Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR.

Applied and Environmental Microbiology
M T Suzuki, S J Giovannoni

Abstract

The PCR is used widely for the study of rRNA genes amplified from mixed microbial populations. These studies resemble quantitative applications of PCR in that the templates are mixtures of homologs and the relative abundance of amplicons is thought to provide some measure of the gene ratios in the starting mixture. Although such studies have established the presence of novel rRNA genes in many natural ecosystems, inferences about gene abundance have been limited by uncertainties about the relative efficiency of gene amplification in the PCR. To address this question, three rRNA gene standards were prepared by PCR, mixed in known proportions, and amplified a second time by using primer pairs in which one primer was labeled with a fluorescent nucleotide derivative. The PCR products were digested with restriction endonucleases, and the frequencies of genes in the products were determined by electrophoresis on an Applied Biosystems 373A automated DNA sequencer in Genescan mode. Mixtures of two templates amplified with the 519F-1406R primer pair yielded products in the predicted proportions. A second primer pair (27F-338R) resulted in strong bias towards 1:1 mixtures of genes in final products, regardless of the initial proportions ...Continue Reading

References

Oct 1, 1992·Applied and Environmental Microbiology·A L ReysenbachN R Pace
Oct 8, 1992·Nature·P D Siebert, J W Larrick
Jun 1, 1991·Applied and Environmental Microbiology·T B Britschgi, S J Giovannoni
Apr 1, 1990·Proceedings of the National Academy of Sciences of the United States of America·G GillilandH F Bunn
Dec 1, 1989·Proceedings of the National Academy of Sciences of the United States of America·A M WangD F Mark
Feb 14, 1968·Journal of Molecular Biology·J G Wetmur, N Davidson
May 1, 1993·Applied and Environmental Microbiology·J A FuhrmanA A Davis
Oct 18, 1994·Biochimica Et Biophysica Acta·C Morrison, F Gannon
Nov 1, 1993·Analytical Biochemistry·L Raeymaekers
Jun 25, 1993·Nucleic Acids Research·C M DuttonS S Sommer
Aug 1, 1995·Genome Research·L Raeymaekers

❮ Previous
Next ❯

Citations

May 2, 2000·Applied and Environmental Microbiology·R MassanaC Pedrós-Alió
Jul 29, 2000·FEMS Microbiology Letters·L CocolinD A Mills
Jan 3, 2001·Applied and Environmental Microbiology·K RavenschlagR Amann
Aug 30, 2001·Applied and Environmental Microbiology·A SessitschE Kandeler
Jun 1, 2002·Applied and Environmental Microbiology·John DunbarCheryl R Kuske
Jun 13, 2002·Proceedings of the National Academy of Sciences of the United States of America·Scott C Dawson, Norman R Pace
Dec 27, 2002·The Journal of General and Applied Microbiology·Jorge W. Santo Domingo
Dec 17, 2002·The Journal of General and Applied Microbiology·Kousuke IshiiKazuhiro Aoki
Nov 27, 2002·Applied and Environmental Microbiology·Tracy B NorrisTimothy R McDermott
Apr 5, 2003·Journal of Medical Microbiology·K E HillD W Thomas
Mar 7, 2003·Applied and Environmental Microbiology·Yohey SuzukiJillian F Banfield
Jul 4, 2003·Applied and Environmental Microbiology·José R de la TorreNorman R Pace
May 11, 2004·The Journal of Eukaryotic Microbiology·Kathy B SheehanJoan M Henson
Apr 12, 2005·Environmental Microbiology·Mike ManefieldAndrew S Whiteley
Oct 7, 2004·Applied and Environmental Microbiology·Massimiliano CardinaleDaniele Daffonchio
Mar 16, 2005·FEMS Microbiology Letters·Marcell NikolauszKároly Márialigeti
Oct 4, 2005·EMBO Reports·Konrad U FoerstnerPeer Bork
Dec 8, 2005·Applied and Environmental Microbiology·Arlene K RowanIan M Head

❮ Previous
Next ❯

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.