Purification and Characterization of PCR-Inhibitory Components in Blood Cells

Abstract

In a recent study, immunoglobulin G in human plasma was identified as a major inhibitor of diagnostic PCR (W. Abu Al-Soud, L. J. Jönsson, and P. Rådström. J. Clin. Microbiol. 38:345-350, 2000). In this study, two major PCR inhibitors in human blood cells were purified using size exclusion and anion-exchange chromatographic procedures. Based on N-terminal amino acid sequencing and electrophoretic analysis of the purified polypeptides, hemoglobin and lactoferrin were identified as PCR-inhibitor components in erythrocytes and leukocytes, respectively. When different concentrations of hemoglobin or lactoferrin were added to PCR mixtures of 25 microl containing 10 different thermostable DNA polymerases and 1 ng of Listeria monocytogenes DNA as template DNA, AmpliTaq Gold, Pwo, and Ultma were inhibited in the presence of < or = 1.3 microg of hemoglobin and < or = 25 ng of lactoferrin, while rTth and Tli were found to resist inhibition of at least 100 microg of hemoglobin. In addition, the quantitative effects of seven low-molecular-mass inhibitors, present in blood samples or degradation products of hemoglobin, on real-time DNA synthesis of rTth using the LightCycler Instrument were investigated. A reaction system based on a single-stranded poly(dA) template with an oligo(dT) primer annealed to the 3' end was used. It was found that the addition of 0.25 to 0.1 mg of bile per ml, 2.5 mM CaCl2, 0.25 mM EDTA, 5 microM FeCl3, and 0.01 IU of heparin per ml reduced the fluorescence to approximately 76, 70, 46, 17, and 51%, respectively. Finally, the effects of nine amplification facilitators were studied in the presence of hemoglobin and lactoferrin. Bovine serum albumin (BSA) was the most efficient amplification facilitator, so that the addition of 0.4% (wt/vol) BSA allowed AmpliTaq Gold to amplify DNA in the presence of 20 instead of 1 microg of hemoglobin and 500 instead of 5 ng of lactoferrin. Including 0.02% (wt/vol) gp32, a single-stranded-DNA binding protein, in the reaction mixture of AmpliTaq Gold was also found to reduce the inhibitory effects of hemoglobin and lactoferrin.

Synergy between DNA polymerases increases polymerase chain reaction inhibitor tolerance in forensic DNA analysis

“…Furthermore, DNA polymerases may have different abilities to retain activity in different ion concentration ranges [8]. Humic and fulvic acids in soil are examples of inhibitors with DNA polymerase binding properties [26], lactoferrin and heme in blood release iron ions [27] disturbing the ion Note. The samples were amplified in duplicate using five different DNA polymerase-buffer systems: standard AmpliTaq Gold (2.5 U), AmpliTaq Gold (5 U), ExTaq Hot Start (2.5 U), PicoMaxx High Fidelity (2.5 U), and a DNA polymerase blend containing equal amounts of ExTaq Hot Start and PicoMaxx High Fidelity (2.5 U in total).…”

Comparison of four methods of extracting DNA fromD. gallinae(Acari: Dermanyssidae)

“…First, amplification of PUC plasmid DNA with M13 primers was regularly inhibited when some negative Chelex samples were added to this control PCR mix. In addition, the inhibitory effect of these samples was overcome, by extracting them a second time with the Qiamp DNA extraction kit, or by using BSA, known for reducing the inhibitory effect of haemoglobin [1]. However, the authors of studies using this Chelex resin did not notice any difficulties but they worked on non hematophagous mites (Tetranychidae and Phytoseiidae) [14,21,25,29].…”

Newborn Screening for Severe Combined Immunodeficiency in Taiwan

“…We found that the combination of 0.01% Triton X-100 in phosphate-buffered saline (PBS) and Qiagen Neutralization Solution performed the best in our system, along with an additional 0.4 µg/µL Bovine serum albumin (BSA) in the PCR mixture. Since the anticoagulants affect the PCR amplification efficiency [21,22], we tested parallel DBS samples using EDTA or heparin as the anticoagulants and compared the Ct (threshold cycle) of TREC (corresponding to the TREC copy numbers) and the Ct of RNase P. The median Ct values for TREC and RNase P without anticoagulant were 32.9 (range 32.4-33.8) and 26.71 (range 24.9-27.2), respectively. Compared to these medians, using EDTA in the blood slightly increased the Ct values of both TREC and RNase P (by 2% and 1%, respectively), while using heparin in the blood slightly decreased the values by 1% and 4%.…”