Determination of nucleic acid methylation

Abstract

The present invention relates to methods and kits for determining the methylation status of a target nucleic acid molecule in a sample.

What is claimed is: 
     
         1 . A method of determining the methylation status of a target nucleic acid molecule in a sample, wherein the method comprises the steps of:
 (a) bisulfate treatment of the sample for converting the unmethylated cytosine bases in the target nucleic acid molecule to uracil;   (b) amplifying at least part of the converted target nucleic acid molecule, said part comprising a target region the methylation status of which is to be analyzed, in a polymerase chain reaction (PCR), using a pair of methylation-specific primers under conditions allowing such amplification to generate PCR amplification products (amplicons);   (c) contacting the amplicons with a methylation-specific plasmonic nanoprobe comprising a plasmonic nanoparticle and a non-ionic oligonucleotide analog probe covalently coupled thereto, the oligonucleotide analog probe comprising a base sequence that is complementary to the target region, under conditions that allow the oligonucleotide analog probe and the amplicons comprising the target region to hybridize to each other, wherein the probe generates a detectable signal if hybridized to the target nucleic acid molecule that is distinguishable from the signal of the unhybridized probe;   (d) determining the methylation status of the target nucleic acid molecule based on the determination of the melting temperature T m  of the hybrid of the nanoprobe and the target nucleic acid.   
     
     
         2 . The method according to  claim 1 , wherein the methylation status of one or more CpG islands within the target nucleic acid molecule is determined. 
     
     
         3 . The method according to  claim 1 , wherein asymmetric PCR (aPCR) is used in step (b) of the method. 
     
     
         4 . The method according to  claim 1 , wherein at least one of the methylation-specific primers hybridizes to the converted target nucleic acid molecule at a region comprising one or more CpG islands, such that the primer pair preferably hybridizes to the converted methylated nucleic acid molecule over the converted unmethylated molecule during the PCR. 
     
     
         5 . The method according to  claim 1 , wherein a blocker sequence is added during the PCR to minimize the amplification of the converted unmethylated nucleic acid molecule. 
     
     
         6 . The method according to  claim 1 , wherein the plasmonic nanoparticle used in step (c) of the method is a plasmonic gold nanoparticle. 
     
     
         7 . The method according to  claim 1 , wherein the non-ionic oligonucleotide analog probe used in step (c) of the method is a morpholino oligonucleotide probe. 
     
     
         8 . The method according to  claim 1 , wherein the detectable signal in step (c) of the method is the color of the assay solution that is indicative of whether the probe is hybridized to the amplicons or not. 
     
     
         9 . The method according to  claim 1 , wherein the melting temperature in step (d) of the method is indicated by a color change caused by nanoprobe dissociation and subsequent aggregation. 
     
     
         10 . The method according to  claim 1 , wherein the method further comprises isolating genomic DNA from the sample prior to step (a) of the method. 
     
     
         11 . The method according to  claim 1 , wherein the method further comprises using a nucleic acid molecule comprising the unmethylated target nucleotide sequence as a negative control, and/or using a nucleic acid molecule comprising the methylated target nucleotide sequence as a positive control. 
     
     
         12 . The method according to  claim 1 , wherein the target nucleic acid molecule is the human Septin 9 (SEPT9) gene promoter, the methylation-specific primers have the nucleic acid sequences 5′-ATTAGTTATTATGTCGGATTTCGC-3′ (SEQ ID NO: 1) and 5′-CAACACGTCCGCGACCG-3′ (SEQ ID NO: 2), the blocker has the nucleic acid sequence 5′-GTTATTATGTTGGATTTTGTGGTTAATGTGTAG-3′ and is labelled with C3 spacer at the 3′ end (SEQ ID NO: 3), and the oligonucleotide analog probe used is a morpholino oligonucleotide having the nucleic acid sequence 5′-CAACTACGCGTTAACCGCGATTTTT-3′ (SEQ ID NO: 4). 
     
     
         13 . The method according to  claim 12 , wherein the method is used to determine the methylation status of the human Septin 9 (SEPT9) gene promoter, preferably in the cell-free DNA (cfDNA) fragments circulating in the blood released from colorectal cancer cells.