Bulsulfite DNA Sequencing Determines Base Methylation in Epigenetics Research

Bulsulfite DNA Sequencing Determines Base Methylation in Epigenetics Research
By Doug Bintzler

Epigenetics involves the study of external factors that affect the genome and gene expression without altering the base sequence. It involves the relationship between base methylation patterns and histones. Methylation causes DNA to wrap tightly around the histone complex. This hides this DNA from the transcription process, thus silencing the gene. Active and silent genes are defined by the function of the cell as cells become more and more specialized. Liver cells have one specific group of active genes while skin cells have a different group of active genes. Methylation and binding to the histone complexes are functions that determine when cells become more differentiated.

External factors could influence the epigenome. Ultraviolet radiation and chemicals found in harmful agents like tobacco affect methylation patterns. Cells that lose their specialized functions may also lose the mechanisms that control cellular division. Rapid cell division leads to cancer and tumor development. A better understanding of the epigenome and base methylation process may provide a better understanding of cancer. In the past, cancer was believed to be caused by mutation or was the result of specific viruses. Now it is understood that epigenetic damage can also lead to cancer. It is one reason cancer may develop in one of two genetically similar people like twins, but not in the other one. Epigenetics research has led to new directions in cancer treatment with varying success. But how do researchers determine methylation patterns when comparing normal and cancerous cells? Basic Sanger sequencing does not differentiate between methylated bases and non-methylated bases.

What Determines Base Methylation

DNA becomes increasingly methylated during development as groups of cells differentiate into cells with specific functions. In general, cytosine bases that are followed by guanosine bases are methylated along the 5-prime carbon, thus forming a methylated CpG. The cellular genetic mechanism provides the information the cell needs to determine which bases eventually become methylated. DNA methyltransferases help maintain proper methylation as cells divide so that the daughter cells perform the same functions as the parent cells.

Bisulfite Treatment of Genomic DNA

Researchers use a process called bisulfite sequencing to identify methylated bases. Genomic DNA undergoes treatment with bisultfite. This converts non-methylated cytosine bases to uracil. Methylated bases are not affected by bisulfite treatment. Once bisulfite treated DNA is amplified by the Polymerase Chain Reaction (PCR), uracil bases are replaced by thymine. Uracil is the RNA equivalent of thymine. Comparison of DNA that is untreated with bisulfite and DNA treated with bisulfite shows bases transitioning from cytosine to thymine as non-methylated bases. The cytosine bases that remain unchanged are methylated. Genomic DNA is the source for bisulfite treatment. Fragments amplified by PCR can not be treated with bisulfite because the methylation patterns are lost during amplification. This is the basic process used by researchers to determine methylation patterns in DNA for epigenetic studies.

Doug Bintzler is the laboratory director for DNA Analysis, LLC. The company provides quality DNA sequencing services and fragment analysis to the research and commercial community. Additional articles and company information are available at http://www.agctsequencing.com.

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