Methods of Analysis of Methylation

Abstract

Methods for determining the methylation status of a plurality of cytosines are disclosed. In some aspects genomic DNA target sequences containing CpGs are targeted for analysis by multiplex amplification using target specific probes that can be specifically degraded prior to amplification. The targets may be modified with bisulfite prior to amplification. In another aspect targets are cut with methylation sensitive or insensitive restriction enzymes and marked with a tag using the target specific probes. The presence or absence of methylation may be determined using methylation sensitive restriction enzyme or bisulfite treatment. Detection in many embodiments employs hybridization to tag arrays, genotyping arrays or resequencing arrays.

Description

RELATED APPLICATIONS [0001] The present application claims priority to U.S. application No. 60 / 862,735 filed Oct. 24, 2006, the disclosure of which is incorporated herein by reference in its entirety.BACKGROUND OF THE INVENTION [0002] The genomes of higher eukaryotes contain the modified nucleoside 5-methyl cytosine (5-meC). This modification is usually found as part of the dinucleotide CpG. [0003] Cytosine is converted to 5-methylcytosine in a reaction that involves flipping a target cytosine out of an intact double helix and transfer of a methyl group from S-adenosylmethionine by a methyltransferase enzyme (Klimasauskas et al. Cell 76:357-369, 1994). This enzymatic conversion is the only epigenetic modification of DNA known to exist in vertebrates and is essential for normal embryonic development (Bird, Cell 70:5-8, 1992; Laird and Jaenisch, Human Mol. Genet. 3:1487-1495, 1994; and Li et al. Cell 69:915-926, 1992). [0004] The frequency of the CpG dinucleotide in the human genome i...

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Benefits of technology

[0009] In some aspects the methods include a step of multiplex amplification of a plurality of regions of interest. The methods provide for the addition of known priming sequences to the 5′ and 3′ ends of the sequences to be amplified so that subsequent amplification may be performed using primers to the known priming sequences. Such multiplexed amplification reactions provide high specificity and uniform amplification of templates.

[0012] The multiplex methods of the present invention may include at least 10 templates of distinct sequence, at least 100 templates of distinct sequence, at least 1000 templates of distinct sequence, or more. Usefully, at least one of the first and second oligonucleotides comprises a bar code sequence, thus allowing concurrent detection of all amplified templates.

Problems solved by technology

Alterations in the normal methylation process have also been shown to be associated with genomic instability (Lengauer et al.

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