pyrosequencing


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pyrosequencing

a real-time DNA sequencing technique based on monitoring DNA synthesis by BIOLUMINESCENCE using four enzymes: DNA POLYMERASE, ATP sulfurylase, LUCIFERASE and apyrase. The NUCLEOTIDES are tested one at a time for incorporation into the nascent DNA strand during synthesis. Each incorporation event, by DNA polymerase, generates pyrophosphate (PPi) as a by-product. The PPi is converted, by ATP sulfurylase, to ATP and this drives the conversion of luciferin to oxyluciferin by luciferase, with the emission of a light pulse. The light produced is detected by a charge coupled device camera and displayed as a peak in a light profile (pyrogram(tm)). Peak height relates to the number of nucleotides incorporated. If not incorporated the nucleotide is degraded by apyrase and no light is emitted. The DNA sequence can be determined from the pyrogram, knowing the sequence of nucleotides added.
References in periodicals archive ?
Shifts in the bacterial communities of leachates from three bovine carcasses, sampled at different decomposition times, were investigated by pyrosequencing of the 16S rRNA.
Detection of antiviral resistance and genetic lineage markers in influenza B virus neuraminidase using pyrosequencing.
The two PRR5L CpGs with closest proximity to each other (12 bp apart) were selected for pyrosequencing analysis (cg00220721 and cg22117805).
The ignored diversity: complex bacteria communities in intensive care units revealed by 16S pyrosequencing.
For the pyrosequencing, the V3-V5 hypervariable region of the 16S rRNA gene was amplified using PCR as previously described (17), with Bact-338F/Bact-909R.
Source LAM resistance M1 2429 1181 mutations detection HBV-RNA Direct YMDD YMDD YVDD sequencing Pyrosequencing YMDD (94%) YMDD YMDD (30%) YIDD (6%) YVDD (70%) HBV-DNA Direct YMDD YMDD YVDD sequencing Pyrosequencing YMDD (96%) YMDD YVDD YIDD (4%)
Quantitative polymerase chain reaction and pyrosequencing assay for long interspersed nuclear element-1 methylation
After amplification, pyrosequencing was performed using the PyroMark Vacuum Prep Workstation and the PyroMark Q24 instrument (Qiagen) following the manufacturer's instructions.
Initial pyrosequencing PCR assays with the validation primers described above for B.
The researcher used pyrosequencing, a relatively new method of rapidly analyzing bacterial DNA markers, to classify the bacteria into different taxonomic groups.
This is one of the first studies to document such diversity using the pyrosequencing technique (a large scale DNA sequencing determination technique) on colostrum samples on the one hand, and breast milk on the other, the latter being collected after one and six months of breastfeeding," co-authors, Maria Carmen Collado from the Institute of Agrochemistry and Food Technology (IATA-CSIC) and Alex Mira, researcher at the Higher Public Health Research Centre (CSISP-GVA), said.
DNA was then subjected to 454 pyrosequencing, a "fingerprinting" technique that provides a snapshot of all the bacterial types present.