It creates many copies of a small portion of DNA, in this case a ~400bp region of the 16S gene
. This gene is present in all bacteria and can be used to identify bacteria based upon sequencing differences in the amplified region.
Amplification of the 16S gene
fragments of total DNA (PCR) was made with a specific primer BoxA1R (Versalovic, Schneider, Bruijn, & Lupski, 1994), synthesized by INVITROGEN--Brazil.
We have already amplified the 16S gene
using standard extraction and PCR techniques.
Sequences of the rRNA 16S gene
from isolates of Euryarchaeota, Crenarchaeota, Thaumarchaeota, and Korarchaeota as well as sequences of uncultured Archaea found in the GenBank dataset were used to construct a phylogenetic tree with the MEGA software , using the maximum-likelihood method, a bootstrap value of 1,000 and the Tamura nucleotide substitution model.
The researchers sequenced the 16S gene
on fecal samples of 114 people, including patients newly diagnosed with rheumatoid arthritis, prior to their treatment; patients with chronic, but treated, RA; patients with psoriatic arthritis; and healthy individuals.
To determine if particular bacterial species correlate with rheumatoid arthritis, the researchers sequenced the so-called 16S gene
on 44 fecal DNA samples from newly diagnosed patients with rheumatoid arthritis prior to immune-suppressive treatment; 26 samples from patients with chronic, treated rheumatoid arthritis; 16 samples from patients with psoriatic arthritis (characterized by red, flaky skin in conjunction with joint inflammation); and 28 samples from healthy individuals.
The 16S gene
is being studied using an Ion Torrent Personal Genome Machine, which can identify ten million bacteria in just three hours.
Amplification of the 16S gene
was based on the following cycling parameters: 2 min at 94[degrees]C for denaturation, followed by 30 cycles of 30 sec at 94[degrees]C, 1 min at 51 [degrees]C for annealing, and 2 min at 72[degrees]C for extension, and then 7 min at 72[degrees]C for the final extension.
We amplified and sequenced a 550-bp region of the 16S gene
using standard amphibian primers 16SA-L and 16SB-H (Vences and others 2005).
rDNA 16S gene
amplification from Klebsiella genus species were performed under the following reaction conditions: Klebrib-1 (5'-GTAATGT CTGGGAAACTGCC-3') [0.5[micro]M] and Klebrib-2A (3'CCACCTTCCTCCAGTTTATC-5') [0.5[micro]M] Taq polymerase [0.25U], MgCl [1.5mM], dNTPs [0.2mM], PCR buffer [1X] and DNA sample [10ng/[micro]l], adjusted to a final volume of 50[micro]l Amplification conditions were 94[degrees]C for 1 min; 40 cycles of 94[degrees]C for 1 min, 58[degrees]C for 1 min, and 72[dergees]C for 1 min; and a final step at 72[degrees]C for 3 min.
data) were used to amplify a 500-bp fragment of the 16S gene
of Anaplasma/Ehrlichia spp.
Genetic variation of Reticulitermes flavipes (Isoptera: Rhinotermitidae) in North America Applying the mitochondrial rRNA 16S Gene