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

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.

Efficient incorporation of positively charged 2', 3'-dideoxynucleoside-5'-triphosphates by DNA polymerases and their application in 'direct-load' DNA sequencing.

A series of charge-modified, dye-labeled 2', 3'-dideoxynucleoside-5'-triphosphates have been synthesized and evaluated as reagents for dye-terminator DNA sequencing. Unlike the commonly used dye-labeled terminators, these terminators possess a net positive charge and migrate in the opposite direction to dye-labeled Sanger fragments during electrophoresis. Post-sequencing reaction purification is not required to remove unreacted nucleotide or associated breakdown products prior to electrophoresis. Thus, DNA sequencing reaction mixtures can be loaded directly onto a separating medium such as a sequencing gel. The charge-modified nucleotides have also been shown to be more efficiently incorporated by a number of DNA polymerases than regular dye-labeled dideoxynucleotide terminators or indeed normal dideoxynucleoside-5'-triphosphates.

TempliPhi, phi29 DNA polymerase based rolling circle amplification of templates for DNA sequencing.

We have developed a novel, isothermal DNA amplification strategy that employs phi29 DNA polymerase and rolling circle amplification to generate high-quality templates for DNA sequencing reactions. The TempliPhi DNA amplification kits take advantage of the fact that cloned DNA is typically obtained in circular vectors, which are readily replicated in vitro using phi29 DNA polymerase by a rolling circle mechanism. This single subunit, proofreading DNA polymerase has excellent processivity and strand displacement properties for generation of multiple, tandem double-stranded copies of the circular DNA, generating as much as 10(7)-fold amplification. Large amounts of product (1-3 microg) can be obtained in as little as 4 hours. Input DNA can be as little as 0.01 ng of purified plasmid DNA, a single bacterial colony, or a 1 microL of a saturated overnight culture. Additionally, the presence of an associated proof reading function within the phi29 DNA polymerase ensures high-fidelity amplification. Once completed, the product DNA can be used directly in sequencing reactions. Additionally, the properties of phi29 DNA polymerase and its use in applications such as amplification ofhuman genomic DNA for genotyping studies is discussed.

Synthesis and application of charge-modified dye-labeled dideoxynucleoside-5'-triphosphates to 'direct-load' DNA sequencing.

A novel series of charge-modified, dye-labeled 2',3'-dideoxynucleoside-triphosphate terminators were synthesized and evaluated as reagents for DNA sequencing. These terminators possess an advantage over existing reagents in that no purification is required to remove unreacted nucleotide or associated breakdown products prior to electrophoretic separation of the sequencing fragments. This obviates the need for a time consuming post-reaction work up, allowing direct loading of DNA sequencing reaction mixtures onto a slab gel. Thermo Sequenase II DNA polymerase poorly incorporates the charge-modified terminators compared with regular dye-labeled terminators. However, extending the linker arm between dye and nucleotide and using a mutant form of a related DNA polymerase can in part mitigate the decrease in substrate efficiency. We also present evidence that these charge-modified terminators can relieve gel compression artefacts when used with dGTP in sequencing reactions.