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

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 ...

Direct sequencing of PCR products using unlabeled primers.

An improved protocol is described for using lambda exonuclease to directly sequence PCR products. It is important not to execute PCR cycles beyond the plateau of amplification. The asymmetric PCR and double-stranded DNA sequencing by a snap-cooling procedure were also performed using the same DNA samples and primers. The improved method was the most reliable and produced the best results.

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

Cycle Sequencing: Dideoxy-mediated Sequencing Reactions Using PCR and End-labeled Primers.

MATERIALS 5x Cycle-sequencing buffer AmpliTaq CS or other exonuclease-deficient versions of Taq DNA polymerase (5 units/μl) EDTA (0.05 M, pH 8.0) Formamide loading buffer Oligonucleotide Primers, radiolabeled at the 5' terminus with 33P or 32P To prepare end-labeled primers, please see Phosphorylating the 5' Termini of Oligonucleotides. Template DNAs Plasmids, cosmids, bacteriophage , and bacte...

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.