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.

The ATM (Ataxia Telangiectasia Mutated) gene encodes a protein kinase that plays a critical role in the DNA damage response. ATM activates various downstream proteins, including p53, to initiate DNA repair or apoptosis. Mutations in the ATM gene are common in chronic lymphocytic leukemia (CLL) and lead to genomic instability, contributing to disease progression and treatment resistance. ATM interacts with several key proteins involved in DNA repair, such as BRCA1 and RAD50, coordinating the cellular response to DNA damage. Identifying ATM mutations helps in understanding the molecular mechanisms of CLL and developing personalized therapeutic strategies. Some important ATM gene variants are listed below:

ATM c.7271T>G / p.V2424G: A DNA point mutation from thymine (T) to guanine (G) at nucleotide position 7271 (c.7271T>G) results in the substitution of valine (V) with glycine (G) at position 2424 of the ATM protein (p.V2424G). This mutation affects the kinase domain of ATM, impairing its ability to respond to DNA damage. The loss of ATM function due to this mutation is associated with genomic instability and progression of chronic lymphocytic leukemia (CLL).
ATM c.6095G>A / p.R2032H: A DNA point mutation from guanine (G) to adenine (A) at nucleotide position 6095 (c.6095G>A) results in the substitution of arginine (R) with histidine (H) at position 2032 of the ATM protein (p.R2032H). This mutation impairs ATM's kinase activity, leading to defective DNA repair mechanisms and contributing to the development of CLL.
ATM c.6161T>C / p.L2054P: A DNA point mutation from thymine (T) to cytosine (C) at nucleotide position 6161 (c.6161T>C) results in the substitution of leucine (L) with proline (P) at position 2054 of the ATM protein (p.L2054P). This mutation disrupts the structural integrity of the ATM protein, compromising its function in DNA damage response and contributing to CLL pathogenesis.

The BCR-ABL gene is created by a translocation between chromosomes 9 and 22. This change produces the Philadelphia chromosome, a hallmark of chronic myeloid leukemia (CML). This fusion gene encodes a constitutively active tyrosine kinase that drives cell proliferation and survival, characteristic of CML. The continuous activation of signaling pathways by BCR-ABL promotes uncontrolled cell growth and resistance to apoptosis. BCR-ABL activates multiple downstream signaling pathways, including the RAS/MAPK, PI3K/AKT and JAK/STAT pathways, which contribute to cell proliferation, survival and differentiation. Targeted therapies, such as tyrosine kinase inhibitors (TKIs), have been developed to inhibit the BCR-ABL kinase, significantly improving outcomes for CML patients.

BCR-ABL1 (p210): The BCR-ABL1 fusion gene creates a constitutively active tyrosine kinase due to the translocation between chromosomes 9 and 22. This fusion protein promotes continuous signaling for cell growth and survival, a hallmark of chronic myeloid leukemia (CML). Targeting BCR-ABL1 with tyrosine kinase inhibitors (TKIs) has transformed the treatment landscape of CML, leading to significant improvements in patient outcomes.
BCR-ABL1 c.944T>C / p.F317L: A DNA point mutation from thymine (T) to cytosine (C) at nucleotide position 944 (c.944T>C) results in the substitution of phenylalanine (F) with leucine (L) at position 317 of the BCR-ABL1 protein (p.F317L). This mutation is associated with resistance to certain tyrosine kinase inhibitors (TKIs), complicating treatment strategies for chronic myeloid leukemia (CML).
BCR-ABL1 c.922A>C / p.T315I: A DNA point mutation from adenine (A) to cytosine (C) at nucleotide position 922 (c.922A>C) results in the substitution of threonine (T) with isoleucine (I) at position 315 of the BCR-ABL1 protein (p.T315I). This mutation causes resistance to multiple TKIs and is known as the "gatekeeper" mutation, posing significant challenges in the treatment of CML.

NOTCH1 is a gene that encodes a transmembrane receptor involved in cell differentiation, proliferation and apoptosis. Mutations in NOTCH1 are frequently observed in chronic lymphocytic leukemia (CLL) and are associated with aggressive disease and poor prognosis. These mutations lead to constitutive activation of the NOTCH1 signaling pathway, promoting uncontrolled cell growth and survival. NOTCH1 signaling interacts with other pathways, such as NF-κB and PI3K/AKT, to regulate cell fate decisions. Targeting the NOTCH1 pathway is a potential therapeutic approach in CLL. Some notable NOTCH1 variants include the following:

NOTCH1 c.7544_7545delCT / p.P2515fs: A deletion of cytosine (C) and thymine (T) at nucleotide positions 7544 and 7545 (c.7544_7545delCT) results in a frameshift mutation, leading to a premature stop codon in the NOTCH1 protein (p.P2515fs). This mutation leads to the constitutive activation of the NOTCH1 signaling pathway, promoting the proliferation and survival of leukemia cells in chronic lymphocytic leukemia (CLL).
NOTCH1 c.6788G>T / p.V2273F: A DNA point mutation from guanine (G) to thymine (T) at nucleotide position 6788 (c.6788G>T) results in the substitution of valine (V) with phenylalanine (F) at position 2273 of the NOTCH1 protein (p.V2273F). This mutation leads to gain-of-function in the NOTCH1 receptor, promoting leukemogenesis in CLL.
NOTCH1 c.6798C>A / p.A2276D: A DNA point mutation from cytosine (C) to adenine (A) at nucleotide position 6798 (c.6798C>A) results in the substitution of alanine (A) with aspartic acid (D) at position 2276 of the NOTCH1 protein (p.A2276D). This mutation causes constitutive activation of NOTCH1, contributing to the aggressive behavior of CLL cells.

SF3B1 (Splicing Factor 3b Subunit 1) is a gene that encodes a core component of the spliceosome, the complex responsible for the precise removal of introns from pre-mRNA. Mutations in SF3B1 are frequently observed in chronic lymphocytic leukemia (CLL) and are associated with distinct clinical features and prognosis. These mutations disrupt normal splicing, leading to the production of aberrant mRNA and proteins that contribute to leukemogenesis. SF3B1 interacts with other splicing factors to ensure accurate splicing, and its mutations can alter the expression and function of numerous genes involved in cell cycle regulation, apoptosis and DNA repair. Understanding SF3B1 mutations is essential for developing targeted therapies and improving treatment outcomes in CLL. Some important SF3B1 variants include the following:

SF3B1 c.2098A>G / p.K700E: A DNA point mutation from adenine (A) to guanine (G) at nucleotide position 2098 (c.2098A>G) results in the substitution of lysine (K) with glutamic acid (E) at position 700 of the SF3B1 protein (p.K700E). This mutation affects the HEAT repeat domains of SF3B1, disrupting normal splicing activity and leading to the dysregulation of multiple genes, contributing to CLL pathogenesis.
SF3B1 c.2101A>G / p.K700Q: A DNA point mutation from adenine (A) to guanine (G) at nucleotide position 2101 (c.2101A>G) results in the substitution of lysine (K) with glutamine (Q) at position 700 of the SF3B1 protein (p.K700Q). Similar to the K700E mutation, this variant disrupts the function of the spliceosome, leading to aberrant mRNA splicing and the promotion of leukemic cell growth in CLL.
SF3B1 c.1793G>T / p.R625L: A DNA point mutation from guanine (G) to thymine (T) at nucleotide position 1793 (c.1793G>T) results in the substitution of arginine (R) with leucine (L) at position 625 of the SF3B1 protein (p.R625L). This mutation alters the spliceosome's ability to accurately process pre-mRNA, resulting in the production of defective proteins that drive CLL progression.

TP53 (Tumor Protein p53) is a crucial tumor suppressor gene involved in regulating the cell cycle, DNA repair and apoptosis. Mutations in TP53 are associated with more aggressive forms of chronic lymphocytic leukemia (CLL) and poor prognosis. Loss of p53 function due to mutations allows cells with damaged DNA to proliferate, contributing to tumor development and progression. TP53 interacts with various proteins, including MDM2, which regulates p53 levels, and ATM, which activates p53 in response to DNA damage. Understanding TP53 mutations is essential for developing targeted therapies and improving treatment outcomes in CLL. Some relevant TP53 variants include the following:

TP53 c.375G>A / p.R125Q: A DNA point mutation from guanine (G) to adenine (A) at nucleotide position 375 (c.375G>A) results in the substitution of arginine (R) with glutamine (Q) at position 125 of the TP53 protein (p.R125Q). This mutation disrupts the DNA-binding domain of p53, impairing its tumor suppressor function. Loss of p53 function due to this mutation contributes to the aggressive nature and treatment resistance of chronic lymphocytic leukemia (CLL).
TP53 c.817C>T / p.R273C: A DNA point mutation from cytosine (C) to thymine (T) at nucleotide position 817 (c.817C>T) results in the substitution of arginine (R) with cysteine (C) at position 273 of the TP53 protein (p.R273C). This mutation affects the DNA-binding domain, leading to loss of p53 function and promoting unchecked cellular proliferation in CLL.
TP53 c.742C>T / p.R248W: A DNA point mutation from cytosine (C) to thymine (T) at nucleotide position 742 (c.742C>T) results in the substitution of arginine (R) with tryptophan (W) at position 248 of the TP53 protein (p.R248W). This mutation disrupts the DNA-binding capability of p53, leading to loss of tumor suppressor activity and contributing to CLL progression.

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