PyroMark Research-Use-Only tests
Biotage introduces the PyroMark™ series, the first genetic tests that deliver the underlying sequence information in real-time. PyroMark offers genetic researchers and service labs two major advantages: the assays are optimized, and each assay result has built-in quality control. PyroMark gives full confidence in assay results by scoring polymorphic sites within the context of the surrounding DNA sequence.
Each test contains quality controlled and tested reagents, ensuring the successful implementation of the tests, and reducing the time to get the assay up and running. You save your single most expensive resource, the time of your people.
CANCER MUTATIONS AND CpG METHYLATION
PyroMark MLH1: simple, quantitative determination of methylation level in the MLH1 promoter, based on real sequence information
The human mutL homologue hMLH1 is located on chromosome 3 (3p21.23). The hMLH1 gene is inactivated by promoter hypermethylation (10-20%) in a number of different cancers, such as colon, endometrial and gastric cancers. PyroMark MLH1 detects the level of methylation in a region -209 to -188 from the transcription start site.
llustration of PyroMark MLH1 assay results. The upper pyrogram shows results from a normal blood DNA
sample and the lower pyrogram from an in vitro methylated DNA sample. Orange columns indicate the measured CpG sites.
Blue columns indicate QC for completion of the bisulfite treatment (a C followed by an A in the original sequence).
For more information, download the PyroMark MLH1 product note.
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PyroMark p16: simple, quantitative determination of methylation level in the p16 promoter, based on real sequence information
The p16 gene (CDKN2A) is located on chromosome 9 (9p21). The gene is frequently mutated or deleted in a wide variety of tumors such as pancreatic cancer, and is known to be an important tumor suppressor gene. It is also inactivated by promoter hypermethylation (5-30%) in a number of different cancers. PyroMark p16 assay detects the level of methylation in a region +148 to +174 in exon 1 of the gene.
Illustration of PyroMark™ p16 assay results. The upper Pyrogram® trace shows results from a normal
blood DNA sample and the lower Pyrogram trace shows results from a DNA sample isolated from a
colon tumor. Orange columns indicate the measured CpG sites, and the individually measured degree
of methylation at each site. Blue columns indicate QC for completion of the bisulfite treatment.
For more information, download the PyroMark p16 product note.
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PyroMark KRAS: Simple and reliable determination of contiguous, multi-variable mutations at codons 12, 13 and 61, based on real sequence information
The K-ras gene is located on chromosome 12 (12p12). PyroMark KRAS can be used to genotype codons 12 and 13 of the K-ras gene, and to perform allele quantification of individual positions in the same codons.
The PyroMark KRAS test includes negative controls in the nucleotide dispensation order in order to detect additional rare mutations. After the run, peak heights from all dispensations can be imported into an Excel worksheet for further analysis by an Excel macro. By this extra evaluation step, rare mutations in codon 12 and 13 can be detected that are not included in the pre-defined “Sequence To Analyze”.
In addition, rare mutations in codon 61 can be analyzed using the second primer set provided in the test.
Illustration of PyroMark KRAS assay results. The left pyrogram shows the wildtype sequence and the right pyrogram heterozygote G to A mutation in the second position of codon 12. Orange columns indicate the analyzed mutations.
For more information, download the PyroMark KRAS product note.
Suggest improvements for future development of PyroMark KRAS
If your require analysis of specific mutations in KRAS, click here
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PyroMark BRAF: Simple and reliable determination of mutation V600E,
based on real sequence information
The B-RAF gene is located on chromosome 7 (7q34). A glutamate (E) for valine (V) substitution at residue 600 in the activation segment accounts for 90% of B-RAF mutations in human cancers. PyroMark BRAF reveals both the V600E mutation as well as other mutations surrounding this position (exon 15) of the B-RAF gene. In addition, rare mutations in exon 11 can be analyzed using the second primer set provided in the test.
Illustration of PyroMark BRAF assay results. The left pyrogram showns a simple dispensation order for the V600Emutation. The right pyrogram shows an extended dispensation order which allows detection of surrounding mutations. Orange columns indicate the analyzed V600E mutation.
For more information, download the PyroMark BRAF product note.
PyroMark LINE-1: Simple, quantitative determination of methylation level in the LINE-1 transposable elements, based on real sequence information
LINE-1 retrotransposable elements make up about 15% of human genome. DNA methylation within the promoter region of human LINE-1 elements is important for maintaining transcriptional inactivation and for inhibiting transposition. Genome-wide losses of DNA methylation within the promoter region of human LINE-1 elements have been regarded as a common epigenetic event in malignancies and may play crucial roles in carcinogenesis. This methylation assay amplifies a region of the LINE-1 element and serves as a marker for global methylation.
Illustration of PyroMark LINE-1 assay results. The upper pyrogram shows results from a normal blood DNA sample and the lower pyrogram from an endocrine tumour sample. Orange columns indicate the measured CpG sites, and the individually measured degree of methylation at each site.
For more information, download the PyroMark LINE-1 product note.
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PyroMark MGMT: Simple, quantitative determination of methylation level in the MGMT gene, based on real sequence information
The O6-Metylguanine DNA methyltransferase (MGMT) gene is located at chromosome 10(10q26). It encodes the DNA repair protein O6-alkylguanine DNA alkyltransferase (AG T). Loss of gene expression frequently occurs via hypermethylation of the CpG sites in the promoter region. MGMT is methylated to 25-50% in a number of different cancers such as brain, colon, lung, breast etc. This methylation assay detects the level of methylation in position 17-39 of exon 1 of the MGMT gene.
Illustration of PyroMark MGMT assay results. The upper pyrogram shows results from a normal blood DNA sample and the lower pyrogram from an in vitro methylated DNA sample. Orange columns indicate the measured CpG sites. Blue columns indicate QC for completion of the bisulfite treatment (a C followed by an A in the original sequence).
For more information, download the PyroMark MGMT product note.
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CLINICAL GENETICS
PyroMark APOE : simple and reliable determination of APOE based on real sequence information
The Apolipoprotein E encoding gene (APOE) is polymorphic with the three common alleles exon 2, exon 3, and exon 4. Two single nucleotide polymorphisms, here designated APOE 112 and APOE 158, result in amino acid substitutions at positions 112 and 158 of the protein. This assay genotypes these two APOE polymorphisms.
Illustration of PyroMark APOE assay results. Orange columns indicate the analyzed SNPs.
For more information, download the PyroMark APOE product note.
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PyroMark HFE : simple and reliable determination of HFE genotype based on real sequence information
These assays are for genotyping of the mutations H63D and S65C in exon 2 and mutation C282Y in exon 4 of the human hereditary hemochromatosis (HFE) gene.
Illustration of PyroMark HFE assay results. Orange columns indicate the analyzed SNPs.
For more information, download the PyroMark HFE product note.
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PyroMark MTHFR : simple and reliable determination of MTHFR genotype based on real sequence information
This assay is for genotyping the C677T variant in the human methylene tetrahydrofolate reductase (MTHFR) gene. The variant replaces the nucleotide cytosine with thymine at position 677 in the MTHFR gene.
Illustration of PyroMark MTHFR assay results. The orange column indicates the analyzed SNP.
For more information, download the PyroMark MTHFR product note.
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PyroMark PWS/AS (Prader-Willi & Angelman Syndromes) : simple, quantitative determination of Prader-Willi and Angelman Syndromes based on real sequence information
We also introduce an innovative test that exploits the quantitative properties of Pyrosequencing, by using CpG methylation analysis to identify Prader-Willi and Angelman Syndromes.
Prader-Willi syndrome (PWS) is caused by loss of expression of genes located on segment q11-13 on the paternally derived chromosome 15. When the expression from the same segment is lost from the maternally derived chromosome 15, Angelman syndrome (AS) arises. This methylation analysis assay detects the effect on methylation of deletion of segment q11-13 on chromosome 15.
Illustration of PyroMark Prader-Willi/Angelman Syndromes assay results. Orange columns indicate
quantified CpG sites. Blue columns indicate QC for completion of the bisulfite treatment (a C followed
by an A in the original sequence). The pyrogram shows the reverse assay of the top strand.
For more information, download the PyroMark PWS/AS product note.
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Find out more about PyroMark
To download PyroMark product notes:
To enquire about present and future PyroMark tests, click here
Disclaimer
PyroMark products are for research use only. Not for use in diagnostic procedures. A patent or patent applications may cover these mutations and/or CpG sites. These products are made available for scientific research only and in no way confer the rights to perform these assays for commercial purposes or profit. Third party licenses may be required and are the responsibility of the reagent purchaser.