Limits...
A localized PCR inhibitor in a porcelain crab suggests a protective role.

El-Maklizi MA, Ouf A, Ferreira A, Hedar S, Cruz-Rivera E - PeerJ (2014)

Bottom Line: By and large, such substances are treated as random nuisances and contaminants with alternate functions; their inhibitory effects on DNA replication being a coincidental property of their molecular structure.Heat treatment was ineffective in arresting inhibition and spectrophotometric techniques suggested that the inhibitor was not a melanin-type compound.The identity of the inhibitory molecule remains unknown.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biology Department, The American University in Cairo , New Cairo , Egypt.

ABSTRACT
A number of polymerase chain reaction (PCR) inhibitors have been identified from biological and environmental samples. By and large, such substances are treated as random nuisances and contaminants with alternate functions; their inhibitory effects on DNA replication being a coincidental property of their molecular structure. Here, we demonstrate the presence of a localized PCR inhibitor in the foregut of the porcelain crab Petrolisthes rufescens (Anomura: Porcellanidae) from the Red Sea. The inhibitor precluded amplification of 28s, 16s and 18s gene sequences effectively but lost activity at 10(-2) dilutions from initial concentration. Heat treatment was ineffective in arresting inhibition and spectrophotometric techniques suggested that the inhibitor was not a melanin-type compound. The compound was not detected from midgut, hindgut, or gills of the crab. Activity of the inhibitor was precluded when samples were treated with suspensions from the midgut, suggesting that enzymatic degradation of the inhibitor likely happens at that part of the gut. As many microbial pathogens invade their hosts via ingestion, we suggest the presence of the localized inhibitor could carry a defensive or immunological role for P. rufescens. The identity of the inhibitory molecule remains unknown.

No MeSH data available.


Related in: MedlinePlus

DNA amplification at different concentrations in foreguts and fish extracts.Amplification products of 28s primers from different Petrolisthes rufescens foreguts (FG) and fish muscle (see Materials and Methods). Extracted DNA aliquots were added to reactions at different amounts in order to assess potential inhibition due to DNA template concentrations. Undiluted foregut DNA masses in PCR reactions were 18.7 ng (FG1), 4.1 ng (FG2), and 0.93 ng (FG3). From these, 50% (0.5) and 25% (0.25) dilutions were also tested. Fish DNA controls contained 1–5 ng per PCR reaction.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4260131&req=5

fig-1: DNA amplification at different concentrations in foreguts and fish extracts.Amplification products of 28s primers from different Petrolisthes rufescens foreguts (FG) and fish muscle (see Materials and Methods). Extracted DNA aliquots were added to reactions at different amounts in order to assess potential inhibition due to DNA template concentrations. Undiluted foregut DNA masses in PCR reactions were 18.7 ng (FG1), 4.1 ng (FG2), and 0.93 ng (FG3). From these, 50% (0.5) and 25% (0.25) dilutions were also tested. Fish DNA controls contained 1–5 ng per PCR reaction.

Mentions: In addition to the crab organs, fish muscle DNA (from the goatfish Upeneus nigromarginatus) was extracted using the same protocols and served as positive control in various experiments as explained below. Henceforth, the use of the word “extract” will refer to aliquots resulting from DNA extraction procedures. A NanoDrop 3300 fluorospectrometer (Thermo Scientific, Waltham, Massachusetts, USA) was used to quantify DNA extracted using the Quant-iT PicoGreen dsDNA assay kit (Life Technologies cat # P11496; Thermo Fisher, Waltham, Massachusetts, USA). As per manufacturer specifications, this instrument and technique can detect DNA concentrations down to 0.001 ng/µl. To assess the efficiency of our DNA extraction protocols, preliminary quantification was performed on parts from 6 randomly selected crabs and on the fish DNA used as control. Two serial dilutions per sample were done, 10−1 and 10−2, and an equal volume of the dye was added to each dilution before measuring absorbance 530. We also used NanoDrop fluorospectrometry throughout the study to standardize the amount of DNA in our PCR reactions to 2 ng of extracted crab DNA and 2 ng of control fish DNA. Early experiments (e.g., Fig. 1) showed the best amplification of fish DNA in reactions using this amount (see below). This is also consistent with other studies using 2 ng of DNA per 20–25 µl PCR reaction, although amplification can be observed at much lower concentrations (Andrade et al., 2012; Jin et al., 2012; Bernal-Martínez et al., 2013; Batmalle et al., 2014; Okeke et al., 2014).


A localized PCR inhibitor in a porcelain crab suggests a protective role.

El-Maklizi MA, Ouf A, Ferreira A, Hedar S, Cruz-Rivera E - PeerJ (2014)

DNA amplification at different concentrations in foreguts and fish extracts.Amplification products of 28s primers from different Petrolisthes rufescens foreguts (FG) and fish muscle (see Materials and Methods). Extracted DNA aliquots were added to reactions at different amounts in order to assess potential inhibition due to DNA template concentrations. Undiluted foregut DNA masses in PCR reactions were 18.7 ng (FG1), 4.1 ng (FG2), and 0.93 ng (FG3). From these, 50% (0.5) and 25% (0.25) dilutions were also tested. Fish DNA controls contained 1–5 ng per PCR reaction.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4260131&req=5

fig-1: DNA amplification at different concentrations in foreguts and fish extracts.Amplification products of 28s primers from different Petrolisthes rufescens foreguts (FG) and fish muscle (see Materials and Methods). Extracted DNA aliquots were added to reactions at different amounts in order to assess potential inhibition due to DNA template concentrations. Undiluted foregut DNA masses in PCR reactions were 18.7 ng (FG1), 4.1 ng (FG2), and 0.93 ng (FG3). From these, 50% (0.5) and 25% (0.25) dilutions were also tested. Fish DNA controls contained 1–5 ng per PCR reaction.
Mentions: In addition to the crab organs, fish muscle DNA (from the goatfish Upeneus nigromarginatus) was extracted using the same protocols and served as positive control in various experiments as explained below. Henceforth, the use of the word “extract” will refer to aliquots resulting from DNA extraction procedures. A NanoDrop 3300 fluorospectrometer (Thermo Scientific, Waltham, Massachusetts, USA) was used to quantify DNA extracted using the Quant-iT PicoGreen dsDNA assay kit (Life Technologies cat # P11496; Thermo Fisher, Waltham, Massachusetts, USA). As per manufacturer specifications, this instrument and technique can detect DNA concentrations down to 0.001 ng/µl. To assess the efficiency of our DNA extraction protocols, preliminary quantification was performed on parts from 6 randomly selected crabs and on the fish DNA used as control. Two serial dilutions per sample were done, 10−1 and 10−2, and an equal volume of the dye was added to each dilution before measuring absorbance 530. We also used NanoDrop fluorospectrometry throughout the study to standardize the amount of DNA in our PCR reactions to 2 ng of extracted crab DNA and 2 ng of control fish DNA. Early experiments (e.g., Fig. 1) showed the best amplification of fish DNA in reactions using this amount (see below). This is also consistent with other studies using 2 ng of DNA per 20–25 µl PCR reaction, although amplification can be observed at much lower concentrations (Andrade et al., 2012; Jin et al., 2012; Bernal-Martínez et al., 2013; Batmalle et al., 2014; Okeke et al., 2014).

Bottom Line: By and large, such substances are treated as random nuisances and contaminants with alternate functions; their inhibitory effects on DNA replication being a coincidental property of their molecular structure.Heat treatment was ineffective in arresting inhibition and spectrophotometric techniques suggested that the inhibitor was not a melanin-type compound.The identity of the inhibitory molecule remains unknown.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biology Department, The American University in Cairo , New Cairo , Egypt.

ABSTRACT
A number of polymerase chain reaction (PCR) inhibitors have been identified from biological and environmental samples. By and large, such substances are treated as random nuisances and contaminants with alternate functions; their inhibitory effects on DNA replication being a coincidental property of their molecular structure. Here, we demonstrate the presence of a localized PCR inhibitor in the foregut of the porcelain crab Petrolisthes rufescens (Anomura: Porcellanidae) from the Red Sea. The inhibitor precluded amplification of 28s, 16s and 18s gene sequences effectively but lost activity at 10(-2) dilutions from initial concentration. Heat treatment was ineffective in arresting inhibition and spectrophotometric techniques suggested that the inhibitor was not a melanin-type compound. The compound was not detected from midgut, hindgut, or gills of the crab. Activity of the inhibitor was precluded when samples were treated with suspensions from the midgut, suggesting that enzymatic degradation of the inhibitor likely happens at that part of the gut. As many microbial pathogens invade their hosts via ingestion, we suggest the presence of the localized inhibitor could carry a defensive or immunological role for P. rufescens. The identity of the inhibitory molecule remains unknown.

No MeSH data available.


Related in: MedlinePlus