Inhibition mechanisms of hemoglobin, immunoglobulin G, and whole blood in digital and real-time PCR.

Sidstedt, Maja; Hedman, Johannes; Romsos, Erica L; Waitara, Leticia; Wadsö, Lars; Steffen, Carolyn R; Vallone, Peter M; Rådström, Peter

Sidstedt, Maja; Hedman, Johannes; Romsos, Erica L; Waitara, Leticia; Wadsö, Lars; Steffen, Carolyn R; Vallone, Peter M; Rådström, Peter.

Afiliación

Sidstedt M; Applied Microbiology, Department of Chemistry, Lund University, P.O. Box 124, 221 00, Lund, Sweden.
Hedman J; Swedish National Forensic Centre, 581 94, Linköping, Sweden.
Romsos EL; Applied Microbiology, Department of Chemistry, Lund University, P.O. Box 124, 221 00, Lund, Sweden. johannes.hedman@tmb.lth.se.
Waitara L; Swedish National Forensic Centre, 581 94, Linköping, Sweden. johannes.hedman@tmb.lth.se.
Wadsö L; Materials Measurement Laboratory, National Institute of Standards and Technology, Gaithersburg, MD, 20899-8314, USA.
Steffen CR; Applied Microbiology, Department of Chemistry, Lund University, P.O. Box 124, 221 00, Lund, Sweden.
Vallone PM; Government Chemist Laboratory Authority, P.O. Box 164, Dar es Salaam, Tanzania.
Rådström P; Division of Building Materials, Lund University, 221 00, Lund, Sweden.

Anal Bioanal Chem ; 410(10): 2569-2583, 2018 Apr.

Article en En | MEDLINE | ID: mdl-29504082

RESUMEN

Blood samples are widely used for PCR-based DNA analysis in fields such as diagnosis of infectious diseases, cancer diagnostics, and forensic genetics. In this study, the mechanisms behind blood-induced PCR inhibition were evaluated by use of whole blood as well as known PCR-inhibitory molecules in both digital PCR and real-time PCR. Also, electrophoretic mobility shift assay was applied to investigate interactions between inhibitory proteins and DNA, and isothermal titration calorimetry was used to directly measure effects on DNA polymerase activity. Whole blood caused a decrease in the number of positive digital PCR reactions, lowered amplification efficiency, and caused severe quenching of the fluorescence of the passive reference dye 6-carboxy-X-rhodamine as well as the double-stranded DNA binding dye EvaGreen. Immunoglobulin G was found to bind to single-stranded genomic DNA, leading to increased quantification cycle values. Hemoglobin affected the DNA polymerase activity and thus lowered the amplification efficiency. Hemoglobin and hematin were shown to be the molecules in blood responsible for the fluorescence quenching. In conclusion, hemoglobin and immunoglobulin G are the two major PCR inhibitors in blood, where the first affects amplification through a direct effect on the DNA polymerase activity and quenches the fluorescence of free dye molecules, and the latter binds to single-stranded genomic DNA, hindering DNA polymerization in the first few PCR cycles. Graphical abstract PCR inhibition mechanisms of hemoglobin and immunoglobulin G (IgG). Cq quantification cycle, dsDNA double-stranded DNA, ssDNA single-stranded DNA.

Asunto(s)

ADN/sangre; Hemoglobinas/metabolismo; Inmunoglobulina G/metabolismo; Reacción en Cadena de la Polimerasa/métodos; Proteínas Bacterianas/genética; ADN/genética; ADN/metabolismo; ADN Bacteriano/sangre; ADN Bacteriano/genética; ADN Bacteriano/metabolismo; ADN Polimerasa Dirigida por ADN/metabolismo; Humanos; Proteínas de Unión a Retinoblastoma/genética; Salmonella typhimurium/genética; Ubiquitina-Proteína Ligasas/genética

Palabras clave

Blood; DNA polymerase; Digital PCR; PCR inhibition; PCR inhibitors; Real-time PCR

Texto completo

Añadir a Mi BVS

Imprimir

XML

PubMed Links

Buscar en Google

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: ADN / Hemoglobinas / Inmunoglobulina G / Reacción en Cadena de la Polimerasa Límite: Humans Idioma: En Revista: Anal Bioanal Chem Año: 2018 Tipo del documento: Article País de afiliación: Suecia Pais de publicación: Alemania

Texto completo

Añadir a Mi BVS

Imprimir

XML

PubMed Links

Buscar en Google