Methylation sequencing from limiting DNA: embryonic, fixed, and microdissected cells
Introduction
The analysis of DNA methylation can be achieved by a number of different methods, each of which ideally requires a large amount of high-quality DNA, such as cultured cells or fresh tissue samples. However, DNA from early embryonic cells or from tumor cells isolated by microdissection or cells isolated from slides or paraffin sections is often limited in quantity or degraded. For limited or degraded DNA, it is often possible to employ methylation techniques based on polymerase chain reaction (PCR), for example, methylation-sensitive restriction enzyme PCR analysis (MSRE-PCR) [1], or bisulfite/PCR-based methods such as combined bisulfite restriction analysis (COBRA) [2], methylation-sensitive PCR (MSP) [3] or methylation-sensitive single-nucleotide primer extension (Ms-SnuPE) [4]. However, all of these techniques are not informative for studying the detailed methylation profile of individual cells. As described in Fig. 1, the methylation state of a heterogeneously methylated DNA region can often be misinterpreted depending on the method employed for analysis. Restriction-based analysis such as Southern blots, MSRE-PCR, and COBRA can determine only the methylation state of those CpG sites located within restriction enzyme sites and therefore often result in methylation being missed. Alternatively, MSP analysis identifies only regions that are extensively methylated within the priming sites. Bisulfite sequencing is the method of choice if a detailed methylation profile is required, as every cytosine is scored for its methylation state [5].
In this paper we present protocols that adapt the standard bisulfite sequencing reaction to analyze the detailed pattern of methylation in limited or degraded samples. We also discuss the theoretical and practical limits of sensitivity possible using the bisulfite technique and some additional experimental considerations that are necessary when using “difficult” samples as source material.
Section snippets
DNA isolation from preimplantation embryos
To monitor detailed changes in the methylation pattern of individual genes during development it is important to ensure the maximum yield of DNA is isolated from embryos to obtain optimal bisulfite sequencing results [6], [7]. We have evaluated various methods for isolating DNA from embryos or small (<103) numbers of cultured cells. Fig. 2 shows the sensitivity of PCR amplification from bisulfite-treated DNA after isolation using guanidine hydrochloride/proteinase K [8], proteinase K/sodium
References (23)
- et al.
Genomics
(1998) - et al.
Dev. Biol.
(2001) - et al.
Anal. Biochem.
(1995) - et al.
Nucleic Acids Res.
(1990) - et al.
Nucleic Acids Res.
(1997) - et al.
Proc. Natl. Acad. Sci. USA
(1996) - et al.
Nucleic Acids Res.
(1997) - et al.
Nucleic Acids Res.
(1994) - et al.
Mol. Cell. Biol.
(1999) - et al.
Biotechniques
(1997)
Nat. Genet.
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