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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

Results of the PCR amplification of cox2 fragments from up to 129 years old herbarium specimens (leaves infected by biotrophic oomycete pathogens), from 10 ng of extracted DNA.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). Upper half of each row: ∼620 bp fragment, lower half: ∼350 bp fragment.
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pone-0003584-g002: Results of the PCR amplification of cox2 fragments from up to 129 years old herbarium specimens (leaves infected by biotrophic oomycete pathogens), from 10 ng of extracted DNA.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). Upper half of each row: ∼620 bp fragment, lower half: ∼350 bp fragment.

Mentions: When using 10 ng of extracted DNA per reaction (Fig. 2), eight protocols gave only PCR amplicons of the cox2 region for one or two of the specimens, and with none of these protocols (AncB, RisN, RisNC, RisP, Ris PC, FerG, CheX, NaOH), amplification of the target sequence was obtained for samples more than 100 years old. Only the DNA extracts from five protocols gave 350 bp amplification products for the 129 year old sample (SigP, AnaP, EznF, EznP, EriP) in either absolute (i.e. extract amount) or relative (i.e. specific amount in ng) PCR. The larger fragment (∼620 bp) could only be amplified from the samples less than 100 years old, when using the Fermentas Taq DNA polymerase. It was observed that with increasing age of the samples PCR amplification performance decreased.


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)

Results of the PCR amplification of cox2 fragments from up to 129 years old herbarium specimens (leaves infected by biotrophic oomycete pathogens), from 10 ng of extracted DNA.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). Upper half of each row: ∼620 bp fragment, lower half: ∼350 bp fragment.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0003584-g002: Results of the PCR amplification of cox2 fragments from up to 129 years old herbarium specimens (leaves infected by biotrophic oomycete pathogens), from 10 ng of extracted DNA.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). Upper half of each row: ∼620 bp fragment, lower half: ∼350 bp fragment.
Mentions: When using 10 ng of extracted DNA per reaction (Fig. 2), eight protocols gave only PCR amplicons of the cox2 region for one or two of the specimens, and with none of these protocols (AncB, RisN, RisNC, RisP, Ris PC, FerG, CheX, NaOH), amplification of the target sequence was obtained for samples more than 100 years old. Only the DNA extracts from five protocols gave 350 bp amplification products for the 129 year old sample (SigP, AnaP, EznF, EznP, EriP) in either absolute (i.e. extract amount) or relative (i.e. specific amount in ng) PCR. The larger fragment (∼620 bp) could only be amplified from the samples less than 100 years old, when using the Fermentas Taq DNA polymerase. It was observed that with increasing age of the samples PCR amplification performance decreased.

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