HIGH-THROUGHPUT SEQUENCING DETECTION METHOD FOR METHYLATED CPG ISLANDS

摘要

A high-throughput sequencing method for detecting methylated CpG islands includes: processing a DNA sample by using a modifier, and converting cytosine in the DNA sample into uracil, and keeping 5′methylcytosine unchanged; amplifying the obtained segment by using a primer A and DNA polymerase, to obtain a segment having one end being capable of anchoring a junction primer C; amplifying the obtained segment by using a primer B and DNA polymerase, to obtain a segment gathering methylated CpG islands and having two ends being capable of separately anchoring junction primers C and D; amplifying the obtained segment at a PCR exponent by using the junction primers C and D and the DNA polymerase, to obtain the amplified product; and separating and purifying the amplified product, to form a high-throughput sequencing library and perform computer sequencing, and data analysis.

主权项

1. A high-throughput sequencing method for detecting methylated CpG islands comprising:
(a) treating a DNA sample with a modifying agent to form the modified DNA wherein cytosine bases but not 5′-methyl-cytosine bases of the DNA sample are modified to uracil bases; (b) providing Primer A and DNA polymerase to the modified DNA to allow at least one round of linear amplification to form the semi-amplicon capable of anchoring Adapter Primer C at one end, wherein Primer A is composed of a 3′ portion and a 5′ portion, wherein the 3′ portion contains 4 or more nucleotides capable of binding to the modified DNA and allowing amplification, wherein the 5′ portion allows Adapter Primer C to bind to its reverse complementary sequence for PCR amplification; (c) amplifying the semi-amplicon by using Primer B and DNA polymerase to form the full-amplicon enriched with methylated CpG islands and capable of anchoring Adapter Primer C at one end and Adapter Primer D at the other end, wherein Primer B is composed of a 3′ portion and a 5′ portion, wherein the 3′ portion contains 7 or more nucleotides and is a high-CpG-density sequence capable of binding to the semi-amplicon and allowing amplification and enrichment of the methylated CpG islands, wherein the high-CpG-density sequence is a sequence of which the 3′-terminal 7 nucleotides contain 2 or 3 CpG dinucleotides, wherein the 5′ portion allows Adapter Primer D to bind to its reverse complementary sequence for PCR amplification; (d) amplifying the full-amplicon by using Adapter Primer C, Adapter Primer D and DNA polymerase to form the final-amplicon via PCR exponential amplification; (e) separating and purifying the final-amplicon to form the library for high-throughput sequencing, sequencing the library and analyzing the data.