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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), using an amount of DNA equalling 0.05 mg of starting material.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-g001: Results of the PCR amplification of cox2 fragments from up to 129 years old herbarium specimens (leaves infected by biotrophic oomycete pathogens), using an amount of DNA equalling 0.05 mg of starting material.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: The 19 different DNA extraction methods yielded highly diverging amounts of DNA, as presented in Table 1. In all cases, the DNA was highly fragmented and gave a smear on agarose gels, revealing mostly fragment sizes below 500 bp (data not shown). DNA extracts were colourless to brownish, depending on the method used. All DNA-amplicons sequenced revealed the target sequence and no contamination was observed. In an initial test, an amount of DNA extracted which equalled 0.05 mg of dried plant material was used in the PCR reactions (Fig. 1). Only 13 DNA extraction protocols gave PCR amplicons for one or more specimens. From these, 3 DNA extracts (gained by the methods AnaP, EznF, EriP) gave a 350 bp amplification product from a 129 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)

Results of the PCR amplification of cox2 fragments from up to 129 years old herbarium specimens (leaves infected by biotrophic oomycete pathogens), using an amount of DNA equalling 0.05 mg of starting material.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-g001: Results of the PCR amplification of cox2 fragments from up to 129 years old herbarium specimens (leaves infected by biotrophic oomycete pathogens), using an amount of DNA equalling 0.05 mg of starting material.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: The 19 different DNA extraction methods yielded highly diverging amounts of DNA, as presented in Table 1. In all cases, the DNA was highly fragmented and gave a smear on agarose gels, revealing mostly fragment sizes below 500 bp (data not shown). DNA extracts were colourless to brownish, depending on the method used. All DNA-amplicons sequenced revealed the target sequence and no contamination was observed. In an initial test, an amount of DNA extracted which equalled 0.05 mg of dried plant material was used in the PCR reactions (Fig. 1). Only 13 DNA extraction protocols gave PCR amplicons for one or more specimens. From these, 3 DNA extracts (gained by the methods AnaP, EznF, EriP) gave a 350 bp amplification product from a 129 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