Preparation of PCR Products for DNA Sequencing

Preparation of PCR products for DNA sequencing.

We demonstrate that routine PCR product analytical agarose gels can also serve as preparative gels for quick DNA template purification before sequencing. The band of interest is excised, placed into a Gel Nebulizer inside a Micropure separator and rapidly purified in a single centrifugation step. Gel-purified PCR product, suitable for manual and automated sequencing, is delivered within 10 min.

PCR template preparation for capillary DNA sequencing.

Fluorescence-based capillary DNA sequencing has facilitated the early completion of several complex sequencing projects. While capillary systems offer great benefits in terms of ease of use and automation, we find that they are sufficiently different from slab gel separation methodologies, demanding re-examination of the protocols used to generate and use DNA sequencing templates. We have recen...

Sequencing of PCR-amplified DNA.

Alternatives for sequencing of PCR products essentially fall into one of two categories; generation of single-stranded DNA for sequencing or the direct sequencing of double-stranded product. Of the two alternatives, sequencing of double-stranded PCR products is likely to be of greatest immediate significance in terms of general applicability and rapidity. Double-stranded sequencing allows the u...

Rapid sequencing of unpurified PCR products by thermal asymmetric PCR cycle sequencing using unlabeled sequencing primers.

Numerous methods have been developed for direct sequencing of single or double-stranded DNA amplified by PCR, and most require template purification steps prior to sequencing to remove excess unincorporated primers and dNTPs. Purification methods can be cumbersome, laborious and time consuming, or yield low DNA recoveries. Strategies for direct sequencing of doublestranded DNA products without ...

5'-tailed sequencing primers improve sequencing quality of PCR products.