Quantification of Marker Mutations in Acute Leukemia

Limiting dilution polymerase chain reaction (PCR) was developed for quantifying PCR targets. This method was used for the quantification of marker mutations in acute leukemia.

By diluting DNA samples so that only one or two copies per well were present and then amplifying those copies with PCR, Prof. Morley and his team at Flinders University and Medical Center (Adelaide, SA, Australia) were able to detect two copies of leukemic DNA against a background of 160,000 normal genomes.

The team discovered that the outcome of acute leukemia can be predicted by measuring the response to treatment using limiting dilution PCR to quantify the leukemic cells at high sensitivity. Subsequently Prof. Morley's Lab used real-time quantitative polymerase chain reaction (qPCR) to develop a highly sensitive method for isolating and quantifying the chromosomal translocation that is typically associated with Chronic Myelogenous Leukemia (CML). Because the translocation point for each patient is different in CML, real-time PCR conditions may vary from patient to patient and may therefore produce different results. The lab has now returned to digital PCR.

“Advancements in digital PCR have given us the ability to overcome variations in real-time PCR amplification efficiency and have also enabled us to do away with using a standard curve,” Prof. Morley said.

Monoquant (Adelaide, SA, Australia), a company associated with Flinders University, used the Bio-Rad (Hercules, CA, USA) QX100 system to refine the new clinical test for CML. Not only does the instrument offer high sensitivity, it also removes variability in amplification efficiency that results from using patient-specific PCR primers, a traditional sticking point for the US Food and Drug Association (FDA; Silver Spring, MD, USA). Monoquant hopes the results from the QX100 system will fast track the FDA approval process for its test.


“It’s a great feeling knowing that something we helped create is propelling our work today,” Prof. Morley said. “We are hoping that this new test we’re developing will offer a better degree of monitoring and better disease management for patients by tracking the progression or remission of CML.”

Related Links:
Flinders University and Medical Center
Monoquant
Bio-Rad