EGFR Pyro Kit

For sequencing-based detection and quantitation of mutations in the EGFR gene

Features

  • Comprehensive results in real time
  • Accurate quantification of mutations in the EGFR gene
  • Easy detection of complex mutations
  • Sequence context provides built-in control of the assay
  • Flexible post-run analysis
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EGFR Pyro Kit (24)

Cat. No. / ID: 970480

For 24 reactions: Sequencing Primers, PCR Primers, Unmethylated Control DNA, PyroMark PCR Master Mix, CoralLoad Concentrate, Buffers, and Reagents
The EGFR Pyro Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Product Details

The EGFR Pyro Kit is a molecular detection kit for the identification of mutations and deletions in the EGFR gene. The kit provides primers and reagents for amplification of the EGFR gene, plus buffers, primers, and reagents for detection and quantification of mutations in real time using Pyrosequencing technology on the PyroMark Q24 System.

Principle

The EGFR Pyro Kit is used for quantitative measurements of mutations in codons 719, 768, 790, 858, and 861, as well as deletions and complex mutations in exon 19 of the human EGFR gene in real time using Pyrosequencing technology on the PyroMark Q24 System. The EGFR gene encodes the epidermal growth factor receptor (EGFR) protein. Mutations in the tyrosine kinase domain of the EGFR gene can enable tumor growth and progression. EGFR mutations are found in approximately 10% of non-small cell lung cancer incidences in the US and 35% in East Asia. Additionally, EGFR mutations are found in 6% of brain tumors.

The following mutations are detected.

  • Exon 18 (719): G719A, G719C, G719S
  • Exon 19 (Del): 20 deletions and complex mutations 
  • Exon 20 (768 and 790): S768I, T790M
  • Exon 21 (858–861): L858R, L861Q, L861R

Procedure

The kit consists of 4 PCR assays: one for detecting mutations in codon 719 (exon 18), one for detecting mutations in codons 768 and 790 (exon 20), one for detecting mutations in codons 858 to 861 (exon 21), as well as one for detecting deletions in exon 19. The 4 regions are amplified separately by PCR and sequenced through the defined region (see figure " Illustration of the EGFR assay"). The amplicon covering codons 768 and 790 is divided into 2 sequencing reactions. Sequences surrounding the defined positions serve as normalization and reference peaks for quantification and quality assessment of the analysis.

After PCR using primers targeting exons 18, 19, 20, and 21, the amplicons are immobilized on Streptavidin Sepharose High Performance beads. Single-stranded DNA is prepared, and the corresponding sequencing primers anneal to the DNA. The samples are then analyzed on the PyroMark Q24 System using a run setup file and a run file. It is recommended to use the EGFR Plug-in Report to analyze the run. This report ensures that the correct LODs are used and different sequences to analyze are automatically used to detect all mutations and deletions. However, the run can also be analyzed using the analysis tool integral to the PyroMark Q24 System (see figures " Pyrogram trace of a normal genotype in codon 719", " Pyrogram trace of a normal genotype in codon 768", " Pyrogram trace of a normal genotype in exon 19", and " Pyrogram trace of a GGAATTAAGAGAAGC deletion in exon 19"). The "Sequence to Analyze" can be then adjusted for detection different deletions in exon 19 and for rare mutations in the other exons after the run.

See figures

Applications

The EGFR Pyro Kit is used for measuring mutations in codons 719, 768, 790, 858, and 861, as well as deletions and complex mutations in exon 19 of the human EGFR gene.

The following mutations are detected.

  • Exon 18 (719): G719A, G719C, G719S
  • Exon 19 (Del): 20 deletions and complex mutations 
  • Exon 20 (768 and 790): S768I, T790M
  • Exon 21 (858–861): L858R, L861Q, L861R

Supporting data and figures

Resources

Software User Guides (1)
For installation and use with PyroMark Q24 Instruments and PyroMark Q24 Software version 2.0
Analysis Software (1)
EGFR Plug-in 1.3
SOFTWARE (2MB)
Version 1.3.0.7
For use with PyroMark Q24 Software version 2.0.8

FAQ

Can I use more than 10ng DNA for the PCR reaction for the Pyro kits?
The NRAS Pyro, EGFR Pyro. BRAF PyroKRAS Pyro, and UGT1A1 Pyro Kits all require that no more than 10ng of DNA be used for the PCR reaction.  Too much DNA can sometimes lead to inhibition problems in the PCR reaction.
FAQ ID -2379
Which purification kits are recommended for the Pyro kits?

The purification kits recommended for the BRAF Pyro, EGFR Pyro, KRAS Pyro, and NRAS Pyro kits are:

Paraffin-embedded tissue

Blood

FAQ ID -2380
What plug-ins are available for the Pyrosequencing kits?
Plug-Ins are available for:
  • EGFR Pyro Kit 
  • KRAS Pyro Kit
  • NRAS Pyro Kit
  • RAS extension Kit
  • BRAF Pyro Kit
  • These plug-ins are available for download on the respective catalog page under the Product Resources tab in the Analysis Software section. Note: PyroMark Q24 software version 2.0.7 is needed for the usage of the Plug-ins.
    FAQ ID -2381
    Can the insertions in Exon 20 be detected with the EGFR Pyro Kit?
    No, there is no specific assay for the insertions and the codon 768 assay does not allow detection of the insertions starting at amino acid 770.
    FAQ ID -2383
    Can PyroMark Gold reagents be vortexed?
    Reconstiuted enzyme and substrate of PyroMark Gold Reagents, should not be vortexed since this could lead to conformational changes which affect the activity.
    FAQ ID -2844