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

Dilution of midgut aliquots and foregut inhibition of PCR.Tests of the activity limits of midgut (MG) suspensions to counteract the foregut (FG) PCR inhibitor (N = 3). Fish DNA (3 µl ) was mixed with FG extracts and one of five concentrations of MG extract (baseline concentration to 10−4) from the same crab before amplifying with 18s primers. Presence of PCR product indicates the neutralization of the inhibitor. Positive and negative controls are as in Fig. 4. Empty lanes are not labeled. Though not completely clear from the picture, a very faint band, denoting amplification, was observed for FG3 + MG3(10−4).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig-6: Dilution of midgut aliquots and foregut inhibition of PCR.Tests of the activity limits of midgut (MG) suspensions to counteract the foregut (FG) PCR inhibitor (N = 3). Fish DNA (3 µl ) was mixed with FG extracts and one of five concentrations of MG extract (baseline concentration to 10−4) from the same crab before amplifying with 18s primers. Presence of PCR product indicates the neutralization of the inhibitor. Positive and negative controls are as in Fig. 4. Empty lanes are not labeled. Though not completely clear from the picture, a very faint band, denoting amplification, was observed for FG3 + MG3(10−4).

Mentions: Amplification of control DNA with 18s in the presence of foregut extracts was possible if midgut suspensions were present in the mix, thus suggesting regulation of the inhibitor in the midgut (Fig. 5). The effect of the midgut component (or components) counteracting the activity of the inhibitor was detected even at 10−4 dilutions of the original midgut aliquot (Fig. 6). For the sample FG3 + MG3(10−4) there was only a faint band, suggesting that the dilution of the midgut was approaching the limit of activity against the inhibitor. For these two experiments, both positive and negative controls showed the expected patterns (Figs. 5 and 6).


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)

Dilution of midgut aliquots and foregut inhibition of PCR.Tests of the activity limits of midgut (MG) suspensions to counteract the foregut (FG) PCR inhibitor (N = 3). Fish DNA (3 µl ) was mixed with FG extracts and one of five concentrations of MG extract (baseline concentration to 10−4) from the same crab before amplifying with 18s primers. Presence of PCR product indicates the neutralization of the inhibitor. Positive and negative controls are as in Fig. 4. Empty lanes are not labeled. Though not completely clear from the picture, a very faint band, denoting amplification, was observed for FG3 + MG3(10−4).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig-6: Dilution of midgut aliquots and foregut inhibition of PCR.Tests of the activity limits of midgut (MG) suspensions to counteract the foregut (FG) PCR inhibitor (N = 3). Fish DNA (3 µl ) was mixed with FG extracts and one of five concentrations of MG extract (baseline concentration to 10−4) from the same crab before amplifying with 18s primers. Presence of PCR product indicates the neutralization of the inhibitor. Positive and negative controls are as in Fig. 4. Empty lanes are not labeled. Though not completely clear from the picture, a very faint band, denoting amplification, was observed for FG3 + MG3(10−4).
Mentions: Amplification of control DNA with 18s in the presence of foregut extracts was possible if midgut suspensions were present in the mix, thus suggesting regulation of the inhibitor in the midgut (Fig. 5). The effect of the midgut component (or components) counteracting the activity of the inhibitor was detected even at 10−4 dilutions of the original midgut aliquot (Fig. 6). For the sample FG3 + MG3(10−4) there was only a faint band, suggesting that the dilution of the midgut was approaching the limit of activity against the inhibitor. For these two experiments, both positive and negative controls showed the expected patterns (Figs. 5 and 6).

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