TempliPhi: A sequencing template preparation procedure that eliminates overnight cultures and DNA purification

Abstract

Preparing plasmid templates for DNA sequencing is the most time-consuming step in the sequencing process. Current template preparation methods rely on a labor-intensive, multistep procedure that takes up to 24 h and produces templates of varying quality and quantity. The TempliPhi DNA Sequencing Template Amplification Kit eliminates the requirement for extended bacterial growth prior to sequencing and saves laboratory personnel hands-on time by eliminating the centrifugation and transfer steps currently required by older preparatory methods. In addition, costly purification filters and columns are not necessary, as amplified product can be added directly to a sequencing reaction. Starting material can be any circular template from a colony, culture, glycerol stock, or plaque. Based on rolling circle amplification and employing bacteriophage Phi29 DNA polymerase, the method can produce 3-5 microg of template directly from a single bacterial colony in as little as 4 h. Implementation of these procedures in a laboratory or core sequencing facility can decrease cost on tips, plates, and other plasticware, while at the same time increase throughput.

FIGURE 1

TempliPhi amplification of circular DNA template. Random hexamer (~) and Phi29 DNA polymerase (•) (A) anneal to single-stranded or denatured double-stranded circular DNA template(B). Phi29 DNA polymerase synthesizes DNA from the template displacing strands produced by downstream replicating events (C). Amplification continues, resulting in complementary tandem repeats of the original template. Random hexamer binds to the concatamer strands priming for addition amplification (D). Continuous strand displacement results in the exponentially increasing release of double stranded DNA (E).

FIGURE 2

Agarose gel of TempliPhi product. Lane 1: 125 ng KiloBASE^™(Amersham Biosciences) molecular weight marker. Lane 2: 1 μL of uncut TempliPhi product from a 20-μL reaction amplified from 1 ng pUC19 demonstrating large molecular weight product too large to migrate from the loading well. Lane 3: 1 μL of the pUC19 TempliPhi product digested by BamHI.

FIGURE 3

Histogram of DNA amounts in 384 16-h TempliPhi reactions. The amount of TempliPhi DNA was determined by PicoGreen assay and plotted within a gradient divided into 1-μg regions.

FIGURE 4

Workflow comparison between alkaline lysis DNA preparation and TempliPhi DNA sequencing template amplification.