QClamp® Gene Mutation Detection Tests | DiaCarta, Inc.

QClamp® Gene Mutation Detection Tests are highly sensitive qPCR-based assays for tissue biopsy (FFPE) and liquid biopsy (ctDNA) applications. QClamp® assays detect reliably 0.1% to 0.5% mutant DNA out of wild-type DNA for targeted mutations, providing lower detection limit compared with competition due to strict enrichment of mutant sequences while suppressing amplification of wild-type sequences.

QClamp® assays provide a rapid, reproducible and affordable solution which employs a simple workflow and PCR machines that are commonly used in research and clinical labs. The tests can be performed on standard real-time qPCR instruments such as ABI QuantStudio 5, Roche LightCycler® 480 and Bio-Rad CFX384. 

QClamp® Gene Mutation Detection Test Offerings

QClamp® KRAS Mutation Detection Test

Detects codons 12, 13, 59, 61, 117 and 146 for FFPE and plasma samples.

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QClamp® NRAS Mutation Detection Test

Detects codons 12, 13, 59, 61, 117 and 146 for FFPE and plasma samples.

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QClamp® EGFR Mutation Detection Test

Detects codons 719, 861, Ex19del, Ex20insASV and S768I, T790M and L858R mutations for FFPE and plasma samples.

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QClamp® BRAF Mutation Detection Test

Detects codon 600 for FFPE and plasma samples.

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QClamp® JAK2 Mutation Detection Test

Detects codon 617 for plasma samples.

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QClamp® PIK3CA Mutation Detection Test

Detects codons 542, 545 and 1047 for FFPE and plasma samples.

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Advantages of QClamp® Gene Mutation Detection Test

ULTRA-SENSITIVE

Detect reliably 0.1% to 0.5% VAF mutant DNA out of wild-type DNA for targeted mutations

SAMPLE READY

Suitable for plasma and FFPE samples

LOW INPUT DNA

Minimum 5ng input DNA per reaction. Less than 2 tubes of blood (10mL each) needed for cfDNA

COMPREHENSIVE COVERAGE

Covering all relevant somatic mutations in KRAS, NRAS, EGFR, BRAF, JAK2 and PIK3CA oncogenes

FAST RESULTS

Less than 4 hours of assay run time

GREAT VERSATILITY

Validated on the most common qPCR machines with minimized variability

Gene Mutation Detection and Cancer

Cancer diagnostics is critical to accurately diagnose cancer for optimal therapy to save or extend cancer patients’ lives. With the development of different types of biomarkers, including genetic biomarkers, cancer patients can get much more detailed profiling based on these biomarkers, rather just cancer types, and personalized medicine can be applied for targeted therapy.

One great example is targeted therapies using companion diagnostics to identify EGFR mutations among non-small cell lung cancer (NSCLC) at different stages of the therapy and apply different tyrosine inhibitor drugs.

Challenges for Detecting Cancer Mutations

Due to the heterogeneity of tumor tissues, it is challenging to be able to detect the mutations residing in a small population of tumor cells within a large number of normal cells.

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The first challenge is to get the tumor tissue that contains more tumor cells. Pathologists can use micro-dissection to get the samples for testing to increase the chance of getting more tumor cells in the testing samples.

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The next challenge is the testing method. Multiple testing methods have been used in the labs to detect cancer gene mutations, but low sensitivity has been a major concern to identify mutations in limited tumor samples. An optimal method is not only time- and cost-saving, but also has sufficient analytical sensitivity.

Cancer Gene Mutation Detection Powered by XNA Technology

ULTRA-SENSITIVE

XNA is the Optimal Choice for Cancer Gene Mutation Detection Compared to other Technologies

XNA, xenonucleic acids, are innovative new nucleic acid molecular oligomers that hybridize by Watson-Crick base pairing to target DNA sequences yet have a modified chemical backbone. XNA oligomers are highly effective at hybridizing to targeted normal DNA sequences and can be employed as molecular clamps in quantitative real-time polymerase chain reactions (PCR) or as highly specific molecular probes for detection of nucleic acid target sequences. The XNA tightly binds to the wildtype sequence that is 100% complementary in sequence and blocks DNA polymerase from DNA elongation; only the mutant target sequence gets amplified because the XNA:mutant DNA duplex is not stable due to mismatch and fall off from the template in PCR reactions.

Sanger Sequencing

Advantages: accurate result and is, therefore, the gold standard
Disadvantages: low sensitivity (20% to 25% VAF)

Pyrosequencing Assays

Advantages: better sensitivity and throughput than Sanger sequencing,  the early form of NGS
Disadvantages: low sensitivity (5% to 8% VAF)

NGS Sequencing

Advantages: high-throughput and good sensitivity – 1% to 5% VAF, or even better
Disadvantages: costly and time consuming (7 to 10 days)

Digital Droplet PCR (ddPCR)

Advantages: high sensitivity and claimed to be 0.001% VAF
Disadvantages: much less sensitivity observed in testing than claimed  and suffers false-positive results

qPCR Analysis

Sensitivity can reach 1% VAF for some targets. Rapid and little hands-on work. Multiple methods for qPCR and a lot of variations in sensitivity. Some of them are only 10% VAF

QClamp® Assays Limit of Detection (LoD)

The LoD for the qPCR assay is determined by running the QClamp® assay using a serial dilution of mutant DNA in wild-type background at different total DNA inputs and several mutation frequencies for each target. To determine if a sample is positive (contains mutation) or negative (does not contain mutation) for a particular target sequence, the ΔCq of the sample at this target sequence and the validated standard ΔCq for this target sequence for a positive and negative control will need to be compared.

Cq difference (ΔCq) = Sample Assay Cq – Internal Control Assay (beta-actin) Cq

If further sequencing is required, the qPCR reactions can be sequenced directly by Sanger sequencing using target-specific primers.

Supporting Data

Below 0.1% Detection Sensitivity of Mutant DNA

Amplification Plot from ABI QuantStudio 5 (JAK2)

Amplification Plot from Bio-Rad CFX384 (JAK2)

Amplification Plot from Roche LightCycler® 480 (JAK2)

Streamlined Workflow for QClamp® Gene Mutation Detection Tests

Step 1: DNA Isolation & Quantification

Extract DNA from FFPE or plasma using a commercial DNA extraction kit followed by measuring the concentration using fluorometric analysis

Step 2: set up qpcr

Mix the assay reagents, load into PCR plate, add controls and extracted DNA ~ 30-60 minutes

Step 3: Amplification parameters

Enter amplification parameters on
qPCR instrument, load PCR plate
and start the run ~ 2.5 hours

Step 4: Data analysis

Determine the presence or absence
of mutations according to the Cq
value cutoffs ~ 15 minutes

Utilizing QClamp® on Clinical Sample Mutation Detection

CLINICAL SAMPLES

NSCLC Patients Plasma Samples

Detecting L858R in Plasma from 4 NSCLC Patients with and without XNA. In the presence of XNA, only wild-type sequence is detected and mutant DNA is only detected in the presence of XNA

 

Without XNA Technology – Mainly Wild-Type (WT) L858 Detected

With XNA Technology – Wild-Type Blocked, only L858R Mutant Detected

L858R Detected by Sanger Sequencing after XNA Blocking (WT = CTG; L858R = CGG)

CLINICAL SAMPLES

Human Tumor FFPE Samples

Human tumor FFPE samples for EGFR gene mutation detection. EGFR mutations (Exon 19 deletion and T790M) in human tumor FFPE samples are detected using QClamp® EGFR detection kit. Abundant wild-type DNA is detected at early qPCR cycles when no XNA is present. With XNA, only mutant DNA is detected at late qPCR cycles and wild-type DNA amplification is blocked.

Customer Testimonial

“QClamp® XNA technology is the most sensitive and detected mutations missed by …”
“Our lab did a good comparison study for BRAF mutations with all the technologies available in the market and found that QClamp® XNA technology is the most sensitive and detected mutations missed by all current methods including NGS and ddPCR. I strongly recommend this technology!”

Dr. Shidong Jia

Scientist, Genentech

QClamp® Gene Mutation Detection Test Product Specifications

Intended Use

For in vitro diagnostic use (CE/IVD) or for research use

Sample Type

FFPE and Plasma

Input DNA

5-10ng/Reaction

Pack Size

30 Samples

Instruments Validated

Roche LightCycler® 480, Bio-Rad CFX384 and ABI QuantStudio 5

Detection Chemistry

TaqMan

Turnaround Time

Less Than 4 hours

Stability

Stable for 12 Months at -25°C  to -15°C

Resources

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Call us: +1 (800) 878-6662

Email us: information@diacarta.com

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