PyroMark™KRAS v2.0 test

The human KRAS gene is located at chromosome 12p12, and encodes a 21-kD protein (p21ras or K-Ras). The K-Ras protein functions as a binary molecular switch that controls intracellular signalling networks in a number of signalling pathways. Of particular interest is the involvement in the signal transduction pathway involving Epidermal Growth Factor Receptor (EGFR) where K-Ras serves as a downstream signalling affector.

Certain mutations in the KRAS gene result in a K-Ras protein that is constitutively active leading to an uncontrolled cell proliferation. Such oncogenic activation has been suggested to be involved in many aspects of the development and progressions of cancer, including abnormal cell growth and differentiation, as well as increased invasiveness and metastasis. Consequently, mutations in the KRAS gene are found in many types of human cancers, with the most common mutations being found in codons 12, 13 and 61.

PyroMark™ KRAS v2.0 test – Assay characteristics
PyroMark™ KRAS v2.0 test are optimized to be used on any Pyrosequencing^® instrument for analysis of the most frequently observed mutations in KRAS. The assay is intended for use on human genomic DNA prepared from fresh, frozen or paraffin-embedded tissues.

The assay enables fast detection of all common KRAS mutations in codons 12, 13 and 61 (Figure 1). In addition, post-run analysis of peaks using an Excel macro enables identification of additional rare mutations in codons 12, 13, and 61. These mutations can be quantified using PyroMark™Q96 MD and PyroMark™Q96 ID systems by running an identical sample with changed ”Sequence to Analyse”. The application software for PyroMark™Q24 instrument contains a function that enables post-run quantification of rare mutations simply by changing the sequence to analyse.

Figure 1. Illustration of KRAS mutations in codons 12, 13 and 61 detected and quantified by PyroMark KRAS v2.0.

The assay is available as two products: PyroMark™Q96 KRAS 2.0 test for PyroMark™Q96 MD and PyroMark™Q96 ID instruments, and PyroMark™Q24 KRAS 2.0 test for PyroMark™Q24.

Assay description
The primers included in PyroMark™ KRAS v2.0 test were designed using Pyrosequencing Assay Design Software v 1.0 to obtain optimal amplification and sequencing results. The final design comprised a forward assay design for analysis of codons 12+13 and a reverse assay design for the analysis of codon 61 (See Figure 2).

Figure 2. Illustration of PyroMark™ KRAS v2.0 assay set up. The sequence indicated is the analyzed sequence for a normal sample. Boxed sequences indicate codons of particular interest for mutation analysis. FP or FPB, forward PCR primer; RP or RPB, reverse PCR primer; B, biotinylated; Seq, sequencing primer.

Results obtained from mutational analysis of KRAS using PyroMark™Q24 KRAS v2.0 test and PyroMark™Q24 instrument.

Figure 3. Pyrogram traces obtained by Pyrosequencing analysis on PyroMark™Q24 using the PyroMark™Q24 KRAS v2.0 test. The upper Pyrogram trace illustrates the peak pattern obtained by Pyrosequencing analysis of a sample with normal genotype in codons 12 and 13. The middle Pyrogram trace illustrates the peak pattern obtained after analysis of a sample containing a mutation in position 2, codon 12. The nucleotide change from G to A results in substituting glycine with aspartic acid. The Pyrogram trace at the bottom illustrates the peak pattern obtained after analysis of a sample with mutations in position 1, codon 12. Here the nucleotide change results in substituting glycine with cysteine.

Figure 4. Pyrogram traces obtained after Pyrosequencing analysis of codon 13 in KRAS on PyroMark™Q24 using the KRAS™Q24 v2.0 test. Upper Pyrogram trace illustrates the peak pattern obtained after Pyrosequencing analysis of a sample with normal genotype in codons 12 and 13. Lower Pyrogram trace illustrates peak pattern obtained after Pyrosequencing analysis of a sample containing mutations in position 2 codon 13. The nucleotide change of a guanine to adenosine results in substituting the amino acid glycine with aspartic acid .

Figure 5. Pyrogram traces obtained after Pyrosequencing analysis of codon 61 in KRAS using PyroMark Q24 KRAS 2.0 test and PyroMark Q24 instrument. The upper Pyrogram trace illustrates the peak pattern obtained after Pyrosequencing analysis of a sample with normal genotype. The lower Pyrogram trace illustrates the peak pattern obtained after Pyrosequencing analysis of a sample with mutation in position 2 codon 61. This nucleotide exchange results in substituting glutamine with leucine.

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*For Research Use Only. Not for use in diagnostic procedures. No claim or representation is intended to provide information for the diagnosis, prevention, or treatment of a disease.

**The product is intended for molecular biology applications. This product is neither intended for the diagnosis, prevention, or treatment of a disease, nor has it been validated for such use either alone or in combination with other products.