Inhibition of PCR by sample contaminants

A negative PCR can mean the target was absent, or it can mean something in the tube stopped the enzyme. Heme, humic acids, ethanol, guanidine, heparin, bile salts and too much DNA are the contaminants this page is about. The decision is how to tell a true negative from a dead tube, using dilution and a spike-in, before you change the extraction. Where those contaminants come from is how DNA extraction methods differ. The cycle they interrupt is how polymerase chain reaction works.

This is reasoning, not a cleanup recipe. When you do need a different polymerase or a mix aimed at dirty templates, name the matrix in the quote request and look up enzyme classes in the molecular biology catalogue.

When to open this question, and when to stop

Open it when a sample is negative or strangely weak, the positive control worked, the no-template control is clean, and the matrix is one of the dirty ones below. Also open it when an extract smells of ethanol, looks brown, or came from a heparin tube. Do not open it first when the no-template control already shows the product. That is contamination, and dilution will not explain it.

Stop calling the sample negative when you have not diluted and you have not spiked. "No band" is then only "no band". Stop again when a spike into the sample fails: the tube is dead, and a biological negative is not available. Stop well short of a clinical or diagnostic negative. A research reading with a spike that worked is as far as this application goes.

What each contaminant is doing

Heme, from blood and from some tissues rich in blood, inhibits polymerase. A red or brown extract is a hint that heme or related porphyrins may be present. The colour is not a measurement of inhibition. It is a reason to run the spike.

Humic acids are brown, polyphenolic material from soil, sediment and some waters. They bind protein and nucleic acid, and they can quench fluorescence in a real-time assay as well as block extension. A soil extract that is the colour of tea belongs in this class until a spike says the polymerase is happy.

Ethanol is the carry-over from wash steps on columns and beads. It reaches the eluate when the matrix was not dried as that protocol asks. It inhibits polymerase at residual levels the extraction card is written to avoid. Follow that drying step. This article does not replace it. If you suspect ethanol, a dried aliquot or a fresh elution is a preparation change. The reasoning test is still whether a spike amplifies in the eluate you have now.

Guanidine stands here for guanidinium salts used as chaotropes to bind DNA to silica. Carry-over inhibits the enzyme. A drop in A260/A230 often accompanies leftover chaotrope, and it also falls when phenol or carbohydrate remains. Treat the ratio as a hint. Pair it with a spike before you condemn the extract or clear it.

Heparin is an anticoagulant in some blood-collection tubes. It co-purifies with DNA and inhibits polymerase. The tube type belongs in the sample note. If the blood was collected in heparin, a negative PCR is not interpretable until a spike has succeeded or the DNA has been cleaned by a method your institution has already accepted for heparin. Do not assume an EDTA tube and a heparin tube are the same matrix.

Bile salts come with faecal samples and with some intestinal contents. They disrupt polymerase activity. A stool extract is inhibitor-rich by default in this list. Plan the spike before you plan the sentence about absence of a target.

Excess DNA is the contaminant that is the nucleic acid itself. Too much template can suppress amplification, occupy primers on off-target sites, or drag inhibitors along in proportion to the mass you added. Absorbance at 260 nanometres will also over-call the mass when free nucleotides are present. A dye selective for double-stranded DNA, with a standard curve, is the fairer check on how much template you added. Follow the enzyme's input range. "More DNA" is a common way to make a weak reaction worse.

Dilution and spike-in are different questions

Dilution uses the same extract and less of it. If a diluted tube amplifies and the neat tube does not, inhibitor concentration was high enough to block, and target was still abundant enough to see after dilution. You may say the neat reaction was inhibited. You may then choose, as a later step, to repeat from a cleaner preparation. You may not name the chemical with certainty from the dilution pattern alone.

If the neat tube and the dilutions are all blank, dilution has not separated a true negative from a tube so inhibited that even the diluted aliquot is dead, or from a target so scarce that dilution removed it. Stop. Do not average the blanks into a confident absence.

A spike-in puts a known template into an aliquot of the sample extract. Use an amount your assay should see easily in a clean buffer, and run that clean spike beside it so you know the spike itself works. If the spike is silent inside the extract and loud in clean buffer, the extract is inhibitory. The sample result is blocked. If the spike is loud inside the extract and the sample target is absent, a true research negative is the better reading, limited to this assay and this input.

When you need to see the spike and the sample target in one tube, use a spike you can tell apart: a different length, or a probe the sample target does not light. Spiking the same target and cheering a band cannot tell you which DNA the band came from.

An internal amplification control is the planned version of the spike, designed into the assay from the start. If you do not have one yet, a one-off spike on the samples that matter is the application. Once the matrix is familiar and spikes have a history of working, you can reduce how often you spike. You cannot reduce it to zero on the first day you meet a new matrix.

Do not confuse inhibition with a bad primer or a bad cycler

A positive control in clean buffer catches the enzyme, the programme and the primers. If that positive fails, you are not in this article. Fix the reaction. If the positive works and only the dirty matrix fails, you are here. Annealing temperature and primer design still matter for specificity, but they are not the first explanation of a sample that kills a spike.

Excess cycles will not digest heme. They drive a living reaction into plateau and grow non-specific products. They do not revive a dead tube.

Safety

Blood, stool and soil are biosafety and chemical problems before they are PCR problems. Institutional rules decide containment, disinfection and waste. The reasoning in this page assumes you are already allowed to hold the extract. It does not grant that permission. Guanidine and related chaotropes are hazardous chemicals. Follow the extraction product's safety data. Do not casual-extract your way out of inhibition with phenol and chloroform because a spike failed. That is a different hazard and a different method, owned by the laboratory's SOP.

What to put in an enquiry

Name the matrix and the contaminant you suspect, the amplicon length, endpoint or real-time, and the fact that a spike failed or that dilution restored a band. Ask for an enzyme or mix class intended for that matrix, and say you will keep a no-template control and a spike. The nucleic acid analysis pathway connects the extraction to the PCR. The multiplex PCR enquiry reference is only a prompt if several targets share the inhibited tube. It is not a statement that an assay is running. Ask whether a quotation is possible. Leave the cleanup protocol to the extraction decision once the spike has told you the tube is dead.