Manifold sequencing: efficient processing of large sets of sequencing reactions.

Abstract

Automated instruments for DNA sequencing greatly simplify data collection in the Sanger sequencing procedure. By contrast, the so-called front-end problems of preparing sequencing templates, performing sequencing reactions, and loading these on the instruments remain major obstacles to extensive sequencing projects. We describe here the use of a manifold support to prepare and perform sequencing reactions on large sets of templates in parallel, as well as to load the reaction products on a sequencing strument. In this manner, all reaction steps are performed without pipetting the samples. The strategy is applied to sequencing PCR-amplifled clones of the human mitochondrial D-loop and for detection of heterozygous positions in the human major histocompatibility complex class H gene HLA-DQB, amplified from genomic DNA samples. This technique will promote sequencing in a clinical context and could form the basis of more efficient genomic sequencing strategies.Large-scale genomic sequencing projects now under way are paralleled by an increasing interest to determine the nucleotide sequences of specific genes in patient samples. These efforts have prompted the development of a number of automated instruments to record nucleotide sequence data (1, 2). Other time-consuming aspects of collecting nucleotide sequence information, besides recording the information, include preparing the sequencing templates and generating and loading the sequencing reaction products on gels. Solidphase sequencing of PCR products is an efficient method of preparing sequencing templates that results in high-quality sequence data (3, 4). We demonstrate in this paper a solidphase-based method to process, in parallel, large sets of reactions up to and including the point where the sequencing reaction products are loaded on an automated sequencing instrument. In this strategy, sequencing templates are isolated and handled by using a solid support with 96 prongs projecting into individual reaction wells. Of crucial importance for our approach was the development of a very efficient means of immobilizing amplified sequencing templates on the limited surface available on the manifold support. This effect was achieved by creating a porous surface layer on the polystyrene device (5). In this manner, sufficient avidin can be coupled to individual prongs of the support in order to bind and sequentially transfer sets of biotin-labeled templates for fluorescence sequencing reactions through the series of reaction steps preceding gel analysis (Fig. 1). MATERIALS AND METHODSSolid Supports. Comb-shaped devices, similar to those used when casting sequencing gels to create slots for loading samples, were cut out of sheets of 100-,m biaxial polystyrene (Nordbergs Tekniska AB, Sundbyberg, Sweden) (Fig. 1A). As an alternative, a commercially available polystyrene device was used. This support has a set of 96 ball-and-stick extensions, projecting into individual wells of a microtiter plate located underneath (Falcon).Avidin-coated supports were constructe...

Worse survival for TP53 (p53)-mutated breast cancer patients receiving adjuvant CMF

“…The fluorescence-labelled primers were this time labelled with Cy5, owing to the fact that the ALF express sequencer was used instead of the original ALF version. A manifold-based version of solid phase sequencing was utilised, essentially as described by Lagerkvist et al [16]. The sequencing products generated were analysed using an automated laser fluorescence sequencer (ALFexpress TM ; Amersham-Pharmacia Biotech).…”

Determination of Single-Nucleotide Polymorphisms by Real-time Pyrophosphate DNA Sequencing

“…For this purpose test samples of each SNP in this panel were employed that harbored the two alternative homozygous and the heterozygous configurations, respectively, as determined either by the semiautomated solid-phase Sanger DNA technique (Ståhl et al 1988;Lagerkvist et al 1994) or by solid-phase single-base extension reactions (Syvänen et al 1990(Syvänen et al , 1993. To obtain reference luminescence peaks for each of the variable bases, several SNP-succeeding bases were read.…”

“…Manifold sequencing reactions of PCR-amplified and biotinlabeled DNA-fragments, employing streptavidin-coupled Sepharose HP attached to teeth of plastic combs (Amersham Pharmacia Biotech AB, Uppsala, Sweden), were conducted essentially as outlined (Ståhl et al 1988;Lagerkvist et al 1994). Resulting products were loaded on a sequencing gel and detected using an ALFexpress automated laser fluorescence sequencer.…”

Mutational analysis of the CDKN2 gene in metastases from patients with cutaneous malignant melanoma

“…Mutational alterations of CDKN2 detected in the germ line of familial melanoma cases (Hussussian et al, 1994;Kamb et al, 1994b) 4,683,195 and 4,683,202 owned by HoffmanLa Roche). The sequencing reactions were carried out by direct sequencing of the PCR products using Pharmacia Biotech's (Uppsala, Sweden) combs solid phase DNA sequencing (Autoload kit) (Lagerkvist et al, 1994). One of the PCR primers was biotinylated for the isolation of single-strand DNA and FITC-modified sequencing primers were used for the sequencing reactions.…”