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Amplification of cox2 (approximately 620 bp) from 2 mg of up to 129 years old herbarium specimens, comparing 19 extraction methods and 15 polymerases.

Telle S, Thines M - PLoS ONE (2008)

Bottom Line: We conclude that DNA extraction and the choice of DNA polymerase are crucial factors for successful PCR amplification from small samples of historic herbarium specimens.Through a combination of suitable DNA extraction protocols and DNA polymerases, only a fraction of the preserved plant material commonly used is necessary for successful PCR amplification.This facilitates the potential use of a far larger number of preserved specimens for molecular phylogenetic investigation and provides access to a wealth of genetic information in preserved in specimens deposited in herbaria around the world without reducing their scientific or historical value.

View Article: PubMed Central - PubMed

Affiliation: University of Hohenheim, Institute of Botany 210, Stuttgart, Germany.

ABSTRACT
During the past years an increasing number of studies have focussed on the use of herbarium specimens for molecular phylogenetic investigations and several comparative studies have been published. However, in the studies reported so far usually rather large amounts of material (typically around 100 mg) were sampled for DNA extraction. This equals an amount roughly equivalent to 8 cm(2) of a medium thick leaf. For investigating the phylogeny of plant pathogens, such large amounts of tissue are usually not available or would irretrievably damage the specimens. Through systematic comparison of 19 DNA extraction protocols applied to only 2 mg of infected leaf tissue and testing 15 different DNA polymerases, we could successfully amplify a mitochondrial DNA region (cox2; approximately 620 bp) from herbarium specimens well over a hundred years old. We conclude that DNA extraction and the choice of DNA polymerase are crucial factors for successful PCR amplification from small samples of historic herbarium specimens. Through a combination of suitable DNA extraction protocols and DNA polymerases, only a fraction of the preserved plant material commonly used is necessary for successful PCR amplification. This facilitates the potential use of a far larger number of preserved specimens for molecular phylogenetic investigation and provides access to a wealth of genetic information in preserved in specimens deposited in herbaria around the world without reducing their scientific or historical value.

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Related in: MedlinePlus

Comparison of the performance of various DNA polymerases.Square fields indicate amplification of the ∼350 bp fragment with different intensity. Black fields: amplicon amount >90 ng, dark grey: amplicon amount 30–90 ng, grey: amplicon amount 10–30 ng, light grey: amplicon amount <10 ng, white: no amplicon detectable, asterisks: very faint band visible (≪10 ng); white dots indicate additional amplification of the ∼620 bp fragment. +: positive control, −: negative (water) control.
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pone-0003584-g003: Comparison of the performance of various DNA polymerases.Square fields indicate amplification of the ∼350 bp fragment with different intensity. Black fields: amplicon amount >90 ng, dark grey: amplicon amount 30–90 ng, grey: amplicon amount 10–30 ng, light grey: amplicon amount <10 ng, white: no amplicon detectable, asterisks: very faint band visible (≪10 ng); white dots indicate additional amplification of the ∼620 bp fragment. +: positive control, −: negative (water) control.

Mentions: Of the DNA extraction methods applied in this study, four gave consistent cox2 amplification throughout all samples tested (SigP, AnaP, EznF, EriP). For subsequent experiments, three of these were selected, representing different methodologies (AnaP, EznF, EriP). To these, the extraction method of May and Ristaino (15: RisNC), which was reported to be suitable for amplification from minute amounts of a more than 150 year old herbarium specimen, a commercial DNA extraction kit especially designed for extraction of ancient DNA (AncA), and a simple genomic DNA extraction kit (Fermentas, FerG) were added. The DNA obtained by these protocols from the three oldest samples (82, 101, 129 years) was used to test the amplification performance of 15 DNA polymerases, encompassing 5 conventional Taq polymerases, one hot-start Taq polymerase, a DNA- Repair Kit, 3 Taq polymerase based blends, 8 proofreading enzymes, and a genetically engineered DNA polymerase. All polymerases tested gave amplification in the positive control, which was represented by a herbarium specimen less than three years old. For the herbarium specimens tested, the DNA polymerases tested showed highly different amplification performances (Fig. 3). Only four of the polymerases tested yielded the ∼620 bp fragment from at least one of the DNA extracts obtained by at least one DNA protocol (BioTaqRed, Mango-Taq, peqGoldTaq, BIO-X-ACT short). In addition, also the PreCR-mix gave amplification of the ∼620 bp fragment for one of the DNA extracts tested. Only two polymerases were able to amplify the ∼620 bp fragment from the oldest sample (BioTaq Red, Mango-Taq). The best performance was exhibited by the Mango-Taq DNA polymerase, which was the only polymerase which was able to amplify the ∼620 bp amplification product from the 102 year old sample.


Amplification of cox2 (approximately 620 bp) from 2 mg of up to 129 years old herbarium specimens, comparing 19 extraction methods and 15 polymerases.

Telle S, Thines M - PLoS ONE (2008)

Comparison of the performance of various DNA polymerases.Square fields indicate amplification of the ∼350 bp fragment with different intensity. Black fields: amplicon amount >90 ng, dark grey: amplicon amount 30–90 ng, grey: amplicon amount 10–30 ng, light grey: amplicon amount <10 ng, white: no amplicon detectable, asterisks: very faint band visible (≪10 ng); white dots indicate additional amplification of the ∼620 bp fragment. +: positive control, −: negative (water) control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2572190&req=5

pone-0003584-g003: Comparison of the performance of various DNA polymerases.Square fields indicate amplification of the ∼350 bp fragment with different intensity. Black fields: amplicon amount >90 ng, dark grey: amplicon amount 30–90 ng, grey: amplicon amount 10–30 ng, light grey: amplicon amount <10 ng, white: no amplicon detectable, asterisks: very faint band visible (≪10 ng); white dots indicate additional amplification of the ∼620 bp fragment. +: positive control, −: negative (water) control.
Mentions: Of the DNA extraction methods applied in this study, four gave consistent cox2 amplification throughout all samples tested (SigP, AnaP, EznF, EriP). For subsequent experiments, three of these were selected, representing different methodologies (AnaP, EznF, EriP). To these, the extraction method of May and Ristaino (15: RisNC), which was reported to be suitable for amplification from minute amounts of a more than 150 year old herbarium specimen, a commercial DNA extraction kit especially designed for extraction of ancient DNA (AncA), and a simple genomic DNA extraction kit (Fermentas, FerG) were added. The DNA obtained by these protocols from the three oldest samples (82, 101, 129 years) was used to test the amplification performance of 15 DNA polymerases, encompassing 5 conventional Taq polymerases, one hot-start Taq polymerase, a DNA- Repair Kit, 3 Taq polymerase based blends, 8 proofreading enzymes, and a genetically engineered DNA polymerase. All polymerases tested gave amplification in the positive control, which was represented by a herbarium specimen less than three years old. For the herbarium specimens tested, the DNA polymerases tested showed highly different amplification performances (Fig. 3). Only four of the polymerases tested yielded the ∼620 bp fragment from at least one of the DNA extracts obtained by at least one DNA protocol (BioTaqRed, Mango-Taq, peqGoldTaq, BIO-X-ACT short). In addition, also the PreCR-mix gave amplification of the ∼620 bp fragment for one of the DNA extracts tested. Only two polymerases were able to amplify the ∼620 bp fragment from the oldest sample (BioTaq Red, Mango-Taq). The best performance was exhibited by the Mango-Taq DNA polymerase, which was the only polymerase which was able to amplify the ∼620 bp amplification product from the 102 year old sample.

Bottom Line: We conclude that DNA extraction and the choice of DNA polymerase are crucial factors for successful PCR amplification from small samples of historic herbarium specimens.Through a combination of suitable DNA extraction protocols and DNA polymerases, only a fraction of the preserved plant material commonly used is necessary for successful PCR amplification.This facilitates the potential use of a far larger number of preserved specimens for molecular phylogenetic investigation and provides access to a wealth of genetic information in preserved in specimens deposited in herbaria around the world without reducing their scientific or historical value.

View Article: PubMed Central - PubMed

Affiliation: University of Hohenheim, Institute of Botany 210, Stuttgart, Germany.

ABSTRACT
During the past years an increasing number of studies have focussed on the use of herbarium specimens for molecular phylogenetic investigations and several comparative studies have been published. However, in the studies reported so far usually rather large amounts of material (typically around 100 mg) were sampled for DNA extraction. This equals an amount roughly equivalent to 8 cm(2) of a medium thick leaf. For investigating the phylogeny of plant pathogens, such large amounts of tissue are usually not available or would irretrievably damage the specimens. Through systematic comparison of 19 DNA extraction protocols applied to only 2 mg of infected leaf tissue and testing 15 different DNA polymerases, we could successfully amplify a mitochondrial DNA region (cox2; approximately 620 bp) from herbarium specimens well over a hundred years old. We conclude that DNA extraction and the choice of DNA polymerase are crucial factors for successful PCR amplification from small samples of historic herbarium specimens. Through a combination of suitable DNA extraction protocols and DNA polymerases, only a fraction of the preserved plant material commonly used is necessary for successful PCR amplification. This facilitates the potential use of a far larger number of preserved specimens for molecular phylogenetic investigation and provides access to a wealth of genetic information in preserved in specimens deposited in herbaria around the world without reducing their scientific or historical value.

Show MeSH
Related in: MedlinePlus