Interference of PCR amplification by the polyamines, spermine and spermidine.

Ahokas, Hannu; Erkkilä, Maria

doi:10.1101/gr.3.1.65

Cited by 37 publications

(17 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…In addition, we investigated the effect of ethanol fixation on DNA extraction and subsequent PCR, as fixation is often used in the field to preserve cell morphology and community composition when samples cannot be immediately frozen. Ethanol is the simplest and safest fixative, which has previously yielded PCR products from some marine algae (Marin et al, 2001), in contrast to other common fixatives such as formalin and Lugol's solution, which in some cases have been shown to interfere with subsequent PCR reactions (Wilson, 1997;Marin et al, 2001;Godhe et al, 2002;Ahokas and Erkkila, 1993). …”

Section: Introductionmentioning

confidence: 97%

Evaluation of DNA extraction methods for freshwater eukaryotic microalgae

2012

View full text Add to dashboard Cite

Section: Introductionmentioning

confidence: 97%

Evaluation of DNA extraction methods for freshwater eukaryotic microalgae

2012

View full text Add to dashboard Cite

“…For example, in barley (Ahokas and Erkkila, 1993), cotton (Wan and Wilkins, 1993), pomegranate (Ono et al, 2012) and human stool samples (Roperch et al, 2015, Kikuchi et al, 2010. However, the quantity per reaction varies in different applications reported.…”

Section: Resultsmentioning

confidence: 99%

“…The polyamines such as spermine and spermidine have been found to increase the success of PCR for DNA extracted from plants such as barley (Ahokas and Erkkila, 1993), cotton (Wan and Wilkins, 1993) and pomegranates (Ono et al,2012). Although these studies reported different concentrations of PCR enhancers, it would be beneficial to optimize a protocol applicable for range of plant species with phytochemical diversity.…”

Section: Introductionmentioning

confidence: 99%

Optimization of DNA extraction and PCR protocols for plants with high Phenolics: Bael, Mango, Pomegranate as examples

Pathirana¹,

Chandrasekara²,

Attanayake³

et al. 2018

Ceylon J. Sci.

View full text Add to dashboard Cite

Abstract:The genetic studies of tropical tree species containing high amount of phenols are greatly hampered by the inability to extract sufficient quantities of high quality DNA and the presence of PCR inhibitors in extracted DNA samples. While there are some DNA extraction and PCR protocols for such species, no consistency in results. Hence, optimization of such protocols is a prerequisite for conducting research studies on tropical flora. This study reports the optimization of cheaper DNA extraction and PCR procedures for plants having high amount of phenolic compounds and PCR inhibitors. Three fruit crop species, Bael (Aegle marmelos L. Correa), Pomegranate (Punica granatum L.) and Mango (Mangifera indica L.), with distinctly diverse secondary metabolic profiles, were used as examples. The CTAB DNA extraction method, with some modifications, was compared with two commercially available DNA extraction kits namely; Promega Wizard® Genomic DNA purification kit and QIAGEN DNeasy® Plant Mini kit. DNA from three to five genotypes from each species was extracted from each method and the quality and quantity were assessed. Spermidine was added to the PCR mix at the rate of 0.8 µM to block the PCR inhibitors and the DNA samples were amplified using universal plant barcoding primer pair rbcL, and SSR or ISSR primers. The modified CTAB method resulted significantly higher quantity of quality DNA in all samples compared to two commercial kits. Henceforth DNA extracted from CTAB method, and the two commercial kits were used to precede PCR. However, expected bands were not generated in regular PCR. Interestingly, the inclusion of spermidine amplified the relevant band/s in relatively easy PCR reactions such as rbcL, as well as trickier reactions such as SSR and ISSR. These results suggest that cheaper alternative procedures used in this research study could be used successfully for the range of applications in plants with array of secondary metabolic profiles.

show abstract

“…In addition, several substances, such as bovine serum albumin (McKeown 1994;Kreader 1996;Abu Al-Soud and Radstrom 2000) and T4 gene 32 protein (Panaccio and Lew 1991;Kreader 1996), have been used to alleviate the effects of inhibitory substances during the PCR steps. Ahokas and Erkkila (1993) reported that spermidine shows a concentrationdependent effect on the yield and specificity of PCR in plant material. Spermidine is a type of polyamine that interacts with DNA phosphate groups (Ouameur and Tajmir-Riahi 2004), and this interaction has been shown to protect small DNA molecules from common damaging agents, such as ionizing radiation and reactive oxygen species (Khan et al 1992;Spotheim-Maurizot et al 1995).…”

Section: Introductionmentioning

confidence: 98%