Amplification and direct sequence analysis of the 23S rRNA gene from thermophilic bacteria.
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.
Technical University of Denmark, Lyngby, Denmark.
A
A
Ibrahim
J
J
Hofman-Bang
B K
BK
Ahring
D001419:Bacteria / Q000235:genetics*; D004275:DNA, Ribosomal / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D012338:RNA, Ribosomal, 23S / Q000235:genetics*; D017422:Sequence Analysis, DNA
D004275:DNA, Ribosomal; D012338:RNA, Ribosomal, 23S