CHAPTER 8 NOTES

This chapter uses the word "weight" in place of the term "mass". This is not correct and reflects a lack of scientifically-literate copy editors.

Please make sure to show real examples from the literature.

Look to see if there are new techniques that are not discussed in the chapter.

For each technique, it is important to not the disadvantages as well as the advantages.

Some of the techniques that you will discuss are older and are not currently used. It is still important to discuss these since one needs to understand them so that you can read the older literature. Also older approaches teach principles that are often (re-)used to engineer new techniques.

page 126 methylation-specific restriction endonucleases

Some restriction enzymes won't cut a recognition site that contains 5-meC and some will only cut methylated targets.

This can be exploited to identify the methylation state of a DNA target(s).

The easiest way to imagine how this works is the variation in which two enzymes that recognize the same site but that differ only on their methylation sensitivity are used. Example MspI cleaves CCGG regardless of methylation state but HpaII ONLY cleaves the unmethylated DNA targets.

Here is a list of enzymes and their methylation sensitivity:

http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/dam_dcm_cpg_methylation.asp

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COBRA - Figure 8.2B bisulfite conversion of genomic DNA, then PCR with flanking primers. The product contains a restriction enzyme cut site that contains a C that might or might not be methylated. If it is NOT methylated then bisulfite conversion destroys the restriction site because it converts the C-->U. Because of this, the PCR product made from UNmethylated bisulfite-treated is NOT cut by the enzyme.

It is important to remember that the 5meC is NOT bisulfite converted to U. This means that the restriction site survives and the enzyme cuts the PCR product made from bisulfite-treated DNA.

The products are run on a gel and a the unique banding patterns and relative abundance of each band provides a measure of the methylation state of the DNA (see figure 8.2B).

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The Mass-Array figure in this chapter does not make sense. Read about it elsewhere. This technique could be left to the semi-genomic section. You guys decide.

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Genomephi is mentioned but not discussed. It is not essential for understanding the techniques but more annoying that the author mentions it just once.

See

http://www.biocompare.com/Articles/ProductReview/235/GenomiPhi-DNA-Amplification-Kit-by-GE-Healthcare-formerly-Amersham-Bioscience.html

This is a whole genome amplification technique (strand displacing polymerase) that is supposed to let you in vitro replicate DNA without altering the relative abundance the fragments. In my opinion, one should never assume that the technique or the kit really does this. You must independently verify all whole genome amplification procedures.

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Figure 8.4 Panel A. The sequence is mis-aligned and the shading is nonsensical. Please see this figure: Figure 8 point 4.pdf

MBD protein (methyl CpG binding domain protein) is usually restricted to genomic assays.

To talk about this technique as presented you must include a discussion of the function and consequences of annealing temperature. You may need a PCR figure just to refresh everyones' mind. You will also need to describe what is meant by a melting curve.

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CHAPTER 9 GENOMIC METHYLATION ASSAYS

Figure 9.2 is tough to understand. Original references are usually the solution. Please see this paper:

Hatada I, Hayashizaki Y, Hirotsune S, Komatsubara H, Mukai T (1991) A genomic scanning method for higher organisms using restriction sites as landmarks. Proc Natl Acad Sci U S A 88:9523–9527

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Figure 9.3 "Arbitrary primers will amplifiy from a great many DNA fragments.

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MS-RDA is a subtraction approach and is not really described in the text. Look it up elsewhere if you want to figure it out pg 137.

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Figure 9.4 Methylation CpG island amplification coupled microarray (MCAM) uses the fact that linkers cannot be ligated to SmaI ends but that they can be ligated to XmaI ends.

Based on the fact that SmaI is blocked by methylation.

XmaI is impaired but not blocked, so given time it will work.

SmaI and XmaI recognize the same DNA target (CCCGGG).

The blunt end generated by SmaI almost never ligates to a linker.

The sticky end generated by XmaI ligates will to the sticky-ended linker.

The PCR primers recognize the linker.

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Methylation-specific digital karyotyping (MSDK) derived from SAGE. Please see the SAGE figure. SAGE.pdf

Use this PDF to help you understand the approach. SAGE was a technique to identify differential gene expression. Really it can be used to detect any differences between DNA samples.

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Sequencing-based approaches

Major problem is that it is more difficult to ID the chromosomal origins of DNA sequences when almost all of the C's in the genome have been converted to U's!

Padlock probes for CpG islands have been used to help with these. page 140

What are are padlock probes?

See Deng J, Shoemaker R, Xie B, Gore A, LeProust EM, Antosiewicz-Bourget J, Egli D, Maherali N, Park IH, Yu J, Daley GQ, Eggan K, Hochedlinger K, Thomson J, Wang W, Gao Y, Zhang K (2009) Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming. Nat Biotechnol 27:353–360. Figure 1

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page 144: 5-hydroxymethylcytosine <-- A new modification!!! Can we assay it? See below.

5-hmC and 5mC analysis

http://www.neb.com/nebecomm/products/productE3317.asp