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Effect of primer mismatch, annealing temperature and PCR cycle number on 16S rRNA gene-targetting bacterial community analysis

Abstract In the attempt to explore complex bacterial communities of environmental samples, primers hybridizing to phylogenetically highly conserved regions of 16S rRNA genes are widely used, but differential amplification is a recognized problem. The biases associated with preferential amplification... Full description

Journal Title: FEMS microbiology ecology 2007-05-01, Vol.60 (2), p.341-350
Main Author: Sipos, Rita
Other Authors: Székely, Anna J , Palatinszky, Márton , Révész, Sára , Márialigeti, Károly , Nikolausz, Marcell
Format: Electronic Article Electronic Article
Language: English
Subjects:
DNA
Quelle: Alma/SFX Local Collection
Publisher: Oxford, UK: Blackwell Publishing Ltd
ID: ISSN: 0168-6496
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recordid: cdi_swepub_primary_oai_DiVA_org_uu_224290
title: Effect of primer mismatch, annealing temperature and PCR cycle number on 16S rRNA gene-targetting bacterial community analysis
format: Article
creator:
  • Sipos, Rita
  • Székely, Anna J
  • Palatinszky, Márton
  • Révész, Sára
  • Márialigeti, Károly
  • Nikolausz, Marcell
subjects:
  • Amplification
  • Annealing
  • Annealing temperature
  • Bacteria
  • Bacteria - classification
  • Bacteria - genetics
  • Bacteria - growth & development
  • Bacteriology
  • Biological and medical sciences
  • Biological Sciences
  • Biologiska vetenskaper
  • Deoxyribonucleic acid
  • DNA
  • DNA Primers - genetics
  • DNA, Bacterial - chemistry
  • DNA, Bacterial - genetics
  • Ecosystem
  • Electrophoresis, Capillary
  • Environmental Microbiology
  • Environmental testing
  • Fundamental and applied biological sciences. Psychology
  • Microbial communities
  • Microbiology
  • Mikrobiologi
  • Miscellaneous
  • Molecular Sequence Data
  • Natural Sciences
  • Naturvetenskap
  • PCR bias
  • Phylogeny
  • Polymerase chain reaction
  • Polymerase Chain Reaction - methods
  • Polymorphism
  • Polymorphism, Restriction Fragment Length
  • Restriction fragment length polymorphism
  • RNA, Ribosomal, 16S - genetics
  • rRNA 16S
  • Sequence Analysis, DNA
  • Sequences
  • Temperature
  • Temperature effects
  • Temperature preferences
  • Template matching
  • Universal diversity
ispartof: FEMS microbiology ecology, 2007-05-01, Vol.60 (2), p.341-350
description: Abstract In the attempt to explore complex bacterial communities of environmental samples, primers hybridizing to phylogenetically highly conserved regions of 16S rRNA genes are widely used, but differential amplification is a recognized problem. The biases associated with preferential amplification of multitemplate PCR were investigated using ‘universal’ bacteria-specific primers, focusing on the effect of primer mismatch, annealing temperature and PCR cycle number. The distortion of the template-to-product ratio was measured using predefined template mixtures and environmental samples by terminal restriction fragment length polymorphism analysis. When a 1 : 1 genomic DNA template mixture of two strains was used, primer mismatches inherent in the 63F primer presented a serious bias, showing preferential amplification of the template containing the perfectly matching sequence. The extent of the preferential amplification showed an almost exponential relation with increasing annealing temperature from 47 to 61°C. No negative effect of the various annealing temperatures was observed with the 27F primer, with no mismatches with the target sequences. The number of PCR cycles had little influence on the template-to-product ratios. As a result of additional tests on environmental samples, the use of a low annealing temperature is recommended in order to significantly reduce preferential amplification while maintaining the specificity of PCR.
language: eng
source: Alma/SFX Local Collection
identifier: ISSN: 0168-6496
fulltext: fulltext
issn:
  • 0168-6496
  • 1574-6941
  • 1574-6941
url: Link


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descriptionAbstract In the attempt to explore complex bacterial communities of environmental samples, primers hybridizing to phylogenetically highly conserved regions of 16S rRNA genes are widely used, but differential amplification is a recognized problem. The biases associated with preferential amplification of multitemplate PCR were investigated using ‘universal’ bacteria-specific primers, focusing on the effect of primer mismatch, annealing temperature and PCR cycle number. The distortion of the template-to-product ratio was measured using predefined template mixtures and environmental samples by terminal restriction fragment length polymorphism analysis. When a 1 : 1 genomic DNA template mixture of two strains was used, primer mismatches inherent in the 63F primer presented a serious bias, showing preferential amplification of the template containing the perfectly matching sequence. The extent of the preferential amplification showed an almost exponential relation with increasing annealing temperature from 47 to 61°C. No negative effect of the various annealing temperatures was observed with the 27F primer, with no mismatches with the target sequences. The number of PCR cycles had little influence on the template-to-product ratios. As a result of additional tests on environmental samples, the use of a low annealing temperature is recommended in order to significantly reduce preferential amplification while maintaining the specificity of PCR.
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subjectAmplification ; Annealing ; Annealing temperature ; Bacteria ; Bacteria - classification ; Bacteria - genetics ; Bacteria - growth & development ; Bacteriology ; Biological and medical sciences ; Biological Sciences ; Biologiska vetenskaper ; Deoxyribonucleic acid ; DNA ; DNA Primers - genetics ; DNA, Bacterial - chemistry ; DNA, Bacterial - genetics ; Ecosystem ; Electrophoresis, Capillary ; Environmental Microbiology ; Environmental testing ; Fundamental and applied biological sciences. Psychology ; Microbial communities ; Microbiology ; Mikrobiologi ; Miscellaneous ; Molecular Sequence Data ; Natural Sciences ; Naturvetenskap ; PCR bias ; Phylogeny ; Polymerase chain reaction ; Polymerase Chain Reaction - methods ; Polymorphism ; Polymorphism, Restriction Fragment Length ; Restriction fragment length polymorphism ; RNA, Ribosomal, 16S - genetics ; rRNA 16S ; Sequence Analysis, DNA ; Sequences ; Temperature ; Temperature effects ; Temperature preferences ; Template matching ; Universal diversity
ispartofFEMS microbiology ecology, 2007-05-01, Vol.60 (2), p.341-350
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descriptionAbstract In the attempt to explore complex bacterial communities of environmental samples, primers hybridizing to phylogenetically highly conserved regions of 16S rRNA genes are widely used, but differential amplification is a recognized problem. The biases associated with preferential amplification of multitemplate PCR were investigated using ‘universal’ bacteria-specific primers, focusing on the effect of primer mismatch, annealing temperature and PCR cycle number. The distortion of the template-to-product ratio was measured using predefined template mixtures and environmental samples by terminal restriction fragment length polymorphism analysis. When a 1 : 1 genomic DNA template mixture of two strains was used, primer mismatches inherent in the 63F primer presented a serious bias, showing preferential amplification of the template containing the perfectly matching sequence. The extent of the preferential amplification showed an almost exponential relation with increasing annealing temperature from 47 to 61°C. No negative effect of the various annealing temperatures was observed with the 27F primer, with no mismatches with the target sequences. The number of PCR cycles had little influence on the template-to-product ratios. As a result of additional tests on environmental samples, the use of a low annealing temperature is recommended in order to significantly reduce preferential amplification while maintaining the specificity of PCR.
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18Environmental Microbiology
19Environmental testing
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21Microbial communities
22Microbiology
23Mikrobiologi
24Miscellaneous
25Molecular Sequence Data
26Natural Sciences
27Naturvetenskap
28PCR bias
29Phylogeny
30Polymerase chain reaction
31Polymerase Chain Reaction - methods
32Polymorphism
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34Restriction fragment length polymorphism
35RNA, Ribosomal, 16S - genetics
36rRNA 16S
37Sequence Analysis, DNA
38Sequences
39Temperature
40Temperature effects
41Temperature preferences
42Template matching
43Universal diversity
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authorSipos, Rita ; Székely, Anna J ; Palatinszky, Márton ; Révész, Sára ; Márialigeti, Károly ; Nikolausz, Marcell
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atitleEffect of primer mismatch, annealing temperature and PCR cycle number on 16S rRNA gene-targetting bacterial community analysis
jtitleFEMS microbiology ecology
addtitleFEMS Microbiol Ecol
date2007-05-01
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volume60
issue2
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pages341-350
issn
00168-6496
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eissn1574-6941
notesEditor: Christoph Tebbe
abstractAbstract In the attempt to explore complex bacterial communities of environmental samples, primers hybridizing to phylogenetically highly conserved regions of 16S rRNA genes are widely used, but differential amplification is a recognized problem. The biases associated with preferential amplification of multitemplate PCR were investigated using ‘universal’ bacteria-specific primers, focusing on the effect of primer mismatch, annealing temperature and PCR cycle number. The distortion of the template-to-product ratio was measured using predefined template mixtures and environmental samples by terminal restriction fragment length polymorphism analysis. When a 1 : 1 genomic DNA template mixture of two strains was used, primer mismatches inherent in the 63F primer presented a serious bias, showing preferential amplification of the template containing the perfectly matching sequence. The extent of the preferential amplification showed an almost exponential relation with increasing annealing temperature from 47 to 61°C. No negative effect of the various annealing temperatures was observed with the 27F primer, with no mismatches with the target sequences. The number of PCR cycles had little influence on the template-to-product ratios. As a result of additional tests on environmental samples, the use of a low annealing temperature is recommended in order to significantly reduce preferential amplification while maintaining the specificity of PCR.
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pmid17343679
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