification

Abstract

MULTIGEN technology (T. Vinayagamoorthy, U.S. patent 6,197,510, March 2001) is a modification of conventional sequencing technology that generates a single electropherogram consisting of short nucleotide sequences from a mixture of known DNA targets. The target sequences may be present on the same or different nucleic acid molecules. For example, when two DNA targets are sequenced, the first and second sequencing primers are annealed to their respective target sequences, and then a polymerase causes chain extension by the addition of new deoxyribose nucleotides. Since the electrophoretic separation depends on the relative molecular weights of the truncated molecules, the molecular weight of the second sequencing primer was specifically designed to be higher than the combined molecular weight of the first sequencing primer plus the molecular weight of the largest truncated molecule generated from the first target sequence. Thus, the series of truncated molecules produced by the second sequencing primer will have higher molecular weights than those produced by the first sequencing primer. Hence, the truncated molecules produced by these two sequencing primers can be effectively separated in a single lane by standard gel electrophoresis in a single electropherogram without any overlapping of the nucleotide sequences. By using sequencing primers with progressively higher molecular weights, multiple short DNA sequences from a variety of targets can be determined simultaneously. We describe here the basic concept of MULTIGEN technology and three applications: detection of sexually transmitted pathogens (Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasma urealyticum), detection of contaminants in meat samples (coliforms, fecal coliforms, and Escherichia coli O157:H7), and detection of single-nucleotide polymorphisms in the human N-acetyltransferase (NAT1) gene (S. Fronhoffs et al., Carcinogenesis 22:1405-1412, 2001).

FIG. 1.

Schematic representation of novel features of MULTIGEN technology. (A) Generation of amplicons α, β, and δ from three different targets by MPCRs; (B) possible PCR primer sites for generating target amplicons and possible sites for sequencing primers; (C) relative molecular sizes and position of sequencing primers a, b, and d to the targets A, B, and D; (D) relative electrophoretic migration of truncated DNA molecular species produced from primer extension of targets A, B, and D.

FIG. 2.

Electropherogram showing electrophoretic separation and sequences from multiple targets. (A) Electropherogram of raw data showing two distinct regions of fluorescence signals representing two stretches of sequences, (B) electropherogram of raw data showing distinct regions of fluorescence signals representing three stretches of sequences; (C) electropherogram of analyzed data showing two sequences—a 15-base nucleotide sequence generated by sequencing primer HPV18, followed immediately by a nonsignal, which is then followed by a 41-nucleotide sequence generated by sequencing primer HPV31; (D) electropherogram analyzed data showing three sequences—a 13-base nucleotide sequence generated by sequencing primer HPV18, followed immediately by a 20-nucleotide sequence generated by sequencing primer HPV33, followed immediately by a 41-nucleotide sequence generated by sequencing primer HPV31.

FIG. 3.

Agarose gel electrophoresis showing amplicons from MPCRs. (A) Pathogens associated with STDs: C. trachomatis (CT; 364 bp), N. gonorrhoeae (NG; CppB gene, 298 bp), and U. urealyticum (UU; ureB gene, 219 bp). (B) Bacterial contaminants associated with meat: fecal coliforms (lamB gene, 306 bp), coliforms (lacZ gene, 275 bp), and E. coli O157: H7 (stx₂ gene, 158 bp). (C) Human N-acetyltransferase (NAT1) gene amplicon one (354 bp) and amplicon two (318 bp).

FIG. 4.

Electropherogram of analyzed data showing U. urealyticum (UU; 28 nucleotides), N. gonorrhoeae (NG; 25 nucleotides), and C. trachomatis (CT; 23 nucleotides) (A); stx₂ gene (30 nucleotides), lacZ gene (25 nucleotides), and lamB gene (24 nucleotides) (B); two segments of human N-acetyltransferase single nucleotide polymorphism showing (G/A) SNP at locus −340 on the first segment and at 445 and 459 in the second segment (C); and homozygous wild type (control) showing cystine at locus 1095 with the test (human) sample showing heterozygocity (cystine/adenine).