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 (“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 breaking 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 adapter to the plurality of restriction fragments, resulting in a mixture of a self-ligation product and a hetero-ligation product, wherein the first RE or the second RE can cleave the self-ligation and is incapable of cleaving the hetero-ligation product; repeating the ligating and digesting steps, to make a the adapter-tagged restriction library substantially free of the self-ligation product.