Amplification and Direct Sequence Analysis of the 23S rRNA Gene from Thermophilic Bacteria

Abstract

We present a simplified and fast method to obtain high-quality sequences directly from PCRs without the traditional gel purification. We also report on an improved method to obtain sequence-quality PCR products from microorganisms that are difficult to lyse with no need for DNA extraction. The technique uses exonuclease 1 and shrimp alkaline phosphatase to degrade residual dNTPs and primers. Our technique is shown to work on both Gram-positive and Gram-negative bacteria.

Starch phosphorylation in potato tubers is influenced by allelic variation in the genes encoding glucan water dikinase, starch branching enzymes I and II, and starch synthase III

“…The PCR conditions were one cycle of 95°C for 2 min, 40 cycles of 95°C for 30 s, 58°C for 30s, 72°C for 1 min, then one cycle of 72°C for 7 min. Unincorporated primers were removed from the PCR products using exonuclease I and shrimp alkaline phosphatase (Ibrahim et al, 2001 ). The PCR products were sequenced in both directions using the amplification primers, except in the case of SBEII where a different forward primer (Supplementary Data 3 ) was used for sequencing in order to avoid an insertion/deletion polymorphism.…”

Systematic analysis of swine leukocyte antigen‐DRB1 nucleotide polymorphisms using genomic DNA‐based high‐resolution genotyping and identification of new alleles

“…For the direct sequencing of PCR products, 3 µl of amplified products were incubated for 30 min at 37°C together with 4 U of exonuclease I (Fermentas, Burlington, Canada), and 0.8 U of shrimp alkaline phosphatase (USB Corporation, Cleveland, OH) in 1.5X reaction buffer to degrade primers and dephosphorylate dNTPs which were not consumed in the reaction for 30 min at 37°C. The reaction was stopped after 15‐min incubation at 80°C (30). Sequencing reactions were performed using ABI PRISM BigDye™ Terminator Cycle Sequencing Kits (Applied Biosystem, Foster City, CA) with the SLADRBi1F9 sequencing primer (Table 1).…”

Sequence variations of the locus‐specific 5′ untranslated regions of SLA class I genes and the development of a comprehensive genomic DNA‐based high‐resolution typing method for SLA‐2

“…To prepare PCR products as DNA templates for direct sequencing, 15 μl of each product was incubated with 10 U of exonuclease I (Fermentas, St. Leon-Rot, Germany) and 4 U of shrimp alkaline phosphatase (USB Corporation, Cleveland, OH) for 30 min at 37 ∘ C to degrade the primers and dephosphorylate the dNTPs that were not consumed in the reaction, respectively (29). The reaction was stopped by incubation at 80 ∘ C for 15 min.…”