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DiaCarta has developed BRAF V600 Mutation Enrichment Kit to improve the Sanger sequencing sensitivity (0.04% vs. standard 20%) by enriching the mutation sequence population within the Sanger sequencing template.

We have developed the BRAF V600 Mutation Enriching Kit based on the XNA technology. The kit can be combined with commercial Sanger sequencing reagents for high-sensitivity mutation-enriching Sanger sequencing. The sensitivity can reach 0.04% VAF, better than the common CDx tests for BRAF V600 detection.

Ease of Use

DiaCarta’s enrichment kit does not add any additional steps in the traditional Sanger sequencing procedure, just changes the PCR step for template preparation.

Lab Developed Test (LDT)

It is easy to validate the BRAF V600 mutation-enriching Sanger sequencing in your own lab with DiaCarta’s enrichment kit.

Pack Sizes and Catalog Numbers

Pack Size: 24-Reaction Kit, DC-10-0198

Pack Size: 96-Reaction Kit, DC-10-0199

Research Use Only (RUO) Product. 

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BRAF V600 Mutation

Introduction

BRAF is an important member of the Ras-Raf-MEK/ERK pathway for the regulation of cell proliferation and apoptosis in normal cells. BRAF V600E mutation (the c.1799 A to T mutation in exon 15) is the most common BRAF mutation in clinical cancer diagnostics. The BRAF mutations are found in multiple types of cancers, mostly in melanoma (50%) and in some colorectal cancer (CRC) (10%), or non-small cell lung cancer (NSCLC) (2-4 %). BRAF V600E allows the enzyme constitutively active with much higher activity than the wildtype enzyme, leading to constant activation of the downstream pathway, cell proliferation, and poor prognosis.

Resources

  • Download the preprint for the BRAF V600 Mutation-enriching Sanger sequencing paper. Download 
  • Download the marketing brochure. Download

Mutation-enriching Sanger Sequencing for BRAF V600E Mutation Detection

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Drawbacks of Sanger Sequencing

Traditional Sanger sequencing is still the gold standard for mutant detection. However, at low sensitivity of 15 to 20% of variant allele frequency (VAF), Sanger sequencing is not approved for BRAF V600E detection used in companion diagnostics for targeted cancer therapy.

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Low-frequency Mutations

If the mutant population is enriched in the patient samples, the low-frequency mutations can be effectively detected in Sanger sequencing with high sensitivity.

The limit of detection of the BRAF V600 in the presence of 500 nM XNA using templates with different VAF. The reverse primer was used for Sanger sequencing and the reverse/complement strand was read. The c1799 BRAF V600E mutation, the T to A mutation, was read as A to T mutation in sequencing results. A as the wildtype and T as the mutant. 

Introducing QClamp® BRAF V600 Mutation Enrichment Kit

We have developed the BRAF V600 Mutation Enriching Kit based on the XNA technology. The kit can be combined with commercial Sanger sequencing reagents for high-sensitivity mutation-enriching Sanger sequencing. The sensitivity can reach 0.04% VAF, better than the common CDx tests for BRAF V600 detection.

The high-sensitivity BRAF V600E mutation-enriching Sanger sequencing has great potential to be used as a Laboratory developed test for BRAF companion diagnostics assay. The mutation-enriching Sanger sequencing does not add any extra steps compared to traditional Sanger sequencing, but only enriches the mutation sequence in the PCR template preparation step. Therefore, this assay is easy to be adopted by standard molecular diagnostics labs that have Sanger sequencing facilities.

The XNA acts as molecular clamps to block the wildtype sequence and selectively amplify the mutant sequence, thereby improving the Sanger sequencing sensitivity. The same technology can also be combined with qPCR technology to improve the traditional qPCR sensitivity in the detection of various cancer mutations and have been developed into QClamps products.

Companion Diagnostic Detection of BRAF V600E is Required for BRAF V600E Target Therapy by FDA

Multiple BRAF V600E inhibitors have been developed and got FDA approval. However, the use of these inhibitors for target therapy requires the companion test to confirm the BRAF V600E mutation in patients. Multiple tests have been approved for the BRAF V600E detection by the FDA. However, these methods are usually expensive and some of the tests may miss the other important mutations, such as BRAF V600K.



 Laboratory Developed Test (LDT) is another important channel for detection of BRAF V600E and can also be used for detection of the mutation as the companion diagnostics test. They are much more cost-effective than the FDA-approved products and are available in multiple clinical labs. We hope to see the mutation-enriching Sanger sequencing soon be used for targeted BRAF cancer therapy in many clinical labs. Contact us if you are thinking about validating the test in your own lab.

CDx Approved by FDAType of cancerInhibitors or Drugs for Target therapyTechnology
Acobas 4800 BRAF V600 Mutation Test by Roche Molecular SystemsMelanomaZelboraf (vemurafenib), or Cotellic (cobimetinib) - in combination with Zelboraf (vemurafenib) qPCR
THXID BRAF Kit by bioMérieuxMelanomaBraftovi (encorafenib) in combination with Mektovi (binimetinib), or Mekinist (tramatenib), or Tafinlar (dabrafenib) ARMS-real time PCR
therascreen BRAF V600E RGQ PCR Kit by Qiagen GmbHColorectal CancerBRAFTOVI (encorafenib) in combination with Erbitux (cetuximab)ARMS-real time PCR
Oncomine™ Dx Target Test by Thermo Fisher Scientific Metastatic non-small cell lung cancer (NSCLC)Combination of dabrafenib and trametinibNGS

Streamlined Workflow for QClamp® BRAF V600 Mutation Enrichment Kit

Resources

Need Help?

Call us: +1 (800) 246-8878

Email us: information@diacarta.com

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