Automated magnetic preparation of DNA templates for solid phase sequencing

TLDR

The study presents an integrated robotic protocol for solid‑phase DNA sequencing that uses magnetic separation and automated electrophoretic detection of fluorescently labeled fragments. The protocol employs magnetic bead–streptavidin–biotin capture of PCR‑generated DNA templates, purifies them in microtiter plates, and automates primer annealing and sequencing reactions to produce 10–20 single‑stranded templates per sample. The method was demonstrated by successfully sequencing HIV genomic DNA from a patient’s blood and a cloned human antibody fragment with fluorescent primers.

Abstract

Abstract An integrated protocol for solid‐phase DNA sequencing using a robotic work station is described involving magnetic separation of DNA and analysis of the sequencing product by electrophoresis with automated detection of the fluorescently labeled fragments. The method, which is based on magnetic beads in combination with streptavidin‐biotin technology, can be used for sequencing both genomic and plasmid DNA. The DNA template is obtained by the polymerase chain reaction (PCR). Protocols to prepare five and ten immobilized samples is described, giving 10 and 20 single‐stranded templates, respectively. The magnetic purification steps are performed in a microtiter plate and this allows for an integrated scheme involving a subsequent procedure for automated primer annealing and sequencing reactions. Here, the procedure is examplified by direct genomic sequencing of DNA in blood sample from a human immunodeficiency virus (HIV)‐ infected patient and a cloned human antibody DNA fragment using fluorescently labeled sequencing primers.