Manifold sequencing: efficient processing of large sets of sequencing reactions.
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 instrument. In this manner, all reaction steps are performed without pipetting the samples. The strategy is applied to sequencing PCR-amplified clones of the human mitochondrial D-loop and for detection of heterozygous positions in the human major histocompatibility complex class II 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.
Lagerkvist
A
A
Beijer Laboratory, Department of Medical Genetics, Uppsala Biomedical Center, Sweden.
Stewart
J
J
Lagerström-Fermér
M
M
Landegren
U
U
D001483:Base Sequence; D004247:DNA; D017931:DNA Primers; D006683:HLA-DQ Antigens / Q000235:genetics*; D059866:HLA-DQ beta-Chains; D006579:Heterozygote; D006801:Humans; D008969:Molecular Sequence Data; D010456:Peptides, Cyclic / Q000235:genetics*; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000295:instrumentation / Q000379:methods*
D017931:DNA Primers; D006683:HLA-DQ Antigens; D059866:HLA-DQ beta-Chains; C057615:HLA-DQbeta antigen; D010456:Peptides, Cyclic; C078763:D-loop peptide, synthetic; D004247:DNA