Frequently asked questions

dPCR LNA Mutation Assays offer significant advantages to cancer researchers working on precise and sensitive mutation detection. These assays are specifically designed for use with the QIAcuity Digital PCR System and are enhanced with Locked Nucleic Acid (LNA) technology. This enhancement greatly improves the specificity and sensitivity of mutation detection, making it possible to identify DNA sequence mutations at very low abundance, with a sensitivity as fine as 0.1% in a single nanoplate well.

The key benefits of dPCR LNA Mutation Assays for cancer researchers include:

High precision and sensitivity: The use of duplex, hydrolysis probe-based assays allows for highly precise detection of mutations. The presence of both mutant and wild-type probes in the same reaction ensures that researchers can detect and quantify minor genetic variations with great accuracy, crucial for studies in heterogeneous cancer samples where only a few cells may carry the mutation.
Enhanced specificity: The integration of LNA into the probes increases the binding affinity and specificity towards the target sequences, minimizing the risk of non-specific bindings and improving the overall reliability of the assays.
Multiplexing capability: Each assay is capable of detecting mutations using two different fluorescent dye combinations, allowing for the simultaneous analysis of mutant and wild-type alleles within the same reaction. This multiplexing ability is particularly useful in applications requiring the analysis of multiple targets, such as assessing co-occurring mutations in cancer.
Flexibility in sample analysis: By dividing the reaction across multiple wells, even greater sensitivity can be achieved, facilitating the detection of extremely rare mutations. This is especially valuable in cancer research, where detecting low-frequency mutations can inform prognosis and treatment strategies.
Streamlined workflow: Supplied in a single-tube format with ready-to-use primer pairs and probes, these assays simplify the experimental setup, enabling efficient and straightforward integration into existing research workflows.

ACVR1 (Activin A Receptor Type 1) encodes a receptor serine/threonine kinase that is involved in the bone morphogenetic protein (BMP) signaling pathway, which regulates cell growth, differentiation and apoptosis. In brain cancer, particularly in diffuse intrinsic pontine gliomas (DIPG), ACVR1 mutations are common and lead to aberrant BMP signaling. These mutations result in constitutive activation of the ACVR1 receptor, promoting tumor growth and resistance to apoptosis. ACVR1 mutations are associated with distinct biological behaviors and can influence the response to targeted therapies in DIPG.

BRAF (B-Raf Proto-Oncogene, Serine/Threonine Kinase) is a proto-oncogene that encodes a protein kinase involved in the MAPK/ERK signaling pathway, which regulates cell growth, differentiation and survival. In brain cancer, particularly in certain pediatric brain tumors such as pilocytic astrocytomas and pleomorphic xanthoastrocytomas, BRAF mutations, especially V600E, are common. These mutations lead to constitutive activation of the BRAF kinase, driving continuous MAPK/ERK pathway signaling and promoting tumor growth. BRAF mutations are associated with distinct biological behaviors and can influence the response to targeted therapies, such as BRAF inhibitors.

EGFR (Epidermal Growth Factor Receptor) is a proto-oncogene that encodes a receptor tyrosine kinase involved in the regulation of cell growth, survival and differentiation. In brain cancer, particularly glioblastoma, EGFR mutations and amplifications are frequently observed and are associated with aggressive tumor behavior and poor prognosis. The EGFR protein interacts with various downstream signaling pathways, including the PI3K/AKT and MAPK/ERK pathways, to promote cell proliferation and survival. Overexpression or mutation of EGFR leads to continuous activation of these pathways, driving tumor growth and resistance to apoptosis. Targeting EGFR with specific inhibitors is a therapeutic strategy in EGFR-mutant glioblastomas.

EGFR c.2573T>G / L858R: A DNA point mutation from thymine (T) to guanine (G) at nucleotide position 2573 (c.2573T>G) results in the substitution of leucine (L) with arginine (R) at position 858 of the EGFR protein (L858R). This mutation occurs in the tyrosine kinase domain of EGFR, leading to constitutive activation of the receptor. The L858R mutation enhances EGFR signaling through the PI3K/AKT and MAPK/ERK pathways, promoting cell proliferation and survival. This variant is commonly found in glioblastomas and is associated with aggressive tumor behavior and poor prognosis. Targeting L858R with specific EGFR inhibitors can be an effective therapeutic strategy.
EGFR c.2235_2249del / Exon 19 Deletion: A deletion of 15 base pairs in exon 19 (c.2235_2249del) results in the loss of five amino acids in the EGFR protein. This exon 19 deletion leads to constitutive activation of the EGFR tyrosine kinase domain, enhancing downstream signaling pathways such as PI3K/AKT and MAPK/ERK. The exon 19 deletion is frequently observed in glioblastomas and contributes to tumor growth and resistance to apoptosis. This variant is a target for EGFR inhibitors, which can inhibit the aberrant signaling and reduce tumor progression.
EGFR c.2369C>T / T790M: A DNA point mutation from cytosine (C) to thymine (T) at nucleotide position 2369 (c.2369C>T) results in the substitution of threonine (T) with methionine (M) at position 790 of the EGFR protein (T790M). This mutation occurs in the tyrosine kinase domain and is often associated with resistance to first- and second-generation EGFR inhibitors. The T790M mutation increases the affinity of EGFR for ATP, reducing the effectiveness of ATP-competitive inhibitors. It is commonly found in glioblastomas that have developed resistance to initial EGFR-targeted therapies. Newer generation inhibitors, such as osimertinib, are designed to target this mutation.
EGFR c.2361G>A / C797S: A DNA point mutation from guanine (G) to adenine (A) at nucleotide position 2361 (c.2361G>A) results in the substitution of cysteine (C) with serine (S) at position 797 of the EGFR protein (C797S). This mutation occurs in the tyrosine kinase domain and is associated with resistance to third-generation EGFR inhibitors, such as osimertinib. The C797S mutation prevents the covalent binding of these inhibitors to the EGFR protein, allowing continued signaling through the PI3K/AKT and MAPK/ERK pathways. This variant is significant in the context of acquired resistance in glioblastomas.
EGFR c.2570T>C / L861Q: A DNA point mutation from thymine (T) to cytosine (C) at nucleotide position 2570 (c.2570T>C) results in the substitution of leucine (L) with glutamine (Q) at position 861 of the EGFR protein (L861Q). This mutation occurs in the tyrosine kinase domain and leads to constitutive activation of EGFR, promoting cell proliferation and survival through downstream signaling pathways. The L861Q mutation is less common but still relevant in glioblastomas, contributing to tumor growth and progression. Targeting this variant with specific EGFR inhibitors can be a therapeutic approach.

H3F3A (H3 Histone Family Member 3A) encodes a variant of the histone H3 protein, which is involved in the packaging of DNA into chromatin and regulation of gene expression. In brain cancer, particularly in pediatric high-grade gliomas and diffuse midline gliomas, mutations in H3F3A, such as K27M and G34R/V, are characteristic. These mutations alter the chromatin structure and epigenetic landscape, leading to dysregulation of gene expression and promoting tumorigenesis. H3F3A mutations are associated with aggressive tumor behavior and poor prognosis, and they define distinct molecular subgroups of brain tumors.

IDH1 (Isocitrate Dehydrogenase 1) encodes an enzyme that is involved in the citric acid cycle, playing a role in cellular metabolism. In brain cancer, particularly lower-grade gliomas and secondary glioblastomas, mutations in IDH1 are common and lead to the production of an oncometabolite called 2-hydroxyglutarate (2-HG). This metabolite interferes with cellular differentiation and promotes tumorigenesis by altering the epigenetic landscape of the cell. IDH1 mutations are associated with distinct metabolic and epigenetic changes that drive tumorigenesis. These mutations are also linked to better prognosis and response to certain therapies compared to IDH1 wild-type gliomas. Some important variants in IGH1 associated with brain cancer include:

IDH1 c.395G>A / R132H: A DNA point mutation from guanine (G) to adenine (A) at nucleotide position 395 (c.395G>A) results in the substitution of arginine (R) with histidine (H) at position 132 of the IDH1 protein (R132H). This mutation occurs at the active site of the enzyme, leading to the production of the oncometabolite 2-hydroxyglutarate (2-HG). The accumulation of 2-HG interferes with cellular differentiation and promotes tumorigenesis by altering the epigenetic landscape. The R132H mutation is the most common IDH1 mutation in gliomas and is associated with better prognosis and response to certain therapies compared to IDH1 wild-type gliomas.
IDH1 c.394C>T / R132C: A DNA point mutation from cytosine (C) to thymine (T) at nucleotide position 394 (c.394C>T) results in the substitution of arginine (R) with cysteine (C) at position 132 of the IDH1 protein (R132C). Similar to R132H, this mutation leads to the production of 2-hydroxyglutarate (2-HG), promoting tumorigenesis through epigenetic alterations. The R132C mutation is less common than R132H but still significant in the context of glioma development and progression. It is associated with distinct metabolic and epigenetic changes that drive tumorigenesis.

NF2 (Neurofibromin 2) is a tumor suppressor gene that encodes the protein merlin, which is involved in regulating cell growth, adhesion and cytoskeletal organization. In brain cancer, particularly in meningiomas and schwannomas, NF2 mutations lead to the loss of merlin function, resulting in dysregulated cell growth and adhesion. The NF2 protein interacts with various signaling pathways, including the Hippo pathway, to control cell proliferation and survival. Loss of NF2 function contributes to the development and progression of these tumors, and NF2 mutations are a hallmark of neurofibromatosis type 2, a genetic disorder that predisposes individuals to multiple nervous system tumors.

PIK3CA (Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Alpha) encodes the p110α catalytic subunit of the PI3K enzyme, which is involved in the PI3K/AKT/mTOR signaling pathway. This pathway regulates cell growth, proliferation and survival. In brain cancer, PIK3CA mutations lead to the activation of the PI3K/AKT pathway, promoting tumor growth and survival. The PIK3CA protein interacts with the regulatory subunit p85 and other signaling molecules to mediate its effects. Dysregulation of this pathway due to PIK3CA mutations contributes to brain cancer progression and resistance to certain therapies. Targeting the PI3K/AKT/mTOR pathway is a potential therapeutic strategy in PIK3CA-mutant brain cancers.

PTCH2 (Patched 2) is a tumor suppressor gene that encodes a receptor involved in the Hedgehog signaling pathway, which regulates cell growth, differentiation and tissue patterning. In brain cancer, particularly in medulloblastomas, PTCH2 mutations lead to dysregulation of the Hedgehog pathway, promoting tumor growth and survival. The PTCH2 protein interacts with the Smoothened (SMO) receptor to inhibit Hedgehog signaling. Loss of PTCH2 function results in continuous activation of the pathway, contributing to the development and progression of medulloblastomas. PTCH2 mutations are associated with distinct molecular subgroups of medulloblastomas and can influence the response to targeted therapies.

PTEN (Phosphatase and Tensin Homolog) is a tumor suppressor gene that encodes a phosphatase involved in the negative regulation of the PI3K/AKT signaling pathway. In brain cancer, particularly glioblastoma, PTEN mutations lead to unregulated activation of the PI3K/AKT pathway, promoting cell growth and survival. The PTEN protein dephosphorylates phosphatidylinositol-3,4,5-trisphosphate (PIP3), antagonizing PI3K signaling. Loss of PTEN function results in increased PIP3 levels, continuous activation of AKT and enhanced cell proliferation and survival. PTEN mutations contribute to the aggressive nature of brain tumors and are associated with poor prognosis.

TP53 (Tumor Protein p53) is a crucial tumor suppressor gene that encodes the p53 protein, which plays a vital role in regulating the cell cycle, DNA repair and apoptosis. In brain cancer, particularly glioblastoma (GBM), TP53 mutations are common and contribute significantly to tumor development and progression. The p53 protein acts as a guardian of the genome, responding to DNA damage by either repairing the damage or initiating cell death if the damage is irreparable. It interacts with various proteins involved in cell cycle control, such as MDM2, which regulates p53 levels through a feedback loop. Loss of p53 function due to mutations leads to uncontrolled cell proliferation, genomic instability and resistance to apoptosis, all of which contribute to the aggressive nature of brain tumors. The TP53 variants below play a critical role in brain cancer by disrupting the normal tumor suppressor functions of p53.

TP53 c.743G>A / R248Q: A DNA point mutation from guanine (G) to adenine (A) at nucleotide position 743 (c.743G>A) results in the substitution of arginine (R) with glutamine (Q) at position 248 of the TP53 protein (R248Q). This mutation occurs in the DNA-binding domain of p53, impairing its ability to bind to DNA and activate transcription of target genes involved in cell cycle arrest and apoptosis. The R248Q mutation leads to loss of tumor suppressor function, contributing to uncontrolled cell proliferation and survival, which are hallmarks of brain cancer. This variant is one of the most common TP53 mutations found in glioblastomas, significantly impacting disease progression and treatment response.
TP53 c.818G>A / R273H: A DNA point mutation from guanine (G) to adenine (A) at nucleotide position 818 (c.818G>A) results in the substitution of arginine (R) with histidine (H) at position 273 of the TP53 protein (R273H). This mutation affects the DNA-binding domain of p53, reducing its ability to regulate genes involved in DNA repair, cell cycle control and apoptosis. The R273H mutation leads to a dominant-negative effect, where the mutant p53 protein interferes with the function of any remaining wild-type p53, further promoting tumorigenesis. This variant is frequently observed in brain cancer and is associated with poor prognosis and resistance to chemotherapy.
TP53 c.524G>A / R175H: A DNA point mutation from guanine (G) to adenine (A) at nucleotide position 524 (c.524G>A) results in the substitution of arginine (R) with histidine (H) at position 175 of the TP53 protein (R175H). This mutation occurs in the core DNA-binding domain, disrupting the structural integrity of p53 and its ability to bind DNA. The R175H mutation leads to loss of tumor suppressor activity, allowing cells with DNA damage to proliferate unchecked. This variant is commonly found in brain cancer and contributes to the aggressive nature of the disease and resistance to standard treatments.
TP53 c.844C>T / R282W: A DNA point mutation from cytosine (C) to thymine (T) at nucleotide position 844 (c.844C>T) results in the substitution of arginine (R) with tryptophan (W) at position 282 of the TP53 protein (R282W). This mutation affects the DNA-binding domain, impairing p53's ability to regulate target genes involved in cell cycle arrest and apoptosis. The R282W mutation results in a loss of function, contributing to the development and progression of brain cancer by allowing cells to evade growth control mechanisms. This variant is associated with poor clinical outcomes and resistance to chemotherapy.
TP53 c.1010G>A / E337K: A DNA point mutation from guanine (G) to adenine (A) at nucleotide position 1010 (c.1010G>A) results in the substitution of glutamic acid (E) with lysine (K) at position 337 of the TP53 protein (E337K). This mutation occurs in the oligomerization domain, affecting p53's ability to form tetramers, which are necessary for its full tumor suppressor activity. The E337K mutation leads to a partial loss of function, contributing to the development and progression of brain cancer by impairing p53-mediated cell cycle arrest and apoptosis. This variant is less common but still significant in the context of brain cancer pathogenesis.

Digital PCR assays for brain cancer gene variants

Revolutionizing brain cancer research with precision dPCR assays

Explore brain cancer related dPCR assays by gene

Discover the QIAcuity family of dPCR instruments

Transform your research capabilities with QIAcuity digital PCR

Streamline your clinical PCR workflows with QIAcuityDx

Frequently asked questions

Disclaimers