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EGFR Pyro Kit

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

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.

Cat No./ID: 970480
EGFR Pyro Kit (24)
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.
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Pyrogram trace of a GGAATTAAGAGAAGC deletion in exon 19.
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Pyrogram trace of a normal genotype in exon 19.
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Pyrogram trace of a normal genotype in codon 719.
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Pyrogram trace of a normal genotype in codon 768.
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Illustration of the EGFR assay.
The sequence indicated is the analyzed sequence for a normal sample. FP: Forward PCR primers; RPB: Reverse PCR primers (B indicates biotinylation); Seq: Sequencing primers.
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.

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
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