TARGETED SEQUENCING TECHNIQUE FOR WHOLE GENOME DNA METHYLATION

摘要

This invention is directed to a guide positioning sequencing technology of whole-genome DNA methylation. The invention provides a new detection method of nucleic acid methylation. In particular, a concept of “positioning” in the detection of nucleic acid methylation is provided. Specifically, a portion of a sequence is used for genome wide positioning and the other portion of the sequence is used for methylation detection in sequencing, thereby solving/defeating previously existing challenges in methylation detection and bioinformatics analysis of a genome.

主权项

1. A method for detection of nucleic acid methylation status, wherein said nucleic acid is double-stranded, said method comprises:
(1) treating the nucleic acid double strands with a polymerase with 3′→5′ cleavage function or a 3′→5′ exonuclease, so that there is a deletion of 80 to 200 bases at the 3′ end of both strands; (2) adding dNTP, wherein cytosine (C) is methylated cytosine (5 mC), so that the deletion at the 3′ end of both strands is end-filled and cytosine thereof is methylated cytosine; (3) treating the double strands in step (2), so that un-methylated cytosine is converted into uracil (U) while the methylated cytosine remains unchanged; (4) PCR amplification, sequencing to determine the methylation status, wherein the un-methylated cytosine has been converted into thymine in a portion of a sequence for determining a methylation site; the other portion of the sequence is the same as the original nucleic acid sequence because the cytosine has been methylated, said portion can be used for sequence positioning in data analysis; comparing the sequence of the two portions to obtain the methylation status of the nucleic acid.