Methylation Analysis
DNA methylation is involved in the regulation of many cellular processes, including X chromosome inactivation, chromosome stability, chromatin structure, embryonic development and transcription. Bisulfite sequencing is the gold standard in validation of methylation experiments as only sequencing provides direct detection of methylation events as well as information across the entire region. Applied Biosystems provides an integrated solution for methylation analysis with optimized protocols for each step.
Experimental Workflow: Find the Products You Need for Every Step
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Step 1:
DNA Purification -
Step 2:
Cycle Sequencing -
Step 3:
PCR Amplification & Cleanup -
Step 4:
Sequencing Reaction Cleanup -
Step 5:
Capillary Electrophoresis -
Step 6:
Data Analysis
Click the arrows above to view products
Step 1: DNA Purification
DNA extraction is a critical first step in the experimental workflow of DNA Sequencing and Fragment analysis. The overall quality, accuracy and length of the DNA sequence read can be significantly affected by characteristics of the sample itself, and the method chosen for nucleic acid extraction. Ideal methods will vary depending on the source or tissue type, how it was obtained from its source, and how the sample was handled or stored prior to extraction.
Products for Step 1: DNA Purification
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MELT™ Total Nucleic Acid Isolation System
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Stop homogenizing tissue samples. With the MELT Total RNA Isolation System, tissue samples are quickly and easily lysed in MELT solution. The RNA in the resulting lysate is stable for days at room temperature or longer at lower temperatures.
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RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE Tissues
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Extract total RNA or DNA from formalin- or paraformalin-fixed, paraffin-embedded (FFPE) tissues. This kit enables the molecular analysis of archived tissue samples at both the genomic and gene expression levels. With this kit, nucleic acid is obtained that is suitable for qRT-PCR, mutation screening, and microarray analyses.
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NucPrep® DNA Chemistry for Tissues
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Starter kit for DNA isolation from animal and plant tissues and cells, etc., using NucPrep® Chemistry.
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MagMAX™ Express Magnetic Particle Processor
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MagMAX™ Express magnetic particle processors are ideal for researchers who want to automate their MagMAX sample preparation kits. The MagMAX Express platform is the premier automated platform to meet your throughput needs.
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MagMAX™ Express-96 Magnetic Particle Processors
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MagMAX™ Express magnetic particle processors are ideal for researchers who want to automate their MagMAX sample preparation kits. The MagMAX Express platform is the premier automated platform to meet your throughput needs.
Step 2: Cycle Sequencing
The methylSEQr™ Bisulfite Conversion Kit converts non-methylated cytosines (C) in the DNA sample to uracil (U). Methylated cytosines remain unchanged. Comparing the sequences of treated vs. non-treated DNA and detection of converted cytosines (now apparent as T base) determine which cytosines in the sample are methylated in CpG dinucleotides.
Products for Step 2: Cycle Sequencing
- methylSEQr™ Bisulfite Conversion Kit
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The methylSEQr™ Bisulfite Conversion Kit converts non-methylated cytosines (C) in your DNA sample to uracil (U). This kit is an initial step in a multi-step workflow that enables you to determine which cytosines in your sample are methylated by comparing the sequences of treated vs. non-treated DNA.
Step 3: PCR Amplification & Cleanup
Design high quality PCR primers for methylation mapping experiments. Simply cut and paste in your region of interest. The tool searches for CpG islands and simulates bisulfite modification of DNA in silico. Perform PCR to amplify region of interest.
Products for Step 3: PCR Amplification & Cleanup
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Methyl Primer Express® Software v1.0
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This free software enables you to design high quality PCR primers for methylation mapping experiments. Simply cut and paste in your region of interest. The tool searches for CpG islands and simulates bisulfite modification of DNA in silico. Applied Biosystems supplies all the tools you need to complete methylation mapping experiments.
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GeneAmp® Gold RNA PCR Reagent Kit and Core Kit
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The GeneAmp® Gold RNA PCR Reagent Kit has been optimized to combine reverse transcription using MultiScribe™ Reverse Transcriptase, with highly sensitive and specific amplification using AmpliTaq Gold® DNA Polymerase.
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Custom Oligo Synthesis - Unlabeled Oligo
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We offer a portfolio of fluorescent dyes for gene expression studies, 5’ nuclease assays, PCR, fragment analysis, and genotyping. When you know the exact sequence of a fluorescent labeled probe or primer, we can synthesize it for you.
Step 4: Sequencing Reaction Cleanup
Amplification products of bisulfite-converted DNA can be analyzed by fragment analysis or sequencing.
Products for Step 4: Sequencing Reaction Cleanup
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BigDye XTerminator® Purification Kit
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The BigDye XTerminator® Purification Kit is a fast, simple purification method for DNA sequencing reactions that removes unincorporated BigDye® terminators. No more dye blobs! Cleanup is complete in under 40 minutes and requires less than 10 minutes of labor.
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GeneScan™ Size Standards
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GeneScan™ Size Standards are fluorescently-labeled DNA fragments required for performing fragment analysis and genotyping applications on our capillary electrophoresis platforms.
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Sequencing Reagents & Kits
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View our full range of Sequencing Reagents & Kits for every budget and application.
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DNA Sequencers
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View our full range of DNA Sequencers for every budget and application.
Step 5: Capillary Electrophoresis
View base-called sequence data and export for specialized downstream applications to assess methylation status. Explore presence of methylation-specific peaks (fragments) with GeneMapper or PeakScanner software.
Products for Step 5: Capillary Electrophoresis
- Sequence Scanner Software
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The free Sequence Scanner Software enables you to view, edit, print and export sequence data generated using the Applied Biosystems Genetic Analyzers. The software generates graphically expressive reports on results.
- Variant Reporter® Software
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The Variant Reporter™ Software performs comparative sequencing, also known as direct sequencing, medical sequencing, PCR sequencing and resequencing with DNA sequencing files. The software is designed for reference based and non-reference based analysis such as mutation detection and analysis, SNP discovery and validation and sequence confirmation.
- SeqScape® Software
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Applied Biosystems SeqScape® software is designed for reference based analysis such as mutation detection and analysis, SNP discovery and validation, pathogen sub-typing, allele identification, and sequence confirmation. This product is also integrated for use with VariantSEQr™ Resequencing Set.
- Sequencing Analysis Software
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This software enables you to basecall, trim, display, edit, and print data from our entire line of capillary DNA sequencing instruments for data analysis and quality control.
- GeneMapper® Software v4.1
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GeneMapper is a flexible genotyping software package that provides DNA sizing and quality allele calls for all Applied Biosystems electrophoresis-based genotyping systems.
- Peak Scanner™ Software v1.0
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Use this free software to perform DNA fragment analysis; separate a mixture of DNA fragments according to their sizes, provide a profile of the separation, and precisely calculate the sizes of the fragments.
Step 6: Data Analysis
After electrophoresis, Data Collection software creates a Sample File of the raw data. Using downstream software applications, further data analysis is required to translate the raw data into the corresponding electropherogram.
Application Note
Product Bulletin
Publication
- Promoter Hypermethylation in Thyroid Cancer Samples Publication: Innovations Newsletter - Issue 09, Oct 2008
Training Courses
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