An optimisation approach to increase DNA amplification success of otter faeces

Simone Lampa, Bernd GRUBER, Klaus Henle, Marion Hoehn

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

Faeces have proved to be a suitable non-invasive DNA source for microsatellite analysis in wildlife research. For the success of such studies it is essential to obtain the highest possible PCR amplification success rate. These rates are still relatively low in most carnivorous species, especially in the otter (Lutra lutra). We therefore optimised the entire microsatellite genotyping process by combining our findings with results from previous studies to gain a high rate of reliable genotypes. We investigated the influence of otter faecal quality in relation to the quantity of slimy secretions and three levels of storage periods at –20C on amplification success. Further, we tested the costeffective and time-saving Chelex extraction method against the profitable QIAamp DNA Stool Kit (Qiagen), and compared three PCR methods - a standard single-step PCR protocol, a single-locus two-step PCR procedure and a multiplex two-step PCR procedure - regarding success rate and genotyping errors. The highest amplification success rate (median: 94%; mean: 78%) was achieved using faecal samples consisting only of jelly extracted with the QIAamp DNA Stool Mini Kit (Qiagen) immediately after collection and amplified following the time and cost efficient multiplex two-step PCR protocol. The two-step procedure, also referred to as pre-amplification approach, turned out to be the main improvement as it increases amplification success about 11% and reduces genotyping errors about 53%, most notably allelic dropouts.
Original languageEnglish
Pages (from-to)201-210
Number of pages10
JournalConservation Genetics
Volume9
Issue number1
DOIs
Publication statusPublished - 2008
Externally publishedYes

Fingerprint

Otters
Feces
feces
amplification
DNA
Polymerase Chain Reaction
microsatellite repeats
Lutra lutra
jellies
Microsatellite Repeats
genotyping
wildlife
storage time
secretion
loci
extraction method
genotype
methodology
rate
Genotype

Cite this

Lampa, Simone ; GRUBER, Bernd ; Henle, Klaus ; Hoehn, Marion. / An optimisation approach to increase DNA amplification success of otter faeces. In: Conservation Genetics. 2008 ; Vol. 9, No. 1. pp. 201-210.
@article{f48bea98325549379e90d6db12f6912a,
title = "An optimisation approach to increase DNA amplification success of otter faeces",
abstract = "Faeces have proved to be a suitable non-invasive DNA source for microsatellite analysis in wildlife research. For the success of such studies it is essential to obtain the highest possible PCR amplification success rate. These rates are still relatively low in most carnivorous species, especially in the otter (Lutra lutra). We therefore optimised the entire microsatellite genotyping process by combining our findings with results from previous studies to gain a high rate of reliable genotypes. We investigated the influence of otter faecal quality in relation to the quantity of slimy secretions and three levels of storage periods at –20C on amplification success. Further, we tested the costeffective and time-saving Chelex extraction method against the profitable QIAamp DNA Stool Kit (Qiagen), and compared three PCR methods - a standard single-step PCR protocol, a single-locus two-step PCR procedure and a multiplex two-step PCR procedure - regarding success rate and genotyping errors. The highest amplification success rate (median: 94{\%}; mean: 78{\%}) was achieved using faecal samples consisting only of jelly extracted with the QIAamp DNA Stool Mini Kit (Qiagen) immediately after collection and amplified following the time and cost efficient multiplex two-step PCR protocol. The two-step procedure, also referred to as pre-amplification approach, turned out to be the main improvement as it increases amplification success about 11{\%} and reduces genotyping errors about 53{\%}, most notably allelic dropouts.",
keywords = "Faecal DNA, Lutra lutra, Microsatellites, Non-invasive samples, Pre-amplification.",
author = "Simone Lampa and Bernd GRUBER and Klaus Henle and Marion Hoehn",
year = "2008",
doi = "10.1007/s10592-007-9328-9",
language = "English",
volume = "9",
pages = "201--210",
journal = "Conservation Genetics",
issn = "1566-0621",
publisher = "Springer",
number = "1",

}

An optimisation approach to increase DNA amplification success of otter faeces. / Lampa, Simone; GRUBER, Bernd; Henle, Klaus; Hoehn, Marion.

In: Conservation Genetics, Vol. 9, No. 1, 2008, p. 201-210.

Research output: Contribution to journalArticle

TY - JOUR

T1 - An optimisation approach to increase DNA amplification success of otter faeces

AU - Lampa, Simone

AU - GRUBER, Bernd

AU - Henle, Klaus

AU - Hoehn, Marion

PY - 2008

Y1 - 2008

N2 - Faeces have proved to be a suitable non-invasive DNA source for microsatellite analysis in wildlife research. For the success of such studies it is essential to obtain the highest possible PCR amplification success rate. These rates are still relatively low in most carnivorous species, especially in the otter (Lutra lutra). We therefore optimised the entire microsatellite genotyping process by combining our findings with results from previous studies to gain a high rate of reliable genotypes. We investigated the influence of otter faecal quality in relation to the quantity of slimy secretions and three levels of storage periods at –20C on amplification success. Further, we tested the costeffective and time-saving Chelex extraction method against the profitable QIAamp DNA Stool Kit (Qiagen), and compared three PCR methods - a standard single-step PCR protocol, a single-locus two-step PCR procedure and a multiplex two-step PCR procedure - regarding success rate and genotyping errors. The highest amplification success rate (median: 94%; mean: 78%) was achieved using faecal samples consisting only of jelly extracted with the QIAamp DNA Stool Mini Kit (Qiagen) immediately after collection and amplified following the time and cost efficient multiplex two-step PCR protocol. The two-step procedure, also referred to as pre-amplification approach, turned out to be the main improvement as it increases amplification success about 11% and reduces genotyping errors about 53%, most notably allelic dropouts.

AB - Faeces have proved to be a suitable non-invasive DNA source for microsatellite analysis in wildlife research. For the success of such studies it is essential to obtain the highest possible PCR amplification success rate. These rates are still relatively low in most carnivorous species, especially in the otter (Lutra lutra). We therefore optimised the entire microsatellite genotyping process by combining our findings with results from previous studies to gain a high rate of reliable genotypes. We investigated the influence of otter faecal quality in relation to the quantity of slimy secretions and three levels of storage periods at –20C on amplification success. Further, we tested the costeffective and time-saving Chelex extraction method against the profitable QIAamp DNA Stool Kit (Qiagen), and compared three PCR methods - a standard single-step PCR protocol, a single-locus two-step PCR procedure and a multiplex two-step PCR procedure - regarding success rate and genotyping errors. The highest amplification success rate (median: 94%; mean: 78%) was achieved using faecal samples consisting only of jelly extracted with the QIAamp DNA Stool Mini Kit (Qiagen) immediately after collection and amplified following the time and cost efficient multiplex two-step PCR protocol. The two-step procedure, also referred to as pre-amplification approach, turned out to be the main improvement as it increases amplification success about 11% and reduces genotyping errors about 53%, most notably allelic dropouts.

KW - Faecal DNA

KW - Lutra lutra

KW - Microsatellites

KW - Non-invasive samples

KW - Pre-amplification.

U2 - 10.1007/s10592-007-9328-9

DO - 10.1007/s10592-007-9328-9

M3 - Article

VL - 9

SP - 201

EP - 210

JO - Conservation Genetics

JF - Conservation Genetics

SN - 1566-0621

IS - 1

ER -