阅读说明：本技术 使用磁阻生物传感器阵列表征dna甲基化 (Characterization of DNA methylation using magnetoresistive biosensor arrays ) 是由 李政録 S·X·王 M·F·汉森 M·杜伏威 G·利兹 于 2018-05-01 设计创作，主要内容包括：一种甲基化检测的方法提供了DNA链中甲基化密度的定量描述。亚硫酸氢盐转化[100]含有甲基化和未甲基化位点的DNA链产生具有错配碱基对的转化DNA链。转化DNA链经PCR扩增[102],并用固定于磁阻(MR)传感器阵列上的互补DNA磁性标记[104]和杂交[106]单链靶DNA链。杂交期间,可以记录结合信号。增加诸如温度或盐浓度之类的严格条件[108],以使磁性标记的单链靶DNA链从固定于磁阻(MR)传感器阵列上的互补DNA链变性。严格条件增加期间,实时记录产生自变性的磁性标记的单链靶DNA链的变性信号[110]并用于确定甲基化和未甲基化的DNA链的严格条件[112]。DNA链可能还包含野生型基因和突变的基因,因此突变位点可以与甲基化位点同时确定。(One method of methylation detection provides a quantitative description of the density of methylation in a DNA strand. Bisulfite conversion [100] of a DNA strand containing methylated and unmethylated sites produces a converted DNA strand with mismatched base pairs. The transformed DNA strand is amplified by PCR [102] and magnetically labeled [104] with complementary DNA immobilized on a Magnetoresistive (MR) sensor array and hybridized [106] to a single stranded target DNA strand. During hybridization, the binding signal can be recorded. Stringent conditions such as temperature or salt concentration are increased [108] to denature the magnetically-labeled single-stranded target DNA strands from complementary DNA strands immobilized on a Magnetoresistive (MR) sensor array. During the increase in stringency conditions, the denaturation signal generated from denatured, magnetically-labeled, single-stranded target DNA strands is recorded in real time [110] and used to determine the stringency conditions of methylated and unmethylated DNA strands [112 ]. The DNA strand may also comprise a wild-type gene and a mutated gene, so that the mutation site can be determined simultaneously with the methylation site.)

1. A method of methylation detection, the method providing a quantitative description of methylation density in a DNA strand, the method comprising:

bisulfite converting a DNA strand containing methylated and unmethylated sites to produce a converted DNA strand having mismatched base pairs;

performing PCR amplification on the transforming DNA strands to produce PCR amplified transforming DNA strands;

magnetically labeling the PCR-amplified transformed DNA strand;

hybridizing the PCR-amplified strands of the transformed DNA to complementary DNA strands immobilized on an array of Magnetoresistive (MR) sensors, wherein the hybridization is performed before or after magnetic labeling;

increasing stringency conditions such that the magnetically-labeled single-stranded target DNA strands are denatured by the complementary DNA strands immobilized on a Magnetoresistive (MR) sensor array;

increasing the stringency conditions, reading in real time the denaturation signal generated from denatured magnetically labeled single-stranded target DNA strands;

from the denaturation signal, the stringency conditions of the methylated and unmethylated DNA strands are determined.

2. The method of claim 1, further comprising reading out binding signals in real time during hybridization of the magnetically labeled single stranded target DNA strands to the complementary DNA strands immobilized on a Magnetoresistive (MR) sensor array.

3. The method of claim 2, wherein the stringent conditions are temperature, wherein increasing the stringent conditions comprises increasing the temperature while salt concentration remains constant, and wherein determining the stringent conditions for the methylated and unmethylated DNA strands comprises determining the melting temperatures of the methylated and unmethylated DNA strands.

4. The method of claim 2, wherein the stringent conditions are salt concentrations, wherein increasing the stringent conditions comprises decreasing the salt concentrations while the temperature remains constant, and wherein determining the stringent conditions for the methylated and unmethylated DNA strands comprises determining melting salt concentrations of the methylated and unmethylated DNA strands.

5. The method of claim 3 or 4, wherein bisulfite converting DNA strands containing methylated and unmethylated sites comprises bisulfite converting DNA strands containing methylated and unmethylated sites and wild type and mutant genes; and wherein determining the stringency conditions for the methylated and unmethylated DNA strands from the denaturation signal comprises determining the stringency conditions for the methylated and unmethylated DNA strands and the wild type gene and mutant gene from the denaturation signal, whereby the mutation site and the methylation site can be determined simultaneously.

6. A method of methylation detection, the method providing a quantitative description of methylation density in a DNA sequence, the method comprising: bisulfite conversion of DNA strands with or without methylation sites; PCR amplification and transformation of DNA chain; the transformed target DNA strand is hybridized to an MR sensor array on which (unmethylated) complementary DNA strands are immobilized; adding a methyltransferase enzyme to methylate a complementary DNA strand corresponding to the target DNA strand methylation site; increasing the temperature until the target DNA strand is denatured from the immobilized DNA strand, leaving the methylated single-stranded DNA if the target DNA is methylated, or leaving the unmethylated single-stranded DNA if the target DNA is unmethylated; adding magnetic nanoparticles coupled to a methyl recognition moiety, such as an anti-methylated lysine antibody, which will bind to methylated DNA strands immobilized to the sensor; reading the binding signal in real time and determining whether the immobilized DNA strand (and the corresponding target DNA strand) is methylated.

Technical Field

The present application relates generally to biosensing technology and devices. More particularly, it relates to the use of biosensor arrays in DNA methylation and mutation analysis.

Background

Cancer is a cellular disease caused by the gradual accumulation of genetic and epigenetic changes. Extensive sequencing work has identified recurrent genetic mutations that can be used as genetic biomarkers for assessing risk of cancer development, classifying disease subtypes, predicting response to treatment, and monitoring treatment efficacy. DNA methylation causes epigenetic silencing of tumor suppressor genes and studies on their direct impact in tumorigenesis and their use as cancer biomarkers. In bladder and colon cancer, a combination of genetic and epigenetic analyses has been shown to be of greater diagnostic value than either method applied alone. However, methylation profiling is not a "yes-no" outcome compared to mutant genotyping, as methylation-driven gene silencing mechanisms are typically sensitive to the overall density of methylation sites, and multiple CpG dinucleotides (the most common methylation sites) are typically present in gene promoters. Finally, methylation density may vary between alleles and cells within a single tumor, leading to heterogeneous characteristics.

Based on amplification, probe hybridization, enzymatic digestion, gel electrophoresis or sequencing, a variety of techniques have been developed to detect single point mutations in DNA. DNA methylation information is lost during Polymerase Chain Reaction (PCR) amplification and DNA hybridization is insensitive to the methylation state of the target region. Therefore, DNA must be subjected to methylation-sensitive pretreatment. Two major DNA methylation analysis techniques are based on methylation sensitive enzymatic digestion, affinity enrichment, using antibodies specific for methylated cytosines, or bisulfite conversion of unmethylated cytosines to uracil. Bisulfite conversion is most widely used because methylation events are converted to single base changes (C/T) that can be detected using techniques derived from mutation detection, including sequencing array hybridization, methylation-sensitive PCR, and methylation-sensitive melting curve analysis. Sequencing bisulfite converted DNA will quantify the methylation status and allow comparison of data in different sequencing runs and batches, but this is expensive and time consuming. Amplification and melting based techniques are not specific for a single methylation site and are not easily scalable to study a large number of methylation sites. Array-based methods, such as Illumina BeadChip (Illumina Inc., illinois, san diego, california), provide highly multiplexed site-specific assays. However, after bisulfite conversion and amplification of the template, the DNA product contains predominantly three bases (guanine, adenine and thymine, plus the residue of methylated cytosine). This reduced sequence complexity complicates the design of probes for endpoint detection, and the reduced sequence variation reduces specificity.

Summary of The Invention

The present invention is directed to simultaneous methylation (and optionally mutation) characterization in a scalable chip platform that provides highly specific and quantitative DNA methylation and mutation data on a compact, easy-to-use, and potentially low-cost platform. Our preferred method is based on the hybridization of magnetically labeled target DNA to tethered DNA probes on the surface of a GMR biosensor array. To improve the specificity of the DNA hybridization assay, we used melting curve measurements of surface tethered DNA hybrids. This avoids routine assay condition optimization, as the target-probe hybrids are exposed to increasing stringency during melting curve measurements. The melting curve of surface tethered DNA probes has also been measured using fluorescence and surface plasmon resonance. Compared to these methods, GMR biosensors have a very high sensitivity, almost no magnetic background signal from the biological sample, and are temperature independent.

In one aspect, the invention provides a method for simultaneously characterizing DNA mutations and methylation events of a site array with a single site specificity. It advantageously employs methylation detection with an array of magnetoresistive sensors and simultaneous characterization of methylation and mutations in DNA sequences. Genomic (mutant) or bisulfite treated (methylated) DNA was amplified using non-discriminatory primers, and the amplicons were then hybridized to an array of Magnetoresistive (MR) biosensors, followed by real-time melting curve measurements. This MR biosensing technology provides scalable DNA hybridization multiplex detection that has been shown to be insensitive to changes in temperature, pH, and bio-fluid matrix. Melting curve methods further enhance assay specificity and tolerance to changes in probe length. Alternatively, the technique may use a method of applying methyltransferases on an array to transfer methylation sites on DNA sequences tethered to the sensor surface and directly target the methylation sites for detection. The method enables characterization of mutations and methylation sites and provides a quantitative assessment of methylation density comparable to bisulfite pyrosequencing.

Embodiments of the present invention advantageously provide epigenetic and mutation analysis that can be easily implemented in magnetic DNA chips. Magnetic detection hybridization provides high sensitivity and little or no magnetic background from the sample and sample matrix. Real-time melting curve measurement of target-probe hybrids improves the specificity of the assay by challenging the hybrids with increasingly stringent conditions. Methods of increasing stringency include, but are not limited to, increasing the temperature of the MR biosensor array and decreasing the salt (Na +) concentration in the sample buffer. Importantly, real-time melting curve measurement eliminates the need for probe optimization required for end-point detection.

In one aspect, a method of methylation detection provides a quantitative description of the methylation density in a DNA strand. The method comprises bisulfite converting a DNA strand containing methylated and unmethylated sites to produce a converted DNA strand having mismatched base pairs; subjecting the transforming DNA strand to PCR amplification to produce a PCR-amplified transforming DNA strand; hybridizing the PCR-amplified transforming DNA strands with complementary DNA strands immobilized on a Magnetoresistive (MR) sensor array; magnetically labeling the PCR-amplified transformed DNA strands before or after hybridization; increasing stringency conditions to denature the magnetically-labeled single-stranded target DNA strands from complementary DNA strands immobilized on a Magnetoresistive (MR) sensor array; reading in real time the denaturation signal generated by the denatured magnetically-labeled single-stranded target DNA strand during the increase of stringent conditions; and determining the stringency conditions of the methylated and unmethylated DNA strands from the denaturation signal.

In one embodiment, the method further comprises reading the binding signal in real time during hybridization of the magnetically labeled single stranded target DNA strands to complementary DNA strands immobilized on a Magnetoresistive (MR) sensor array.

The stringent conditions can be temperature, wherein increasing the stringent conditions comprises increasing the temperature while the salt concentration remains constant, and wherein determining the stringent conditions for methylated and unmethylated DNA strands comprises determining the melting temperatures of methylated and unmethylated DNA strands. Alternatively, the stringent conditions can be salt concentration, wherein increasing the stringent conditions comprises decreasing the salt concentration while the temperature remains constant, and wherein determining the stringent conditions for methylated and unmethylated DNA strands comprises determining the melting salt concentration of methylated and unmethylated DNA. Furthermore, stringent conditions may be a combination of temperature and salt, where increasing stringency includes both increasing temperature and decreasing salt concentration.

In any method that increases stringency conditions, both mutation sites and methylation sites in the DNA can be detected. PCR amplification of the converted DNA strand may include PCR amplification of the DNA strand after bisulfite conversion and without conversion, wherein the input DNA strand may comprise methylated and unmethylated sites and wild-type and mutant genes; and determining the stringency conditions for the methylated and unmethylated DNA strands from the denaturation signal can include determining the stringency conditions for the methylated and unmethylated DNA strands and the wild type gene and mutant gene from the denaturation signal, so that the mutation site and the methylation site can be determined simultaneously.

Accordingly, the present invention provides a methylation detection method using a Magnetoresistive (MR) sensor array. In a preferred embodiment, the DNA strands with methylation sites are bisulfite converted and PCR amplified. They are then hybridized to a temperature-controlled magnetoresistive (e.g., GMR) biosensor array with immobilized complementary DNA strands and magnetically labeled. The target DNA strand is denatured from the immobilized DNA strand by increasing the temperature. Real-time measurement of binding signals from the target DNA was used to determine the melting curve. In another embodiment, salt concentration is used instead of temperature to denature the target DNA strands. This technique can be used in conjunction with gene mutation measurements on the same biosensor chip.

In another aspect, a method of methylation detection provides a quantitative description of the methylation density in a DNA sequence. The method comprises performing bisulfite conversion of a DNA strand with or without a methylation site; PCR amplification and transformation of DNA chain; the transformed target DNA strand is hybridized to an MR sensor array on which (unmethylated) complementary DNA strands are immobilized; adding a methyltransferase enzyme to methylate a complementary DNA strand corresponding to a methylation site of the target DNA strand; increasing the temperature until the target DNA strand is denatured from the immobilized DNA strand, leaving a methylated single-stranded DNA if the target DNA is methylated, or an unmethylated single-stranded DNA if the target DNA is unmethylated; adding magnetic nanoparticles coupled to a methyl recognition moiety, such as an anti-methylated lysine antibody, which will bind to the methylated DNA strand immobilized to the sensor; reading the binding signal in real time and determining whether the immobilized DNA strand (and the corresponding target DNA strand) is methylated.

Different MR techniques can be used for the biosensing array. These sensors include, but are not limited to, Giant Magnetoresistive (GMR) sensors, Magnetic Tunnel Junction (MTJ) sensors, Planar Hall Effect (PHE) sensors.