By Ian Tannock, Richard Hill, Robert Bristow, Lea Harrington

ISBN-10: 0071745203

ISBN-13: 9780071745208

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Here, we focus on the main approaches for studying DNA methylation along with their relative advantages and disadvantages (Table 2–3). A few points are worth emphasizing; sodium bisulfite converts unmethylated cytosines to uracil, while methylated cytosines (mC) remain unchanged (Fig. 2–20). This technique can reveal the methylation status of every cytosine residue, and it is amenable to massively parallel sequencing methods. Affinity-based methods using methyl-specific Methods of Molecular Analysis 23 TABLE 2–3 Methods for analyzing DNA methylation.

In the interim, investigators are using the NGS platforms to target specific regions of interest. This strategy can be used to examine all of the exons in the genome, specific gene families that constitute known drug targets, or megabase-size regions that are implicated in disease or pharmacogenetic effects. Methods to perform the initial first step are known as genomic partitioning and broadly include methods involving PCR, or other hybridization methodologies. These are generally hybridized to target-specific probes either on a microarray surface or in solution.

These vectors allow the manipulation of a gene, most commonly when an exogenous agent (such as tetracycline or estrogen) is added or taken away from culture media: this is achieved with a specific repressor that responds to the exogenous agent, and is fused to domains that activate the gene of interest. One method of transfection uses hydroxyethyl piperazineethanesulfonic acid (HEPES)-buffered saline solution (HeBS) containing phosphate ions combined with a calcium chloride solution containing the DNA to be transfected.

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Basic Science of Oncology by Ian Tannock, Richard Hill, Robert Bristow, Lea Harrington

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