CN101736083A - Gene methylation detection method suitable for fluorescence polarization technique - Google Patents

Abstract

The invention relates to the field of fluorescence polarization detection technique, in particular to a gene methylation detection method suitable for fluorescence polarization technique. The invention aims to solve the problems of complex operation procedure, high cost and low efficiency in the prior art. The invention has the technical scheme that the gene DNA methylation detection method suitable for fluorescence polarization technique is used for amplifying a reagent which comprises MgCl2, a 10* reaction buffer solution, dNTPs, TaqDNA polymerase, a non-methylated region primer of the CpG island of the target gene promotor region, and a DNA probe, in which the 3'end of the methylated sequence or non-methylated sequence in the target region is provided with a fluorescence label. Compared with the prior art, the invention develops a new platform, is comprehensive and accurate, and has the advantages of simple steps, simple operation and simple analysis of results.

Description

A kind of fluorescence polarization technology that is applicable to detects the method for gene methylation

Technical field:

The present invention relates to field of fluorescence polarization detection, be specifically related to a kind of method that detects gene methylation in the fluorescence polarization technology that is applicable to.

Background technology:

Dna methylation is important epigenetic modification, be in genome, to add the monomethyl group on the 5th carbon atom of promoter region CpG island cytosine(Cyt), make it to become the chemically modified process of 5-methylcytosine, do not change base sequence, change its function by influencing genetic expression.It is the characteristic change of human cancer that the dna methylation level changes, and the detection of important dna methylation is the important guiding value of tool aspect tumour early warning, diagnosis, prevention and early stage therapeutic intervention, resistance prediction, science medication.

At present, the dna methylation detection technique mainly contains electrophoretic technique, DHPLC technology, Methylight technology, microarray technology, burnt sequencing technologies both at home and abroad, great majority design, complicated operation, need electrophoresis and multistep aftertreatment more, waste time and energy, the cost height, the plant and instrument costliness, and efficient has much room for improvement, and is difficult to large-scale promotion application.

Summary of the invention:

The present invention will provide a kind of fluorescence polarization technology that is applicable to detect the methylated method of gene DNA, to overcome complex operation step, cost height and the inefficient problem that prior art exists.

For overcoming the problem that prior art exists, technical scheme of the present invention is:

A kind of fluorescence polarization technology that is applicable to detects the methylated method of gene DNA, is that following reagent is increased:

The reagent that is adopted is by MgCl2, and 10 * reaction buffer, dNTPs, TaqDNA polysaccharase, the non-district's primer that methylates in CpG island, target gene promoters district, target region methylate and 3 ' end of the non-sequence that methylates is with fluorescently-labeled dna probe to form;

The pcr amplification reagent that is adopted comprises 10 times PCR damping fluid; Concentration respectively is dGTP, dCTP, dATP and the dTTP of 2.0～2.5mmol/L, concentration is the downstream primer of the promoter region CpG island of target gene of 0.05-0.2 μ mol/L non-methylate district's upstream primer and 3 ' the end target gene that is 0.5-2 μ mol/L with fluorescently-labeled probe, concentration and the TaqDNA polysaccharase of 1-5U/ μ l, and wherein upstream primer is 5～10: 1 with the ratio of downstream primer concentration;

Described amplification reaction condition is: behind the 94-95 ℃ of sex change 4-10min, hatches 5-40 second for 95 ℃, hatches 10-60 second for 55-65 ℃, hatch 10-40 second for 72 ℃, and 35-45 circulation, last circulation does not have 72 ℃ and hatches.

Compared with prior art, advantage of the present invention is:

1, first fluorescence polarization technology is used for the gene DNA detection that methylates, has opened up the new technical platform that gene DNA methylates and detects.

2, first by using the hybridization with the probe and the target region of non-methylation specific of methylating of single-ended mark, cause the increase of fluorescence polarization value, a plurality of sites that methylate of complete detection target region in view of the above, detected result is accurate more comprehensively.

3, present method step is simple, and is simple to operate; Because the present invention uses asymmetric pcr, 3 ' end fluorescently-labeled probe and specific amplification cycles condition, realized that gene DNA methylates to detect in stopped pipe, to finish that therefore be difficult for polluting, the result is accurate.

4, interpretation of result is simple: only need numeral relatively when the result judges, easily stdn, automatization, applied range; Can be used for detecting clinical blood or tissue sample;

Embodiment:

Below in conjunction with embodiment the present invention is described in detail.

Embodiment 1: during cancer suppressor gene P16 promoter methylation fluorescence polarization detects,

1, used reagent is by MgCl2,10 * reaction buffer, and Taq polymerase, dNTPs, cancer suppressor gene P16 promoter primer, target region methylates and 3 ' end of the non-sequence that methylates is with fluorescently-labeled dna probe to form.

2, mentioned reagent is increased: be reflected in the 25uL system and carry out, add PCR damping fluid 2.5 μ L, 1.0U Taq polymerase; Concentration is respectively dGTP, dCTP, dATP and the dTTP of 2.0mmol/L; Respectively to be with fluorescently-labeled probe mixture, concentration be the downstream primer of 0.5uM to concentration for the upstream primer of 0.05uM and 3 ' end, and the template DNA 5ul. that handles of persulphate test kit

Amplification reaction condition is: behind 94 ℃ of sex change 5min, hatched 10 seconds for 95 ℃, hatched 10 seconds for 60 ℃, hatched 10 seconds for 72 ℃, and 45 circulations, last circulation does not have 72 ℃ and hatches.Then amplified production is carried out the detection of fluorescence polarization value, change the detected result that methylates of interpretation blood or tissue sample according to the difference of fluorescence polarization value and control group.

Embodiment 2: in cancer suppressor gene P53 promoter methylation fluorescence polarization is detected

1, used reagent is: by MgCl2,10 * reaction buffer, Taq polymerase, dNTPs, the upstream and downstream primer of P53 promoter gene, target region methylate and 3 ' end of the non-sequence that methylates is with fluorescently-labeled dna probe probe to form.

2, the mentioned reagent PCR method is increased: be reflected in the 25uL system and carry out, add PCR damping fluid 2.5 μ L, 1.0U Taq polymerase; Concentration is respectively dGTP, dCTP, dATP and the dTTP of 2.5mmol/L; Respectively to be with fluorescently-labeled probe mixture, concentration be the downstream primer of 1.5uM to concentration for the upstream primer of 0.2uM and 3 ' end, and the template DNA 2ul. that handles of persulphate test kit

Amplification reaction condition is: behind 94 ℃ of sex change 5min, hatched 5 seconds for 95 ℃, hatched 15 seconds for 61 ℃, hatched 20 seconds for 72 ℃, and 45 circulations, last circulation does not have 72 ℃ and hatches.Then amplified production is carried out the detection of fluorescence polarization value, change the detected result that methylates of interpretation blood or tissue sample according to the difference of fluorescence polarization value and control group.

Claims (1)

1. one kind is applicable to that fluorescence polarization technology detects the methylated method of gene DNA, is that following reagent is increased:

The reagent that is adopted is by MgCl2, and 10 * reaction buffer, dNTPs, TaqDNA polysaccharase, the non-district's primer that methylates in CpG island, target gene promoters district, target region methylate and 3 ' end of the non-sequence that methylates is with fluorescently-labeled dna probe to form;

The pcr amplification reagent that is adopted comprises 10 times PCR damping fluid; Concentration respectively is dGTP, dCTP, dATP and the dTTP of 2.0～2.5mmol/L, concentration is the downstream primer of the promoter region CpG island of target gene of 0.05-0.2 μ mol/L non-methylate district's upstream primer and 3 ' the end target gene that is 0.5-2 μ mol/L with fluorescently-labeled probe, concentration and the TaqDNA polysaccharase of 1-5U/ μ l, and wherein upstream primer is 5～10: 1 with the ratio of downstream primer concentration;

Described amplification reaction condition is: behind the 94-95 ℃ of sex change 4-10min, hatches 5-40 second for 95 ℃, hatches 10-60 second for 55-65 ℃, hatch 10-40 second for 72 ℃, and 35-45 circulation, last circulation does not have 72 ℃ and hatches.