Automated thermal cycling is superior to traditional methods for nucleotide sequencing of bla(SHV) genes

Abstract

Genes encoding SHV-1 and SHV-2 were sequenced by different methods. Nucleotide sequencing of the coding strand by standard dideoxy-chain termination methods resulted in errors in the interpretation of the nucleotide sequence and the derived amino acid sequence in two main regions which corresponded to nucleotide and amino acid changes that had been reported previously. The automated thermal cycling method was clearly superior and consistently resulted in the correct sequences for these genes.

FIG. 1

Sequencing gel of nucleotides 142 through 160 for bla_SHV-1. The autoradiograph shows the compressions that occur in this region following sequencing reactions performed by the dideoxy-chain termination method. The photograph was taken with a Kodak DC120 digital camera and converted to an electronic file by using PhotoEnhancer (Pictureworks).

FIG. 2

Comparison of sequences of nucleotides 142 through 160 of bla_SHV-1. Numbers above the nucleotide sequence indicate the amino acid according to the designation of Ambler et al. (1). Reference Seq is the only published nucleotide sequence for SHV-1 (11). Dideoxy FWD was obtained by dideoxy-nucleotide chain termination sequencing with a forward primer. DiDx REV (RC) is the reverse complement of sequence obtained by standard dideoxy-chain termination sequencing of the noncoding strand with a reverse primer. Auto. Therm was obtained by thermal cycling sequencing reactions followed by running the reactions in an automated sequencing unit. The area of compression that caused a discrepancy is shaded. This compression was such that the three nucleotides corresponding to the glycine were not observed.

FIG. 3

Comparison of sequences of nucleotides 544 through 573 of bla_SHV-1. Numbers above the nucleotide sequence indicate the amino acid according to the designation of Ambler et al. (1). Reference Pro was obtained by sequencing the SHV-1 protein (4). Reference Seq is the only published nucleotide sequence for bla_SHV-1 (11). The DiDx. FWD (days 1 to 4) sequences were obtained by standard (dGTP mix) dideoxy-chain termination sequencing with a forward primer on 4 separate days. DiDx REV (RC) is the reverse complement of sequence obtained by standard dideoxy-chain termination sequencing of the noncoding strand with a reverse primer. DiDx dITP was obtained by substituting dITP for dGTP in the sequencing reaction mixture. 7-deaza dGTP was obtained by substituting 7-deaza-dGTP for dGTP in the sequencing reaction mixture. Auto. Therm was obtained by thermal cycling sequencing reactions followed by running the reactions in an automated sequencing unit. The area of compression which caused a discrepancy is shaded.