DNA restriction library tagging and analysis

摘要

The disclosed methods in the application relates to making an adapter-tagged restriction library. The library or libraries created with the disclosed methods avoid the self-ligation of the restriction fragments. The application further disclosed methods of using the library or libraries to measure the methylation level of a genome, or comparing methylation levels among two or more genomes. The measurement of the global methylation levels can be achieved with quantitative PCR method by measuring the number of restriction fragments in the libraries. It also can be used for next generation Sequencing (NGS), Copy Number Variation (CNV), restriction site mutation, endogenous gene jumping, and exogenous DNA insertion, somatic hypermutation, gene knockout/knock in etc.

主权项

1. A method of making an adapter-tagged restriction library comprising:
providing a double-stranded DNA oligonucleotide which is a twin adapter containing a recognition site of a first restriction enzyme (first RE); digesting the twin adapter with the first RE, resulting in two adapters with the same restriction fragment end; digesting a genomic DNA with at least a second restriction enzyme (second RE), wherein the digestion by the second RE results in the genomic DNA being broken into a plurality of restriction fragments, wherein the second RE has a recognition site different from that of the first RE, yet after digestion leaves the same restriction fragment end as that of the first RE; ligating with a DNA ligase the digested twin adapter to one end or each end of the plurality of restriction fragments, resulting in a mixture of a self-ligation product and a hetero-ligation product; repeating the ligating and digesting steps multiple times as needed with the mixture of a self-ligation product and a hetero-ligation product, to make the adapter-tagged restriction library substantially free of the self-ligation product, wherein the repeated digesting step is the step of digesting the mixture of a self-ligation product and a hetero-ligation product with the first RE or the second RE, wherein the first RE or the second RE can cleave the self-ligation product and is incapable of cleaving the hetero-ligation product, and wherein the repeated digesting and ligating steps result in the hetero-ligation product substantially free of the self-ligation product.