CN1293255A

CN1293255A - Process for detecting gene mutation on glass plate

Info

Publication number: CN1293255A
Application number: CN 00116095
Authority: CN
Inventors: 张先恩; 文继开; 邓教宇; 刘虹; 张治平
Original assignee: Wuhan Institute of Virology of CAS
Current assignee: Wuhan Institute of Virology of CAS
Priority date: 2000-10-25
Filing date: 2000-10-25
Publication date: 2001-05-02

Abstract

A method for detecting gene mutation on glass plate includes such steps as fixing the hybridized complete DNA heteroduplex to glass plate to make the 5' terminal of one of said double strands be cross-linked with mercapto silanized glass, modifying the cytosine in mismatched base with hydroxylamine solution and then with mixed solution of potassium permanganate and tetraethylamine chloride, shearing the modified pyrimidine base with piperidine, digesting the DNA heteroduplex wth (2-5)-unit SI acid enzyme, and mutation detection by linking human colibacillus basic phosphorylase. Its advantages are high correctness, sensitivity and speed, and low cost.

Description

A kind of method that on slide, detects transgenation

The present invention relates to the sudden change of solid phase dna sequence dna and detect, more specifically relate to a kind of method that detects resistance genes involved point mutation in the resistance tubercule bacillus.

Be applied at present the method for the sudden change of check point in the slide common chip micrometering preface and high density oligonucleotide sequence testing chip arranged.Chip micrometering preface method is to utilize fluorescein-labeled dideoxy nucleotide to carry out the solid phase primer extension, measures the sequence of transgenation hot zone, positional mutation exactly.But present method need be synthesized a large amount of primers, causes cost high and sequence that surveyed is very limited, generally is difficult to surpass 100 bases.Detecting point mutation with the high density oligonucleotide sequence testing chip is that original position is synthesized 4 times to testing gene base number 8 (or 9) aggressiveness oligonucleotide on chip, with fluorescently-labeled testing gene is that template is carried out chip hybridization, compares the back at the hybridization signal with wild type gene and determines sudden change.The accuracy of oligonucleotide sequencing chip detection sudden change can't reach 100%, need original position to synthesize a large amount of oligonucleotide fragments, the necessary mark fluorescent element of hybridization template, and chip density height, the analyzing and processing difficulty of the collection of hybridization signal and data is big, therefore must suite of equipment and corresponding software, cost costliness from the last information processing that is fabricated into of beginning chip.Chip capillary cataphoresis is to utilize the variation of dna double chain molecular conformation can cause the change of electrophoretic migration, the dna double chain of sudden change can be distinguished come by highly sensitive capillary electrophoresis.It is very high that but this method requires instrument, and this method is used in many laboratories still incompetence, uses just to seem more difficult.

The purpose of this invention is to provide a kind of method that on slide, detects transgenation, chemical method and zymetology cutting method have been combined, accurate with zymetology detection or fluoroscopic examination means, detect the point mutation that is fixed on the dna sequence dna on the slide easily.

In order to achieve the above object, the present invention adopts following technical measures: the detection method of the sudden change of the dna sequence dna on the solid phase carrier is that hybridization gained heteroduplex DNA is fixed on the carrier, utilize the chemical reagent chemically reactive different with the single stranded DNA tool then to double-stranded DNA, be that chemical reagent can special modification match and the miazines base of mispairing: azanol hydrochloric acid is modified the cytosine(Cyt) in the base mismatch, and potassium permanganate and chlorination triethylammonium tetrakis mixed solution or perosmic anhydride solution are modified the thymus pyrimidine in the base mismatch.Sending the adorned miazines base of identification that pyridine can be special and cut on the phosphide key of this base 3 ' end and cause an otch, because there is base mismatch in a side of otch, therefore is not proper otch and more be similar to a breach.The pairing strand of otch partly can be cut under certain temperature and ionic concn with the S1 nuclease.Owing to produce cutting fully, the fluorescence of introducing or biotin labeling molecule possibility quilt be wash-out fully.Signal by fluorescence or zymetology means certification mark molecule just can judge whether dna sequence dna to be checked exists point mutation.

The method that detects the point mutation of Mycobacterium tuberculosis drug-resistant genes involved on slide is that a DNA chain will determining resistance genes involved in the good tubercule bacillus type strain (H37Rv) is fixed on the slide with mercapto derivatives, then with mutant strain in 5 ' end of obtaining corresponding resistance genes involved of being connected with fluorescence or biotin labeling molecule hybridize, it is characterized in that the complete allogeneic dna sequence DNA two strands that hybridization generates is fixed on slide or other solid phase carriers, 5 of a chain ' end is mutually crosslinked with the solid phase carrier that the slide or the surface of hydrosulphonyl silaneization are connected with Streptavidin in the allogeneic dna sequence DNA two strands, and 5 of another chain ' end carries vitamin H or fluorescent tag molecule; With the cytosine(Cyt) in the hydroxylamine solution modification base mismatch, with the thymus pyrimidine in potassium permanganate and chlorination triethylammonium tetrakis mixed solution or the perosmic anhydride solution modification base mismatch, modify the miazines base with the quilt in the sudden change of piperidines shearing point, the S1 nuclease digestion that adds 2 to 5 units is handled the strand part that occurs in the allogeneic dna sequence DNA two strands; The mark that zymetology or fluoroscopic examination are connected into determines whether to exist sudden change.

The present invention compared with prior art, have the following advantages and effect, the present invention not only can accurate also sensitivity detect the point mutation that exists in the Mycobacterium tuberculosis drug-resistant genes involved fast on slide, but also can detect other sudden changes such as insertion, disappearance, and it is big to detect the dna sequence dna length range, with low cost, easy and simple to handle, be suitable for slide and the sudden change on the solid phase carrier of covalent modification and detect.

Embodiment:

Employing azanol and potassium permanganate or perosmic anhydride are modified cytosine(Cyt) or the thymus pyrimidine in the heteroduplex DNA base mispairing that is fixed on the slide respectively, with adorned base in the piperidines cutting heteroduplex DNA, utilize the S1 nuclease that heteroduplex DNA is thoroughly interrupted then again.Show by having or not of certification mark signal whether this heteroduplex DNA exists the mispairing of single base, determine whether this fragment gene or dna sequence dna exist point mutation.The concrete steps of suddenling change on the Mycobacterium tuberculosis drug-resistant genes involved are as follows detecting on the slide:

1, fluorescein or biotin labeling gene order to be detected: with 6 ' two kinds of primers that end has sulfydryl gene fragment in the tubercule bacillus reference culture that increases respectively; With 5 ' the primer open grave that end has biotin labeling or has fluorescence increases the genes involved fragment of endurance strain.Because two pairs of primer sequences are in full accord, two fragment gene fragments of amplification order except the point mutation site is in full accord.With pillar PCR product purification test kit purified pcr product.

2, the acquisition of allogeneic dna sequence DNA two strands: the 95 ℃ of sex change 5 minutes in annealing buffer of two kinds of PCR products of purifying gained, slowly be annealed to 25 ℃, the dehydrated alcohol sedimentation is also reclaimed the double-stranded DNA of annealing gained.

3, allogeneic dna sequence DNA two strands fixing on the slide: the double-stranded DNA that purifying is good is fixed on through on the slide of silanization, the escherichia coli alkaline phosphatase that adds 1-2 μ l is combined on the allogeneic dna sequence DNA two strands it, the 5-bromo-4-chloro-3-indyl-phosphoric acid and the nitro blue tetrazolium mixed solution that add 3-5 μ l, 37 ℃ were reacted 30-60 minute, detect the fluorescence molecule that is connected on the heteroduplex DNA by the substrate color reaction or with fluorescence detector, whether the allogeneic dna sequence DNA two strands that conclusive evidence resistance genes involved forms fixes successfully.

4, hydroxylamine hydrochloride is to the modification of the pyrimidine bases of mispairing in the fixed allogeneic dna sequence DNA two strands: the 2-4mM hydroxylamine hydrochloride solution PH 6.0 that adds 20 μ l on the slide of fixing allogeneic dna sequence DNA two strands, 37 ℃ are incubated 2 hours, stop this reaction with stop buffer, damping fluid is made of following reagent, the 1-3M sodium acetate, 0.05-0.3mM disodium edta, the tRNA of 15-30mg/ml.Clean this slide with deionized water or distilled water, natural air drying under the aseptic condition.

5, potassium permanganate, chlorination triethylammonium tetrakis mixed solution or perosmic anhydride solution are to the modification of the pyrimidine bases of the double-stranded mispairing of allogeneic dna sequence DNA: at 1-3mM potassium permanganate, 20-30mM chlorination triethylammonium tetrakis mixing solutions or the 15 μ l about adding 20 μ l on the slide that hydroxylamine hydrochloride was handled, the perosmic anhydride solution of 4-5%, 25 ℃ are incubated 1 hour, clean slide with deionized water or distilled water, aseptic air-dry.

6, piperidines and the effect of S1 nuclease and cut the allogeneic dna sequence DNA two strands: on air-dry slide, add 20-50 μ l, the 1-2M piperidines, 90 ℃ of insulations 30 minutes, with distilled water or washed with de-ionized water, air-dry.Add 20 μ l and contain the S1 nuclease of 2-5 unit, 23 ℃ of insulations 15 minutes.

7, the detection of signal mark: clean slide, add the escherichia coli alkaline phosphatase of 1-2 μ l and 5-bromo-4-chloro-3-indyl-phosphoric acid and the nitro blue tetrazolium mixed solution of 3-5 μ l, 37 ℃ were reacted 30-60 minute, the substrate mixed solution is bluish voilet, illustrate and do not have point mutation in the testing gene, the substrate mixed solution is in the faint yellow explanation testing gene sudden change, perhaps directly detecting intensity of fluorescence with fluorescence detector changes and illustrates whether testing gene exists sudden change, constant then explanation of fluorescence intensity do not have point mutation in the testing gene, fluorescence intensity weakens and then illustrates in the testing gene and to have sudden change.

Claims

1, a kind of method that detects point mutation on slide comprises the following steps:

The gene order that A, biotin labeling detect, the primer that 5 ' end is had the sulfydryl gene fragment in the tubercule bacillus reference culture that increases respectively, with 5 ' hold the gene fragment that has biotin labeled primer amplification mutant endurance strain;

The acquisition of B, allogeneic dna sequence DNA two strands with the 95 ℃ of sex change in annealing buffer of two kinds of PCR products of purifying gained, is annealed to 25 ℃, and the dehydrated alcohol sedimentation is also reclaimed;

C, allogeneic dna sequence DNA two strands fixing on slide, the double-stranded DNA of purifying is fixed on through on the slide of silanization, the escherichia coli alkaline phosphatase that adds 1-2 μ l is combined on the allogeneic dna sequence DNA two strands it, the 5-bromo-4-chloro-3-indyl-phosphoric acid and the nitro blue tetrazolium mixed solution that add 3-5 μ l, 37 ℃ were reacted 30-60 minute;

D, azanol hydrochloric acid are to the modification of the pyrimidine bases of mispairing in the fixed allogeneic dna sequence DNA two strands, on the slide of fixing allogeneic dna sequence DNA two strands, add 6.0,37 ℃ of insulations of 2-4mM hydroxylamine hydrochloride solution PH 2 hours of 20 μ l, use the stop buffer termination reaction, clean slide with distilled water, air-dry;

E, potassium permanganate, chlorination triethylammonium tetrakis mixed solution are modified the pyrimidine bases of the double-stranded mispairing of allogeneic dna sequence DNA, the 1-3mM potassium permanganate that on the slide of hydroxylammonium salt acid treatment, adds 20 μ l, 20-30mM chlorination triethylammonium tetrakis mixing solutions, 25 ℃ are incubated 1 hour, clean slide with distilled water, air-dry;

F, usefulness piperidines and S1 nuclease cutting allogeneic dna sequence DNA two strands add 20-50 μ l on air-dry slide, the 1-2M piperidines, and 90 ℃ of insulations are cleaned with distilled water, and are air-dry, add the S1 nuclease that 20 μ l contain 2-5 unit, 23 ℃ of insulations;

G, signal marker detection are cleaned slide, add the escherichia coli alkaline phosphatase of 1-2 μ l and 5-bromo-4-chloro-3-indyl-phosphoric acid and the nitro blue tetrazolium substrate mixed solution of 3-5 μ l, and 37 ℃ were reacted 30-60 minute.