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Hemoglobin and Immunoglobulin G Inhibition Mechanisms in Diagnostic qPCR

Waitara, Leticia LU (2016) KMBM01 20161
Applied Microbiology
Biotechnology
Abstract
Blood samples are extensively used in clinical and forensic fields for genetic material analysis using real-time PCR. Although PCR is a well-established method distinguished for its rapid and sensitive molecular diagnosis, blood components co- purified with nucleic acids have shown to inhibit amplification and detection of PCR products. In this study, IgG and hemoglobin were investigated for their inhibitory mechanisms in qPCR and dPCR.
Using qPCR fluorescence readings, hemoglobin was added to pure amplicons reactions to investigate its fluorescence quenching. IgG was investigated for its nucleic acids capture. Various IgG concentrations were added to PCR mixtures containing either single or double stranded DNA. Heat induced complex... (More)
Blood samples are extensively used in clinical and forensic fields for genetic material analysis using real-time PCR. Although PCR is a well-established method distinguished for its rapid and sensitive molecular diagnosis, blood components co- purified with nucleic acids have shown to inhibit amplification and detection of PCR products. In this study, IgG and hemoglobin were investigated for their inhibitory mechanisms in qPCR and dPCR.
Using qPCR fluorescence readings, hemoglobin was added to pure amplicons reactions to investigate its fluorescence quenching. IgG was investigated for its nucleic acids capture. Various IgG concentrations were added to PCR mixtures containing either single or double stranded DNA. Heat induced complex formation of IgG with ssDNA evaluation involved preheating IgG with DNA, primers or primers- DNA prior their addition to PCR mixtures for amplification. Furthermore, various amounts of whole blood with and without EDTA in their liquid and dry states challenged PCR amplification efficiency.
Hemoglobin caused lowered amplification efficiency and fluorescence quenching in dPCR while as qPCR demonstrated this quenching effect particularly for EG dye. IgG delayed amplification in both qPCR and dPCR. IgG interaction preference towards ssDNA over dsDNA was more noticeable in dPCR than in qPCR. Whole blood lowered PCR amplification efficiency and as little as 0.5%(v/v) resulted in amplification curves with lowered slope. Overall, dPCR was more tolerant than qPCR.
Results obtained here outline and support the plausible mechanisms of IgG and hemoglobin in PCR-based methods. Further investigations to confirm the mechanisms mentioned would give valuable insights that can aid in designing more inhibitor- tolerant reactions. (Less)
Popular Abstract
Real-time polymerase chain reaction, also known as qPCR, is the standard molecular biology technique employed for genetic material (i.e. DNA/RNA) analysis purposes.
PCR amplifies a single target DNA sequence into millions of copies. Meanwhile,these generated copies are detected by fluorescence allowing the analyst to follow the
progress of the reaction in real-time.
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author
Waitara, Leticia LU
supervisor
organization
course
KMBM01 20161
year
type
H2 - Master's Degree (Two Years)
subject
keywords
by Hemoglobin and Immunoglobulin, inhibition, Real-time PCR, applied microbiology, teknisk mikrobiologi
language
English
id
8889137
date added to LUP
2016-08-30 11:10:17
date last changed
2016-08-30 11:10:17
@misc{8889137,
  abstract     = {Blood samples are extensively used in clinical and forensic fields for genetic material analysis using real-time PCR. Although PCR is a well-established method distinguished for its rapid and sensitive molecular diagnosis, blood components co- purified with nucleic acids have shown to inhibit amplification and detection of PCR products. In this study, IgG and hemoglobin were investigated for their inhibitory mechanisms in qPCR and dPCR.
Using qPCR fluorescence readings, hemoglobin was added to pure amplicons reactions to investigate its fluorescence quenching. IgG was investigated for its nucleic acids capture. Various IgG concentrations were added to PCR mixtures containing either single or double stranded DNA. Heat induced complex formation of IgG with ssDNA evaluation involved preheating IgG with DNA, primers or primers- DNA prior their addition to PCR mixtures for amplification. Furthermore, various amounts of whole blood with and without EDTA in their liquid and dry states challenged PCR amplification efficiency.
Hemoglobin caused lowered amplification efficiency and fluorescence quenching in dPCR while as qPCR demonstrated this quenching effect particularly for EG dye. IgG delayed amplification in both qPCR and dPCR. IgG interaction preference towards ssDNA over dsDNA was more noticeable in dPCR than in qPCR. Whole blood lowered PCR amplification efficiency and as little as 0.5%(v/v) resulted in amplification curves with lowered slope. Overall, dPCR was more tolerant than qPCR.
Results obtained here outline and support the plausible mechanisms of IgG and hemoglobin in PCR-based methods. Further investigations to confirm the mechanisms mentioned would give valuable insights that can aid in designing more inhibitor- tolerant reactions.},
  author       = {Waitara, Leticia},
  keyword      = {by Hemoglobin and Immunoglobulin,inhibition,Real-time PCR,applied microbiology,teknisk mikrobiologi},
  language     = {eng},
  note         = {Student Paper},
  title        = {Hemoglobin and Immunoglobulin G Inhibition Mechanisms in Diagnostic qPCR},
  year         = {2016},
}