CN101935689B - Method for identifying telomere DNA fragments and c-kit gene promoter DNA sub-fragments - Google Patents
Method for identifying telomere DNA fragments and c-kit gene promoter DNA sub-fragments Download PDFInfo
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- CN101935689B CN101935689B CN2009100884054A CN200910088405A CN101935689B CN 101935689 B CN101935689 B CN 101935689B CN 2009100884054 A CN2009100884054 A CN 2009100884054A CN 200910088405 A CN200910088405 A CN 200910088405A CN 101935689 B CN101935689 B CN 101935689B
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Abstract
The invention discloses a method for identifying telomere DNA fragments and c-kit gene promoter DNA sub-fragments, which comprises the following steps that: a DNA to be tested is respectively reacted with nitidine chloride or sanguinarine; if the structure of the DNA to be tested subjected to reaction with the nitidine chloride is changed into a mixed G4 structure from a single-stranded structure and the structure of the DNA to be tested subjected to reaction with the sanguinarine is changed into an antiparallel G4 structure from a single-stranded structure, the DNA to be tested is a telomere DNA fragment; and if the structure of the DNA to be tested subjected to reaction with the nitidine chloride is changed into a parallel G4 structure from a single-stranded structure and the structure of the DNA to be tested subjected to reaction with the sanguinarine is still in a single-stranded structure, the DNA to be tested is a c-kit gene promoter DNA sub-fragment. Compared with the prior art, the method of the invention has the following advantages: the telomere DNA fragments and the c-kit gene promoter DNA sub-fragments can be identified effectively; the method can be realized only by mixing compounds and DNA solutions according to the ratio provided in the invention, without tedious post-processing; and the method is simple and feasible in operation and has mild experimental conditions.
Description
Technical field
The present invention relates to a kind of method of discerning telomeric dna fragment and c-kit gene promoter dna fragmentation.
Background technology
Modal DNA is through the formed double-stranded spirane structure of the mode of base pairing by two complementary strands.Yet some DNA that contains a large amount of guanines (G) base repetitive sequence can form four chain spirane structures through Hoogsteen hydrogen bond between the G base, therefore is called as G-four serobilas (being called for short G4).It is found that terminal and some important oncogene transcriptional regulatory districts, can both form G-four serobilas under certain condition at fringes of chromosome.
Research shows that the morbidity of 85-90% malignant tumour is all relevant with the activity of Telomerase, and the G4 structure that forms in the telomere zone can effectively suppress the activity of Telomerase, and then reaches the purpose that suppresses growth of tumour cell.It is made up of one section double-stranded repeat region and one section strand repeat region, for containing the dna fragmentation of a plurality of GGG repeating units.
Except telomere, in the genome of organism, also have many important dna fragmentations can form the G4 structure under given conditions.For example the promoter region of some oncogene and cancer suppressor gene all contains the base sequence that is rich in guanine, can form the G4 structure under given conditions.Wherein, be positioned at the gene fragment (seeing the sequence 4 of sequence table) of proto-oncogene c-kit promotor, can form a kind of special parallel construction G4 under given conditions.The regulation and control that relate to c-kit genetic expression owing to its structure receive much concern.
Therefore, identification telomeric dna fragment and c-kit gene promoter dna fragmentation have very important meaning for the research and development of diagnosing tumor treatment and antitumor drug.
The structure of nitidine chloride is seen formula (I), and the structure of sanguinarine(e) is seen formula (II), and both are four benzene phenanthridine alkaloids.
Formula (I) formula (II)
Summary of the invention
The purpose of this invention is to provide a kind of method of discerning telomeric dna fragment and c-kit gene promoter dna fragmentation.
Identification provided by the invention DNA to be measured comprises the steps: for the telomeric dna fragment still is the method for c-kit gene promoter dna fragmentation
DNA to be measured is reacted with nitidine chloride or sanguinarine(e) respectively; Mix the G4 structure if DNA to be measured is changed into by single-stranded structure with nitidine chloride reaction back, change antiparallel G4 structure with sanguinarine(e) reaction back into by single-stranded structure, DNA then to be measured is the telomeric dna fragment; If DNA to be measured changes parallel G4 structure with nitidine chloride reaction back into by single-stranded structure, constant with sanguinarine(e) reaction back single-stranded structure, DNA then to be measured is a c-kit gene promoter dna fragmentation; Said mixing G4 structure is the mixed structure of parallel G4 structure and antiparallel G4 structure.
The ratio of the amount of substance of said DNA to be measured and said nitidine chloride can be 1: (8-50); The ratio of the amount of substance of said DNA to be measured and said sanguinarine(e) can be 1: (1-50).
The ratio of the amount of substance of said DNA to be measured and said nitidine chloride specifically can be 1: 12; The ratio of the amount of substance of said DNA to be measured and said sanguinarine(e) specifically can be 1: 12.
The reaction of said DNA to be measured and said nitidine chloride can be carried out in the pH value is 6.0～8.0 buffered soln; The reaction of said DNA to be measured and said sanguinarine(e) can be carried out in the pH value is 6.0～8.0 buffered soln.
Said buffered soln specifically can be the Tris buffered soln that concentration is 4-40mM, and preferred concentration is the Tris buffered soln of 10-40mM, is the Tris buffered soln of 10mM like concentration.
In above-mentioned two reaction systems, when reacting initial, the concentration of said DNA to be measured in said buffered soln is 1-5 μ M, is preferably 4 μ M.
The time of said reaction is more than 1 hour, is preferably 12 hours.
In the said method, it is based analyzing method that the method that detects the structural changes of DNA to be measured can be to measure opticity, specifically can be the circular dichroism spectrometry.
Said telomeric dna fragment can be the dna fragmentation that contains four above GGG repeating units.
Said telomeric dna fragment specifically can be the dna fragmentation shown in the sequence 3 of sequence 2 or sequence table of sequence 1 or sequence table of sequence table; Said c-kit gene promoter dna fragmentation specifically can be the dna fragmentation shown in the sequence 4 of sequence table.
The judgement criteria of the structural changes of application circular dichroism spectrometry detection DNA is following: if the signal about 256nm contains single stranded DNA in the reacted solution; If the signal of 265～275nm, contain the DNA of parallel G4 structure in the reacted solution; If the signal of 288～298nm, contain the DNA of antiparallel G4 structure in the reacted solution.
Compared with prior art, method provided by the invention has following advantage: can very effective identification telomeric dna fragment and c-kit gene promoter dna fragmentation; As long as compound is mixed with DNA in ratio provided by the invention, just can realize, and not need loaded down with trivial details aftertreatment, method is simple for this, and experiment condition is gentle.
Description of drawings
Fig. 1 is the circular dichroism spectrum of nitidine chloride and HT4 reaction among the embodiment 1.
Fig. 2 is the circular dichroism spectrum of nitidine chloride and H22 reaction among the embodiment 2.
Fig. 3 is the circular dichroism spectrum of nitidine chloride and H24A reaction among the embodiment 3.
Fig. 4 is the circular dichroism spectrum of nitidine chloride and C23 reaction among the embodiment 4.
Fig. 5 is the circular dichroism spectrum of sanguinarine(e) and HT4 reaction among the embodiment 5.
Fig. 6 is the circular dichroism spectrum of sanguinarine(e) and H22 reaction among the embodiment 6.
Fig. 7 is the circular dichroism spectrum of sanguinarine(e) and H24A reaction among the embodiment 7.
Fig. 8 is the circular dichroism spectrum of sanguinarine(e) and C23 reaction among the embodiment 8.
Fig. 9 is the circular dichroism spectrum of nitidine chloride and HT4 reaction among the embodiment 9.
Figure 10 is the circular dichroism spectrum of nitidine chloride and HT4 reaction among the embodiment 10.
Figure 11 is the circular dichroism spectrum of sanguinarine(e) and HT4 reaction among the embodiment 11.
Figure 12 is the circular dichroism spectrum of sanguinarine(e) and HT4 reaction among the embodiment 12.
Figure 13 is the circular dichroism spectrum of nitidine chloride and C23 reaction among the embodiment 13.
Figure 14 is the circular dichroism spectrum of nitidine chloride and C23 reaction among the embodiment 14.
Figure 15 is the circular dichroism spectrum of sanguinarine(e) and C23 reaction among the embodiment 15.
Figure 16 is the circular dichroism spectrum of sanguinarine(e) and C23 reaction among the embodiment 16.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment like no specified otherwise, is ordinary method.Used test materials among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains.
Used dna sequence dna among the embodiment (be synthetic and identify through order-checking) is as follows:
The telomeric dna fragment:
HT4:5 '-TTAGGGTTAGGGTTAGGGTTAGGG-3 ' (sequence 1 of sequence table).
H22:5 '-AGGGTTAGGGTTAGGGTTAGGG-3 ' (sequence 2 of sequence table).
H24A:5 '-TTGGGTTAGGGTTAGGGTTAGGGA-3 ' (sequence 3 of sequence table).
C-kit gene promoter dna fragmentation:
C23:5 '-AGGGAGGGCGCTGGGAGGAGGG-3 ' (sequence 4 of sequence table).
Nitidine chloride: Chengdu Cisco China ltd, purity is 98%.
Sanguinarine(e): Chengdu Cisco China ltd, purity is 98%.
All adopting concentration among the embodiment is the Tris buffered soln of 10mM.
Solution A: nitidine chloride is added in the Tris buffered soln (pH 7.4), and making its final concentration is 50.0 μ M.
Solution B: HT4 is added in the Tri s buffered soln (pH 7.4), and making its final concentration is 50.0 μ M.
Solution C: Tris buffered soln (pH 7.4).
First group of Ep pipe (three repetitions are set): in the Ep pipe, add 1920 μ l solution A, add 160 μ l solution B then, obtain mixed solution (nitidine chloride in the mixed solution and the molar concentration rate of HT4 are 12: 1); Using Tris buffered soln again is 4 μ M with the concentration that mixed solution is diluted to HT4; After shaking up, reacted 12 hours.
Second group of Ep pipe (three repetitions are set): in the Ep pipe, add 1920 μ l solution C, add 160 μ l solution B then, obtain mixed solution; Using Tris buffered soln again is 4 μ M with the concentration that mixed solution is diluted to HT4; After shaking up, reacted 12 hours.
Respectively two groups of Ep pipes are carried out the circular dichroism spectrum analysis, the result sees Fig. 1.Solution during second group of Ep manages has only single stranded DNA signal (256nm); Solution in first group of Ep pipe occurred parallel G4 structure signal (265～275nm) with the signal of antiparallel G4 structure (288～298nm), nitidine chloride also under the effect of HT4, produces opticity, occurs signal respectively at 374nm and 416nm place.The result shows: under the effect of nitidine chloride, HT4 changes mixing G4 structure into by single-stranded structure.
Solution A: nitidine chloride is added in the Tris buffered soln (pH 7.4), and making its final concentration is 50.0 μ M.
Solution B: H22 is added in the Tris buffered soln (pH 7.4), and making its final concentration is 50.0 μ M.
Solution C: Tris buffered soln (pH 7.4).
First group of Ep pipe (three repetitions are set): in the Ep pipe, add 1920 μ l solution A, add 160 μ l solution B then, obtain mixed solution (nitidine chloride in the mixed solution and the molar concentration rate of H22 are 12: 1); Using Tris buffered soln again is 4 μ M with the concentration that mixed solution is diluted to H22; After shaking up, reacted 12 hours.
Second group of Ep pipe (three repetitions are set): in the Ep pipe, add 1920 μ l solution C, add 160 μ l solution B then, obtain mixed solution; Using Tris buffered soln again is 4 μ M with the concentration that mixed solution is diluted to H22; After shaking up, reacted 12 hours.
Respectively two groups of Ep pipes are carried out the circular dichroism spectrum analysis, the result sees Fig. 2.Solution during second group of Ep manages has only single stranded DNA signal (256nm); Solution in first group of Ep pipe occurred parallel G4 structure signal (265～275nm) with the signal (288～298nm) of antiparallel G4 structure.The result shows: under the effect of nitidine chloride, H22 changes mixing G4 structure into by single-stranded structure.
Embodiment 3, application nitidine chloride identification H24A sequence
Solution A: nitidine chloride is added in the Tris buffered soln (pH 7.4), and making its final concentration is 50.0 μ M.
Solution B: H24A is added in the Tris buffered soln (pH 7.4), and making its final concentration is 50.0 μ M.
Solution C: Tris buffered soln (pH 7.4).
First group of Ep pipe (three repetitions are set): in the Ep pipe, add 1920 μ l solution A, add 160 μ l solution B then, obtain mixed solution (nitidine chloride in the mixed solution and the molar concentration rate of H24A are 12: 1); Using Tris buffered soln again is 4 μ M with the concentration that mixed solution is diluted to H24A; After shaking up, reacted 12 hours.
Second group of Ep pipe (three repetitions are set): in the Ep pipe, add 1920 μ l solution C, add 160 μ l solution B then, obtain mixed solution; Using Tris buffered soln again is 4 μ M with the concentration that mixed solution is diluted to H24A; After shaking up, reacted 12 hours.
Respectively two groups of Ep pipes are carried out the circular dichroism spectrum analysis, the result sees Fig. 3.Solution during second group of Ep manages has only single stranded DNA signal (256nm); Solution in first group of Ep pipe occurred parallel G4 structure signal (265～275nm) with the signal (288～298nm) of antiparallel G4 structure; Nitidine chloride is also under the effect of H24A simultaneously; Produce opticity, occur strong signal respectively at 372nm and 416nm place.The result shows: under the effect of nitidine chloride, H24A changes mixing G4 structure into by single-stranded structure.
Embodiment 4, application nitidine chloride identification C23 sequence
Solution A: nitidine chloride is added in the Tris buffered soln (pH 7.4), and making its final concentration is 50.0 μ M.
Solution B: C23 is added in the Tris buffered soln (pH 7.4), and making its final concentration is 50.0 μ M.
Solution C: Tris buffered soln (pH 7.4).
First group of Ep pipe (three repetitions are set): in the Ep pipe, add 1920 μ l solution A, add 160 μ l solution B then, obtain mixed solution (nitidine chloride in the mixed solution and the molar concentration rate of C23 are 12: 1); Using Tris buffered soln again is 4 μ M with the concentration that mixed solution is diluted to C23; After shaking up, reacted 12 hours.
Second group of Ep pipe (three repetitions are set): in the Ep pipe, add 1920 μ l solution C, add 160 μ l solution B then, obtain mixed solution; Using Tris buffered soln again is 4 μ M with the concentration that mixed solution is diluted to C23; After shaking up, reacted 12 hours.
Respectively two groups of Ep pipes are carried out the circular dichroism spectrum analysis, the result sees Fig. 4.Solution during second group of Ep manages has only single stranded DNA signal (256nm); The signal of parallel G4 structure has appearred in the solution in first group of Ep pipe, and (265～275nm), nitidine chloride also under the effect of C23, produces opticity simultaneously, occurs strong signal respectively at 334nm and 397nm place.The result shows: under the effect of nitidine chloride, C23 changes parallel G4 structure into by single-stranded structure.
Solution A: sanguinarine(e) is added in the Tris buffered soln (pH 7.4), and making its final concentration is 50.0 μ M.
Solution B: HT4 is added in the Tris buffered soln (pH 7.4), and making its final concentration is 50.0 μ M.
Solution C: Tris buffered soln (pH 7.4).
First group of Ep pipe (three repetitions are set): in the Ep pipe, add 1920 μ l solution A, add 160 μ l solution B then, obtain mixed solution (sanguinarine(e) in the mixed solution and the molar concentration rate of HT4 are 12: 1); Using Tris buffered soln again is 4 μ M with the concentration that mixed solution is diluted to HT4; After shaking up, reacted 12 hours.
Second group of Ep pipe (three repetitions are set): in the Ep pipe, add 1920 μ l solution C, add 160 μ l solution B then, obtain mixed solution; Using Tris buffered soln again is 4 μ M with the concentration that mixed solution is diluted to HT4; After shaking up, reacted 12 hours.
Respectively two groups of Ep pipes are carried out the circular dichroism spectrum analysis, the result sees Fig. 5.Solution during second group of Ep manages has only single stranded DNA signal (256nm); The signal (288～298nm) of antiparallel G4 structure has appearred in the solution during first group of Ep manages.The result shows: under the effect of sanguinarine(e), HT4 changes antiparallel G4 structure into by single-stranded structure.
Embodiment 6, application sanguinarine(e) identification H22 sequence
Solution A: sanguinarine(e) is added in the Tris buffered soln (pH 7.4), and making its final concentration is 50.0 μ M.
Solution B: H22 is added in the Tris buffered soln (pH 7.4), and making its final concentration is 50.0 μ M.
Solution C: Tris buffered soln (pH 7.4).
First group of Ep pipe (three repetitions are set): in the Ep pipe, add 1920 μ l solution A, add 160 μ l solution B then, obtain mixed solution (sanguinarine(e) in the mixed solution and the molar concentration rate of H22 are 12: 1); Using Tris buffered soln again is 4 μ M with the concentration that mixed solution is diluted to H22; After shaking up, reacted 12 hours.
Second group of Ep pipe (three repetitions are set): in the Ep pipe, add 1920 μ l solution C, add 160 μ l solution B then, obtain mixed solution; Using Tris buffered soln again is 4 μ M with the concentration that mixed solution is diluted to H22; After shaking up, reacted 12 hours.
Respectively two groups of Ep pipes are carried out the circular dichroism spectrum analysis, the result sees Fig. 6.Solution during second group of Ep manages has only single stranded DNA signal (256nm); The signal (288～298nm) of antiparallel G4 structure has appearred in the solution during first group of Ep manages.The result shows: under the effect of sanguinarine(e), H22 changes antiparallel G4 structure into by single-stranded structure.
Embodiment 7, application sanguinarine(e) identification H24A sequence
Solution A: sanguinarine(e) is added in the Tris buffered soln (pH 7.4), and making its final concentration is 50.0 μ M.
Solution B: H24A is added in the Tris buffered soln (pH 7.4), and making its final concentration is 50.0 μ M.
Solution C: Tris buffered soln (pH 7.4).
First group of Ep pipe (three repetitions are set): in the Ep pipe, add 1920 μ l solution A, add 160 μ l solution B then, obtain mixed solution (sanguinarine(e) in the mixed solution and the molar concentration rate of H24A are 12: 1); Using Tris buffered soln again is 4 μ M with the concentration that mixed solution is diluted to H24A; After shaking up, reacted 12 hours.
Second group of Ep pipe (three repetitions are set): in the Ep pipe, add 1920 μ l solution C, add 160 μ l solution B then, obtain mixed solution; Using Tris buffered soln again is 4 μ M with the concentration that mixed solution is diluted to H24A; After shaking up, reacted 12 hours.
Respectively two groups of Ep pipes are carried out the circular dichroism spectrum analysis, the result sees Fig. 7.Solution during second group of Ep manages has only single stranded DNA signal (256nm); The signal (288～298nm) of antiparallel G4 structure has appearred in the solution during first group of Ep manages.The result shows: under the effect of sanguinarine(e), H24A changes antiparallel G4 structure into by single-stranded structure.
Solution A: sanguinarine(e) is added in the Tris buffered soln (pH 7.4), and making its final concentration is 50.0 μ M.
Solution B: C23 is added in the Tris buffered soln (pH 7.4), and making its final concentration is 50.0 μ M.
Solution C: Tris buffered soln (pH 7.4).
First group of Ep pipe (three repetitions are set): in the Ep pipe, add 1920 μ l solution A, add 160 μ l solution B then, obtain mixed solution (sanguinarine(e) in the mixed solution and the molar concentration rate of C23 are 12: 1); Using Tris buffered soln again is 4 μ M with the concentration that mixed solution is diluted to C23; After shaking up, reacted 12 hours.
Second group of Ep pipe (three repetitions are set): in the Ep pipe, add 1920 μ l solution C, add 160 μ l solution B then, obtain mixed solution; Using Tris buffered soln again is 4 μ M with the concentration that mixed solution is diluted to C23; After shaking up, reacted 12 hours.
Respectively two groups of Ep pipes are carried out the circular dichroism spectrum analysis, the result sees Fig. 8.Solution during second group of Ep manages has only single stranded DNA signal (256nm); Solution during second group of Ep manages has only single stranded DNA signal (256nm), does not have other DNA signal to produce.The result shows: under the effect of sanguinarine(e), the C23 structure does not change.
Embodiment 9, application nitidine chloride identification HT4 sequence
Solution A: nitidine chloride is added in the Tris buffered soln (pH 8.0), and making its final concentration is 50.0 μ M.
Solution B: HT4 is added in the Tris buffered soln (pH 8.0), and making its final concentration is 50.0 μ M.
Solution C: Tris buffered soln (pH 8.0).
First group of Ep pipe (three repetitions are set): in the Ep pipe, add 1000 μ l solution A, add 20 μ l solution B then, obtain mixed solution (nitidine chloride in the mixed solution and the molar concentration rate of HT4 are 50: 1); Using Tris buffered soln again is 1 μ M with the concentration that mixed solution is diluted to HT4; After shaking up, reacted 18 hours.
Second group of Ep pipe (three repetitions are set): in the Ep pipe, add 1000 μ l solution C, add 20 μ l solution B then, obtain mixed solution; Using Tris buffered soln again is 1 μ M with the concentration that mixed solution is diluted to HT4; After shaking up, reacted 18 hours.
Respectively two groups of Ep pipes are carried out the circular dichroism spectrum analysis, the result sees Fig. 9.Solution during second group of Ep manages has only single stranded DNA signal (256nm); Solution in first group of Ep pipe occurred parallel G4 structure signal (265～275nm) with the signal (288～298nm) of antiparallel G4 structure.The result shows: under the effect of nitidine chloride, HT4 changes mixing G4 structure into by single-stranded structure.
Solution A: nitidine chloride is added in the Tris buffered soln (pH 6.0), and making its final concentration is 50.0 μ M.
Solution B: HT4 is added in the Tris buffered soln (pH 6.0), and making its final concentration is 50.0 μ M.
Solution C: Tris buffered soln (pH 6.0).
First group of Ep pipe (three repetitions are set): in the Ep pipe, add 800 μ l solution A, add 100 μ l solution B then, obtain mixed solution (nitidine chloride in the mixed solution and the molar concentration rate of HT4 are 8: 1); Using Tris buffered soln again is 5 μ M with the concentration that mixed solution is diluted to HT4; After shaking up, reacted 1 hour.
Second group of Ep pipe (three repetitions are set): in the Ep pipe, add 800 μ l solution C, add 100 μ l solution B then, obtain mixed solution; Using Tris buffered soln again is 5 μ M with the concentration that mixed solution is diluted to HT4; After shaking up, reacted 1 hour.
Respectively two groups of Ep pipes are carried out the circular dichroism spectrum analysis, the result sees Figure 10.Solution during second group of Ep manages has only single stranded DNA signal (256nm); Solution in first group of Ep pipe occurred parallel G4 structure signal (265～275nm) with the signal (288～298nm) of antiparallel G4 structure.The result shows: under the effect of nitidine chloride, HT4 changes mixing G4 structure into by single-stranded structure.
Embodiment 11, application sanguinarine(e) identification HT4 sequence
Solution A: sanguinarine(e) is added in the Tris buffered soln (pH 8.0), and making its final concentration is 50.0 μ M.
Solution B: HT4 is added in the Tris buffered soln (pH 8.0), and making its final concentration is 50.0 μ M.
Solution C: Tris buffered soln (pH 8.0).
First group of Ep pipe (three repetitions are set): in the Ep pipe, add 1000 μ l solution A, add 20 μ l solution B then, obtain mixed solution (sanguinarine(e) in the mixed solution and the molar concentration rate of HT4 are 50: 1); Using Tris buffered soln again is 1 μ M with the concentration that mixed solution is diluted to HT4; After shaking up, reacted 18 hours.
Second group of Ep pipe (three repetitions are set): in the Ep pipe, add 1000 μ l solution C, add 20 μ l solution B then, obtain mixed solution; Using Tris buffered soln again is 1 μ M with the concentration that mixed solution is diluted to HT4; After shaking up, reacted 18 hours.
Respectively two groups of Ep pipes are carried out the circular dichroism spectrum analysis, the result sees Figure 11.Solution during second group of Ep manages has only single stranded DNA signal (256nm); The signal (288～298nm) of antiparallel G4 structure has appearred in the solution during first group of Ep manages.The result shows: under the effect of sanguinarine(e), HT4 changes antiparallel G4 structure into by single-stranded structure.
Solution A: sanguinarine(e) is added in the Tris buffered soln (pH 6.0), and making its final concentration is 50.0 μ M.
Solution B: HT4 is added in the Tris buffered soln (pH 6.0), and making its final concentration is 50.0 μ M.
Solution C: Tris buffered soln (pH 6.0).
First group of Ep pipe (three repetitions are set): in the Ep pipe, add 1000 μ l solution A, add 1000 μ l solution B then, obtain mixed solution (sanguinarine(e) in the mixed solution and the molar concentration rate of HT4 are 1: 1); Using Tris buffered soln again is 5 μ M with the concentration that mixed solution is diluted to HT4; After shaking up, reacted 1 hour.
Second group of Ep pipe (three repetitions are set): in the Ep pipe, add 1000 μ l solution C, add 1000 μ l solution B then, obtain mixed solution; Using Tris buffered soln again is 5 μ M with the concentration that mixed solution is diluted to HT4; After shaking up, reacted 1 hour.
Respectively two groups of Ep pipes are carried out the circular dichroism spectrum analysis, the result sees Figure 12.Solution during second group of Ep manages has only single stranded DNA signal (256nm); The signal (288～298nm) of antiparallel G4 structure has appearred in the solution during first group of Ep manages.The result shows: under the effect of sanguinarine(e), HT4 changes antiparallel G4 structure into by single-stranded structure.
Embodiment 13, application nitidine chloride identification C23 sequence
Solution A: nitidine chloride is added in the Tris buffered soln (pH 8.0), and making its final concentration is 50.0 μ M.
Solution B: C23 is added in the Tris buffered soln (pH 8.0), and making its final concentration is 50.0 μ M.
Solution C: Tris buffered soln (pH 8.0).
First group of Ep pipe (three repetitions are set): in the Ep pipe, add 1000 μ l solution A, add 20 μ l solution B then, obtain mixed solution (nitidine chloride in the mixed solution and the molar concentration rate of C23 are 50: 1); Using Tris buffered soln again is 1 μ M with the concentration that mixed solution is diluted to C23; After shaking up, reacted 18 hours.
Second group of Ep pipe (three repetitions are set): in the Ep pipe, add 1000 μ l solution C, add 20 μ l solution B then, obtain mixed solution; Using Tris buffered soln again is 1 μ M with the concentration that mixed solution is diluted to C23; After shaking up, reacted 18 hours.
Respectively two groups of Ep pipes are carried out the circular dichroism spectrum analysis, the result sees Figure 13.Solution during second group of Ep manages has only single stranded DNA signal (256nm); The signal of parallel G4 structure has appearred in the solution in first group of Ep pipe, and (265～275nm), nitidine chloride also under the effect of C23, produces opticity simultaneously, occurs strong signal respectively at 334nm and 397nm place.The result shows: under the effect of nitidine chloride, C23 changes parallel G4 structure into by single-stranded structure.
Embodiment 14, application nitidine chloride identification C23 sequence
Solution A: nitidine chloride is added in the Tris buffered soln (pH 6.0), and making its final concentration is 50.0 μ M.
Solution B: C23 is added in the Tris buffered soln (pH 6.0), and making its final concentration is 50.0 μ M.
Solution C: Tris buffered soln (pH 6.0).
First group of Ep pipe (three repetitions are set): in the Ep pipe, add 800 μ l solution A, add 100 μ l solution B then, obtain mixed solution (nitidine chloride in the mixed solution and the molar concentration rate of C23 are 8: 1); Using Tris buffered soln again is 5 μ M with the concentration that mixed solution is diluted to C23; After shaking up, reacted 1 hour.
Second group of Ep pipe (three repetitions are set): in the Ep pipe, add 800 μ l solution C, add 100 μ l solution B then, obtain mixed solution; Using Tris buffered soln again is 5 μ M with the concentration that mixed solution is diluted to C23; After shaking up, reacted 1 hour.
Respectively two groups of Ep pipes are carried out the circular dichroism spectrum analysis, the result sees Figure 14.Solution during second group of Ep manages has only single stranded DNA signal (256nm); The signal of parallel G4 structure has appearred in the solution in first group of Ep pipe, and (265～275nm), nitidine chloride also under the effect of C23, produces opticity simultaneously, occurs strong signal respectively at 334nm and 397nm place.The result shows: under the effect of nitidine chloride, C23 changes parallel G4 structure into by single-stranded structure.
Embodiment 15, application sanguinarine(e) identification C23 sequence
Solution A: sanguinarine(e) is added in the Tris buffered soln (pH 8.0), and making its final concentration is 50.0 μ M.
Solution B: C23 is added in the Tris buffered soln (pH 8.0), and making its final concentration is 50.0 μ M.
Solution C: Tris buffered soln (pH 8.0).
First group of Ep pipe (three repetitions are set): in the Ep pipe, add 1000 μ l solution A, add 20 μ l solution B then, obtain mixed solution (sanguinarine(e) in the mixed solution and the molar concentration rate of C23 are 50: 1); Using Tris buffered soln again is 1 μ M with the concentration that mixed solution is diluted to C23; After shaking up, reacted 18 hours.
Second group of Ep pipe (three repetitions are set): in the Ep pipe, add 1000 μ l solution C, add 20 μ l solution B then, obtain mixed solution; Using Tris buffered soln again is 1 μ M with the concentration that mixed solution is diluted to C23; After shaking up, reacted 18 hours.
Respectively two groups of Ep pipes are carried out the circular dichroism spectrum analysis, the result sees Figure 15.Solution during second group of Ep manages has only single stranded DNA signal (256nm); Solution during second group of Ep manages has only single stranded DNA signal (256nm), does not have other DNA signal to produce.The result shows: under the effect of sanguinarine(e), the C23 structure does not change.
Embodiment 16, application sanguinarine(e) identification C23 sequence
Solution A: sanguinarine(e) is added in the Tris buffered soln (pH 6.0), and making its final concentration is 50.0 μ M.
Solution B: C23 is added in the Tris buffered soln (pH 6.0), and making its final concentration is 50.0 μ M.
Solution C: Tris buffered soln (pH 6.0).
First group of Ep pipe (three repetitions are set): in the Ep pipe, add 1000 μ l solution A, add 1000 μ l solution B then, obtain mixed solution (sanguinarine(e) in the mixed solution and the molar concentration rate of C23 are 1: 1); Using Tris buffered soln again is 5 μ M with the concentration that mixed solution is diluted to C23; After shaking up, reacted 1 hour.
Second group of Ep pipe (three repetitions are set): in the Ep pipe, add 1000 μ l solution C, add 1000 μ l solution B then, obtain mixed solution; Using Tris buffered soln again is 5 μ M with the concentration that mixed solution is diluted to C23; After shaking up, reacted 1 hour.
Respectively two groups of Ep pipes are carried out the circular dichroism spectrum analysis, the result sees Figure 16.Solution during second group of Ep manages has only single stranded DNA signal (256nm); Solution during second group of Ep manages has only single stranded DNA signal (256nm), does not have other DNA signal to produce.The result shows: under the effect of sanguinarine(e), the C23 structure does not change.
Sequence table
< 110>Institute of Chemistry, Academia Sinica
< 120>method of identification telomeric dna fragment and c-kit gene promoter dna fragmentation
<130>CGGNARY92369
<160>4
<210>1
<211>24
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>1
ttagggttag?ggttagggtt?aggg 24
<210>2
<211>22
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>2
agggttaggg?ttagggttag?gg 22
<210>3
<211>24
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>3
ttgggttagg?gttagggtta?ggga 24
<210>4
<211>22
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>4
agggagggcg?ctgggaggag?gg 22
Claims (10)
1. a method of distinguishing telomeric dna fragment and c-kit gene promoter dna fragmentation comprises the steps:
DNA to be measured is reacted with nitidine chloride or sanguinarine(e) respectively; Mix the G4 structure if DNA to be measured is changed into by single-stranded structure with nitidine chloride reaction back, change antiparallel G4 structure with sanguinarine(e) reaction back into by single-stranded structure, DNA then to be measured is the telomeric dna fragment; If DNA to be measured changes parallel G4 structure with nitidine chloride reaction back into by single-stranded structure, constant with sanguinarine(e) reaction back single-stranded structure, DNA then to be measured is a c-kit gene promoter dna fragmentation; Said mixing G4 structure is the mixed structure of parallel G4 structure and antiparallel G4 structure; Said dna fragmentation to be measured is that DNA is telomeric dna fragment or c-kit gene promoter dna fragmentation; Dna fragmentation shown in the sequence 3 of the sequence 1 that said telomeric dna fragment is sequence table or the sequence 2 of sequence table or sequence table; Dna fragmentation shown in the sequence 4 that said c-kit gene promoter dna fragmentation is sequence table.
2. the method for claim 1, it is characterized in that: the ratio of the amount of substance of said DNA to be measured and said nitidine chloride is 1: (8-50); The ratio of the amount of substance of said DNA to be measured and said sanguinarine(e) is 1: (1-50).
3. method as claimed in claim 2 is characterized in that: the ratio of the amount of substance of said DNA to be measured and said nitidine chloride is 1: 12; The ratio of the amount of substance of said DNA to be measured and said sanguinarine(e) is 1: 12.
4. like arbitrary described method in the claim 1 to 3, it is characterized in that: the reaction of said DNA to be measured and said nitidine chloride is in the pH value is 6.0～8.0 buffered soln, to carry out; The reaction of said DNA to be measured and said sanguinarine(e) is in the pH value is 6.0～8.0 buffered soln, to carry out.
5. method as claimed in claim 4 is characterized in that: in the said method, when reacting initial, the concentration of said DNA to be measured in said buffered soln is 1-5 μ M.
6. method as claimed in claim 4 is characterized in that: in the said method, when reacting initial, the concentration of said DNA to be measured in said buffered soln is 4 μ M.
7. method as claimed in claim 6 is characterized in that: the time of said reaction is 1-18 hour.
8. method as claimed in claim 7 is characterized in that: the time of said reaction is 12 hours.
9. method as claimed in claim 8 is characterized in that: in the said method, the method for structural changes that detects DNA to be measured is for being based analyzing method to measure opticity.
10. method as claimed in claim 9 is characterized in that: the method that detects the structural changes of said DNA to be measured is the circular dichroism spectrometry.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005049867A1 (en) * | 2003-11-17 | 2005-06-02 | Cylene Pharmaceuticals, Inc. | Methods for identifying modulators of quadruplex nucleic acids |
| US6979738B2 (en) * | 2003-10-22 | 2005-12-27 | Academia Sinica | Quadruplex stabilizer |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6979738B2 (en) * | 2003-10-22 | 2005-12-27 | Academia Sinica | Quadruplex stabilizer |
| WO2005049867A1 (en) * | 2003-11-17 | 2005-06-02 | Cylene Pharmaceuticals, Inc. | Methods for identifying modulators of quadruplex nucleic acids |
Non-Patent Citations (2)
| Title |
|---|
| Bai Liping.The Noncovalent Binding of Benzophenanthridine Alkaloids to Double-stranded, Bulged and G-quadruplex.《香港浸会大学学位论文》.2008,全文. * |
| BaiLiping.TheNoncovalentBindingofBenzophenanthridineAlkaloidstoDouble-stranded Bulged and G-quadruplex.《香港浸会大学学位论文》.2008 |
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