Polyadenylated Sequencing Primers Enable Complete Readability of PCR Amplicons Analyzed by Dideoxynucleotide Sequencing

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.

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.

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...

Complete sequence of an immunoglobulin mRNA using specific priming and the dideoxynucleotide method of RNA sequencing.

The complete sequence of the mouse immunoglobulin kappa light chain MOPC 21 messenger RNA has been determined using a chain termination method and chemically synthesised deoxyoligonucleotides to initiate the synthesis of a DNA molecule complementary to the mRNA template. Five such oligonucleotide primers have been used for the sequence analysis of this messenger RNA. The approach is excellent f...