How Bicycle PCR Improves Accuracy

Standard qPCR

Standard qPCR is unable to statistically separate a low positive cancer sample compared to a normal DNA sample. This is because the probe will bind to the normal DNA at a rate around 1%. This means the noise is above the signal of a low positive cancer DNA.

Blocking PCR

Blocking PCR can clearly detect a low positive cancer DNA sample. However, blocking PCR can not distinguish between a normal DNA sample and a sample where the DNA was not added. This is because the blocker always prevents the normal DNA from replicating.

Bicycle PCR

Bicycle PCR creates clear and statistically powered separation between a low positive cancer DNA sample and a normal DNA sample. Bicycle PCR can inactivate the blocker. This allows Bicycle PCR to have the very high sensitivity of blocking PCR and the internal control of normal PCR.

Bicycle PCR

Bicycle PCR is the simple solution to greatly improving the accuracy of cancer DNA diagnostics.

Bicycle PCR can accurately detect a single copy of mutant DNA, for a single target mutation. Bicycle PCR accomplishing this by advancing the foundation understanding of PCR. This approach is completely different from most competitors. They claim to increase sensitivity by increasing the number of target genes or by using software (like Artificial Intelligence). These competitors often only mention analytical sensitivity and ignore specificity. Bicycle PCR improves most sensitivity and specificity because it changes the primary source of error (noise) in molecular diagnostics.

Bicycle PCR temporarily prevents the replication of normal DNA during a PCR. Noise, or false positive signal, is caused by probe mismatch hybridization to the normal DNA. Noise is suppressed in Bicycle PCR by temporarily preventing the replication of normal DNA. This creates large statistical separation between a normal DNA sample and a low positive cancer sample. Bicycle PCR has a validated sensitivity of 0.006% variant allele frequency.

Bicycle PCR can easily be incorporated into any standard qPCR or digital PCR assay by adding a blocking oligonucleotide and changing the thermal cycling parameters.

Bicycle PCR versus Blocking PCR

The innovation of Bicycle PCR is the ability to inactivate the blocker. Blocking PCR has been around for 30 years, beginning with PNA clamping. Numerous other blocking technologies have been invented over the years and some have been commercially launched. Thermo Fisher has castPCR and Bio-Rad licensed the NuProbe blocking technology. These blocking methods have consistently shown a sensitivity of 0.01%, however none have been widely adopted. The reason these highly sensitive technologies have not been widely used, despite high sensitivity, is because they are too good at blocking. This problem can be seen in Bio-Rad's ESR1 workflow (#12019648) or in castPCR workflow. The workflow is split into two parts, one with a blocker and one without a blocker. This split workflow highlights how current blocking strategies lack the quality control and internal reference needed to be widely used.

Bicycle PCR overcomes the issues all previous blocking technologies by inventing a method which inactivates the blocker. Bicycle PCR has two different thermal cycles parameters. The first cycle only replicates the mutant DNA, the second cycle replicates the mutant and the normal DNA. This innovation of inactivating a blocker, means that the workflow of blocking PCR no longer has to be split. The single workflow of qPCR or digital PCR can be used, together with Bicycle PCR, to create a test with high sensitivity and high specificity.