XNA Molecular Clamp | DiaCarta, Inc.

XNA Molecular Clamp Technology

Optimal Choice for Early Cancer Detection

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 wild-type 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.

Precision Medicine and Early Cancer Detection

Precision Medicine Initiative defines precision medicine as “an emerging approach for disease treatment and prevention that takes into account individual variability in genes, environment, and lifestyle for each person.” Using this approach, doctors make decisions on treatment and provide suggestions for prevention based on the differences reside in individuals.

In this context of precision medicine for cancer, finding the cancer driver mutations such as genes in growth pathway and tumor suppressor genes is critical, as it is generally recognized that these mutations contribute to early development of different types of cancers. Detection of these mutations not only is essential for early cancer detection, but providing strategies for cancer therapy as a companion diagnostic tool.

The Invasive Way with FFPE Samples

Challenges for Early Cancer Detection – Invasive 

Traditionally, tumor gene mutations have been detected using DNA isolated from formalin-fixed paraffin embedded (FFPE) tumor biopsy tissue samples.

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Surgical tumor biopsies are invasive, sometimes risky, and not always easy to get access to

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Mutations in a distant organ are not found in the biopsy samples and doctors miss the opportunity to increase the patience’s chance for survival

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The mutation information changes as cancer evolves, which demand changing therapeutic strategies that biopsy samples can’t provide.

Challenges for Early Cancer Detection – Non-invasive 

Recently, minimally invasive procedures, commonly called liquid biopsies, such as taking blood and monitoring mutations in patients’ circulating cell-free tumor DNA (ctDNA), have gained traction for early cancer detection.

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The success depends on high sensitivity of the detection technologies, which become a limiting factor. That can also be the major reason that why most of the current cancer studies and detections have been done in malignancy.

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Premaligancy studies or early cancer detection using genomic-wide analysis has been very limited due to the difficulty in detection of cancer gene mutations in a small sub-population of cells before cancer is developed

The Non-invasive Way with Liquid Biopsy

Limitations of Current Technologies for Early Cancer Detection

Finding tumor mutations in ctDNA is like finding the needle in the haystack because very little cancer cell DNA is present in the wild-type DNA from a big population of healthy cells in the circulation. Detection of such minute amount of tumor cell DNA, barely 1% and as little as 0.01% of total ctDNA, requires much more sensitive techniques compared to techniques used in advanced cancers, which may contribute the majority of circulating DNA in the blood.

 

Source:
Genomics, Circuits, and Pathways in Clinical Neuropsychiatry, 2016
Analysis of DNA Methylation by Pyrosequencing, 2016
What is next-generation sequencing? 2013
Droplet Digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data, 2017
Understanding PCR, 2017

Sanger Sequencing

Sanger sequencing is the process of selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication.

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

NGS

Next-generation sequencing (NGS), massively parallel or deep sequencing are related terms that describe a DNA sequencing technology which has revolutionised genomic research.
 

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

qPCR Analysis

Quantitative polymerase chain reaction (Q-PCR) is a method by which the amount of the PCR product can be determined, in real-time, and is very useful for investigating gene expression

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

Pyrosequencing Assays

Pyrosequencing is a technique that uses a sequencing-by-synthesis system which is designed to quantify single-nucleotide polymorphisms (SNPs).

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

ddPCR

Droplet Digital PCR (ddPCR) technology utilizes Taq polymerase in a standard PCR reaction to amplify a target DNA fragment from a complex sample using pre-validated primer or primer/probe assays.

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

XNA Technology

Sensitivity reaches 0.1% to 0.5% VAF, much more sensitive than regular qPCR and other qPCR-derived techniques.

Achieve ultra-sensitivity by only amplifying mutant DNA and block wild-type sequences.
 

 

Introduce XNA Molecular Clamp Technology

XNA molecular clamps assays are highly sensitive using liquid biopsy and FFPE samples. The limit of detection (LoD) can reach as low as 0.1% (7 or 8 copies of mutant DNA) using 5 ng of ctDNA, equivalent to 2 ml of blood from a patient. Since the presence of circulating tumor cells (CTCs) has been found to be associated with poor survival in multiple cancers and lowering the CTC threshold from unfavorable (≥5 CTCs/7.5 ml of blood) to favorable CTCs (<5 CTCs/7.5 ml of blood) improves survival, the number of CTCs in blood can be used as a predictive factor for cancer treatment response. The XNA technology may be used to find the cancer mutations and provide meaningful information for cancer treatment.In this context of precision medicine for cancer, finding the cancer driver mutations such as genes in growth pathway and tumor suppressor genes is critical, as it is generally recognized that these mutations contribute to early development of different types of cancers. Detection of these mutations not only is essential for early cancer detection, but providing strategies for cancer therapy as a companion diagnostic tool.

Features and Benefits for XNA Molecular Clamps Assays

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Regular qPCR reactions on most of common instruments

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Synthetic oligomers (15 to 25 nt long) resistant to any known nucleases

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Regular qPCR reactions on most of common instruments

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Much higher binding affinity for target sequence independent of salt concentration

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Large melting temperature difference (ΔTm = 15-20ºC) in single-nucleotide (SNP’s) and insertion/deletions (indels) (5-7ºC for natural DNA)

Early Cancer detection potential by highly sensitive XNA technology

XNA molecular clamps assays are highly sensitive using liquid biopsy and FFPE samples. The limit of detection (LoD) can reach as low as 0.1% (7 or 8 copies of mutant DNA) using 5 ng of ctDNA, equivalent to 2 ml of blood from a patient. Since the presence of circulating tumor cells (CTCs) has been found to be associated with poor survival in multiple cancers and lowering the CTC threshold from unfavorable (≥5 CTCs/7.5 ml of blood) to favorable CTCs (<5 CTCs/7.5 ml of blood) improves survival, the number of CTCs in blood can be used as a predictive factor for cancer treatment response. The XNA technology may be used to find the cancer mutations and provide meaningful information for cancer treatment.

One of the examples for cancer gene mutation detection using the XNA technology is ColoScape™ colorectal cancer mutation detection kit we developed for FFPE and liquid biopsy, such as blood samples. According to our testing results, for advanced adenomas tissue samples (stage 0), the sensitivity reaches 60%, compared to 42% ColoGuard pre-cancer detection. For stage I to IV colon cancer, the sensitivity is 95.5%. The beauty of using XNA technology is achieving high sensitivity using regular qPCR Taqman assays that can be done in common pathology and genetic biomarker discovery labs without investment of other instrumentation.

Analytical sensitivity in plasma (QClamp® KRAS Mutation Detection Test) powered by XNA technology

Analytical sensitivity in FFPE (QClamp® KRAS Mutation Detection Test) powered by XNA technology

XNA Technology Novel Applications

Single Gene Mutation Detection

Colorectal Cancer Mutation Detection

NGS Targeted Sequencing

CRISPR/Cas9 Gene Editing Test

Adapter Dimer Removal for NGS

Resources

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