Genotyping of Plant and Animal Samples without Prior DNA Purification

Chum, Pak Y.; Haimes, Josh; André, Chas P.; Kuusisto, Pia K.; Kelley, Melissa L.

doi:10.3791/3844

Cited by 10 publications

(7 citation statements)

References 9 publications

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“…Whole spider specimens were used to extract DNA with Extraction Solution and Dilution Solution (Sigma-Aldrich, Saint Louis, MO, USA) according to manufacturer protocol. All samples were macerated using sterilised pipette tips (Chum et al, 2012). Ten taxon specific primer pairs, amplifying 200–300 bp long segment of mitochondrial cytochrome-oxidase I (COI) barcoding region was designed using the NCBI Primer-blast home page (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) following the recommendations of King et al (2008) and tested in silico for Psammotettix specificity against GenBank nucleotid database of Arthropoda taxa with the same application.…”

Section: Methodsmentioning

confidence: 99%

Consuming alternative prey does not influence the DNA detectability half-life of pest prey in spider gut contents

Fülöp

Szita

Gerstenbrand

et al. 2019

PeerJ

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BackgroundKey natural enemy-pest interactions can be mapped in agricultural food webs by analysing predator gut content for the presence of a focal pest species. For this, PCR-based approaches are the most widely used methods providing the incidence of consumption of a focal pest in field sampled predators. To interpret such data the rate of prey DNA decay in the predators’ gut, described by DNA detectability half-life (t1/2), is needed. DNA decay may depend on the presence of alternative prey in the gut of generalist predators, but this effect has not been investigated in one of the major predatory arthropod groups, spiders.MethodsIn a laboratory feeding experiment, we determined t1/2 of the key cereal pest virus vector leafhopper Psammotettix alienus in the digestive tracts of its natural enemy, the spider Tibellus oblongus. We followed the fate of prey DNA in spiders which received only the focal prey as food, or as an alternative prey treatment they also received a meal of fruit flies after leafhopper consumption. After these feeding treatments, spiders were starved for variable time intervals prior to testing for leafhopper DNA in order to establish t1/2.ResultsWe created a PCR protocol that detects P. alienus DNA in its spider predator. The protocol was further calibrated to the digestion speed of the spider by establishing DNA decay rate. Detectability limit was reached at 14 days, where c. 10% of the animals tested positive. The calculated t1/2 = 5 days value of P. alienus DNA did not differ statistically between the treatment groups which received only the leafhopper prey or which also received fruit fly. The PCR protocol was validated in a field with known P. alienus infestation. In this applicability trial, we showed that 12.5% of field collected spiders were positive for the leafhopper DNA. We conclude that in our model system the presence of alternative prey did not influence the t1/2 estimate of a pest species, which makes laboratory protocols more straightforward for the calibration of future field data.

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Section: Methodsmentioning

confidence: 99%

Consuming alternative prey does not influence the DNA detectability half-life of pest prey in spider gut contents

Fülöp

Szita

Gerstenbrand

et al. 2019

PeerJ

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“…Here, a direct PCR approach was applied to analyse a cytochrome P450 marker variability in the Europe populations of Hedera helix, L. Amplification of the target sites of plant genome by the direct PCR is a method where no DNA extraction is needed prior the PCR. It is possible to use both, crude plant extract as well as the small pieces of plant tissues without steps of isolation and purification of total genomic DNA (Chum et al, 2012). Omission of the step of DNA isolation process brings benefits mainly in terms of utilization of samples without loss, saving time and reducing the cost of analysis.…”

Section: Introductionmentioning

confidence: 99%

Direct PCR as the platform of Hedera helix, L. genotypying without the extraction of DNA

Bošeľová¹,

Žiarovská²

2016

JCEA

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Hedera helix, L. is a specie that is used for its ornamental and medicinal properties widely. In spite of its very good biochemical characterization, the knowledge about the DNA variability is very limited and no DNA markers were used to analyse the genomic variability of the ivy populations, up to date. Here, a direct PCR approach was used to evaluate a cytochrome P450 sequences based polymorphism by PBA technique. A set of 4 samples from Slovakia, 2 samples from Czech Republic, 2 samples originated in Poland, 2 samples from Croatia, 2 samples from Scotland and samples from Germany, Austria and Spain were used in analysis. Using a set of three PBA primers and their combinations, the number of amplified fragment levels obtained by individual primer combinations was for both of them 11 and the obtained polymorphism was 91% or 100% respectivelly. The PCoA analysis was performed and the genotypes belonging to a different geographic localities were grouped together.

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“…This makes DNA analysis by direct PCR methods very difficult, so an additional step is usually required to remove the compounds [25,43]. Direct PCR amplification can be overcome by using a quick and easy dilution protocol, coupled with a unique DNA polymerase that has a double-stranded DNA binding domain [44]. The results showed that it is very efficient to use the small leaf disks for the sex determination of papaya.…”

Section: Resultsmentioning

confidence: 99%

Direct LAMP Assay without Prior DNA Purification for Sex Determination of Papaya

Tsai

Shih

et al. 2016

IJMS

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Papaya (Carica papaya L.) is an economically important tropical fruit tree with hermaphrodite, male and female sex types. Hermaphroditic plants are the major type used for papaya production because their fruits have more commercial advantages than those of female plants. Sex determination of the seedlings, or during the early growth stages, is very important for the papaya seedling industry. Thus far, the only method for determining the sex type of a papaya at the seedling stage has been DNA analysis. In this study, a molecular technique—based on DNA analysis—was developed for detecting male-hermaphrodite-specific markers to examine the papaya’s sex type. This method is based on the loop-mediated isothermal amplification (LAMP) and does not require prior DNA purification. The results show that the method is an easy, efficient, and inexpensive way to determine a papaya’s sex. This is the first report on the LAMP assay, using intact plant materials-without DNA purification-as samples for the analysis of sex determination of papaya. We found that using high-efficiency DNA polymerase was essential for successful DNA amplification, using trace intact plant material as a template DNA source.

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Genotyping of Plant and Animal Samples without Prior DNA Purification

Cited by 10 publications

References 9 publications

Consuming alternative prey does not influence the DNA detectability half-life of pest prey in spider gut contents

Consuming alternative prey does not influence the DNA detectability half-life of pest prey in spider gut contents

Direct PCR as the platform of Hedera helix, L. genotypying without the extraction of DNA

Direct LAMP Assay without Prior DNA Purification for Sex Determination of Papaya

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