CN107937485A - A kind of method detected and analyze DNA - Google Patents
A kind of method detected and analyze DNA Download PDFInfo
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- CN107937485A CN107937485A CN201711499798.9A CN201711499798A CN107937485A CN 107937485 A CN107937485 A CN 107937485A CN 201711499798 A CN201711499798 A CN 201711499798A CN 107937485 A CN107937485 A CN 107937485A
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Abstract
A kind of method of detection DNA, it includes the following steps：Obtain the unzipping time fluorescent brightness curve of control group DNA；Obtain the unzipping time fluorescent brightness curve of detection sample DNA；By the unzipping time fluorescent brightness curve of control group DNA compared with detecting the unzipping time fluorescent brightness curve of sample DNA, testing result is obtained.The control group is blank control group or is standard DNA or is the DNA of known mutations type.The present invention detects and analyzes DNA by obtaining the unzipping time fluorescent brightness curve of DNA, compared to the temperature fluorescent brightness curve currently used for detecting and analyzing DNA, heating-up temperature need not be carried out in real time accurately monitoring and calibrating, accelerate its analytic process so that DNA is detected and analytic process becomes easy and quick.
Description
Technical field
The present invention relates to biological technical field, and in particular to a kind of method detected and analyze DNA.
Background technology
In face of the communicable disease of fast propagation, the life and death for concerning patient is accurately diagnosed rapidly, also with patient at one's side
Relatives, friend have greatest relation.Sample is transported into specialized laboratories to find pathogen or potential prominent in order to reduce
Become DNA the time spent in and fund cost, researcher be directed to find more rapidly accurate instrument with meet these diagnosis need
Ask.Molecular diagnosis method based on polymerase chain reaction (PCR) is to receive one of most commonly used diagnostic method, this method
Accurate, comprehensive diagnostic message can be obtained with relatively cheap price.
In test, can be by probe, DNA sequencing, Capillary Electrophoresis or DNA curve analysis (MCA) come into one
The product of step detection PCR, then further excavates more hereditary information.Wherein, the simplicity of MCA and specificity become
Epigenetic difference is quickly detected to single reaction pipe, point mutation and single nucleotide polymorphism (SNP) context of detection most have prospect
Analysis mode.To melt DNA, MCA usually requires temperature to rise to 90 DEG C from 30 DEG C.Dodge team, which analyzes to come from, to be divided
The fusion process of the immobilized oligonucleotide (34-bp) of sub- beacon probe, finds to be required for 5 second time under each temperature gradient
To make temperature stabilization.With regard to same problem, Piunno team also to the oligonucleotides duplex (20-bp) of fixed fluorescent marker into
Liquation is gone；In experimentation, testing crew is while continuously measurement fluorescence also to it with the speed of about 0.005 DEG C/s
Heated.For differentiating sensitive DNA, researcher is necessary necessary by being inserted into heat scan distributed process
Temperature stabilization step keeps the slow rate of heat addition to ensure that sample temperature stablizes the homogeneous reaction with DNA probe.Although sample
The product rate of heat addition may be up to 175 DEG C/s, but these steps can still slow down whole fusion process.From the aforegoing it can be seen that use at present
It is heating-up temperature and the curve of DNA fluorescent brightness in the mode of expression DNA solubility curves, needs observing using this method
Heating-up temperature is monitored and calibrated in real time in journey, DNA dehybridization procedures can be slowed down, analytic process is also comparatively laborious.
The content of the invention
It is an object of the invention to provide a kind of method for the detection DNA that need not observe heating-up temperature.
The purpose of the present invention is what is be achieved through the following technical solutions：
A kind of method of detection DNA, it includes the following steps：
Obtain unzipping time-fluorescent brightness curve of control group DNA；
Obtain unzipping time-fluorescent brightness curve of detection sample DNA；
The unzipping time of control group DNA-fluorescent brightness curve and unzipping time-fluorescent brightness of detection sample DNA is bent
Line is compared, and obtains testing result.
Especially, the control group is blank control group.
Especially, the control group is standard DNA.
Especially, the control group is the DNA of known mutations type.
Especially, molecular beacon probe and DNA target are put into reaction solution, reaction solution is heated, meanwhile, it is right
Its fluorescence signal is recorded, and obtains unzipping time-fluorescent brightness curve of DNA.
Especially, in obtaining control group DNA and obtaining unzipping time-fluorescent brightness curve procedures of detection sample DNA,
Both are consistent the heating temperature curve of heating process.
Especially, its fluorescence signal is recorded in real time.
Especially, this method is used for DNA mutation detection or DNA homology detection or target dna whether there is detection.
A kind of method of detection DNA, it is characterised in that by the unzipping time-fluorescent brightness for obtaining detection sample DNA
Curve obtains the testing result with or without target dna.
It is another object of the present invention to provide a kind of method for the analysis DNA that need not observe heating-up temperature.
Another object of the present invention is achieved by the following technical solution：
A kind of method of unzipping time-fluorescent brightness tracing analysis DNA by obtaining DNA.
Especially, molecular beacon probe and DNA target are put into reaction solution, reaction solution is heated, meanwhile, it is right
Its fluorescence signal is recorded, and obtains unzipping time-fluorescent brightness curve of DNA.
The present invention by obtaining unzipping time-fluorescent brightness curve of DNA detects and analyzes DNA, compared to currently used for
Detection and temperature-fluorescent brightness curve of analysis DNA, it is not necessary to carry out accurately monitoring and calibrating to heating-up temperature in real time, accelerate
Its analytic process so that DNA is detected and analytic process becomes easy and quick.
Brief description of the drawings
Fig. 1 is the principle flow chart for implementing DNA detection and analysis according to the present invention；
Fig. 2 is the flow chart that embodiment one implements DNA detections and analysis.
Fig. 3 is the when m- fluorescent brightness curve of the blank sample and detection sample corresponding to embodiment one；
Fig. 4 is the flow chart that embodiment two implements DNA detections and analysis.
Fig. 5 is the when m- fluorescent brightness curve of the detection sample corresponding to embodiment two；
Fig. 6 is the flow chart that embodiment three implements DNA detections and analysis.
Fig. 7 is the when m- fluorescent brightness curve of the standard DNA and detection sample corresponding to embodiment three；
Fig. 8 is the flow chart that example IV implements DNA detections and analysis.
Fig. 9 is the when m- fluorescent brightness curve of the detection sample (Different Variation bit number of points) corresponding to example IV；
Figure 10 is the flow chart that embodiment five implements DNA detections and analysis.
Figure 11 is the when m- fluorescent brightness curve of the detection sample (variant sites different type) corresponding to embodiment five；
Figure 12 is the heating temperature curve of the heating process corresponding to embodiment one, two, three, four, five.
Embodiment
Describe the specific implementation principle of the present invention in detail next, with reference to attached drawing.
As shown in Figure 1, it is examinations of the present invention and the principle of analysis DNA methods.Left frame 110 shown in Fig. 1, is first
To sample to be tested 111 (sample of the molecular beacon probe of corresponding pairing) according to setting temperature warming curve 112 (can be as
Heating curves shown in Figure 12) heated.During heating, the fluorescent brightness of sample is recorded in real time, to obtain
When m- fluorescent brightness curve 113.
Observation obtain when m- fluorescent brightness curve 113 in fluorescent brightness change with time degree, if change unobvious
Then represent, it is very few without target gene sequence, or target gene sequence quantity in sample to be tested, or target gene sequence genetic mutation
Site excessive 115, represents to contain target gene sequence 116 in test sample if change is obvious.
Right frame 120 as shown in Figure 1 is on the basis of DNA detections, is continued to there is the to be measured of target gene sequence 116
Sample is analyzed.
Standard sample【0】121 be the sample of the molecular beacon probe containing the target dna for whetheing there is any variation and corresponding pairing
Product.To standard sample【0】The heating curves 122 that 121 heating use is bent with being heated to temperature during the heating of detected sample
Line 112 is consistent.During heating, the fluorescent brightness of sample is recorded in real time, m- fluorescent brightness curve during obtaining
123.Standard of comparison【0】The 121 when m- fluorescent brightness curve 123 and sample to be tested containing target gene sequence 116
111 when m- fluorescent brightness curve 113.If two suite line overlaps, then it represents that contained gene order in sample to be tested 111
With standard sample【0】The gene order contained in 121 is consistent, again shows that in gene order contained in sample to be tested 111
There is no genetic mutation, be Wild Type 124.If two suite lines are not overlapping, then it represents that the gene sequence contained in sample to be tested 111
Genetic mutation is shown, is Mutant Type 125.
In the process, if by standard sample【0】121 change blank control sample into, it is possible to for determining to treat test sample
It is very few without target gene sequence, or target gene sequence quantity in product, or target gene sequence genetic mutation site is excessive, if becoming
Change in obvious then expression test sample and contain target gene sequence.
Standard sample【N】126 be the sample of the target dna containing specific variation and the molecular beacon probe of corresponding pairing
Group.Respectively to standard sample【N】Sample heating in 126, heats the heating curves 127 used and the heating to detected sample
During temperature warming curve 112 it is consistent.During heating, the fluorescent brightness of sample is recorded in real time, during obtaining
M- fluorescent brightness curve group 128.Acquisition when m- fluorescent brightness curve group 128 in extract compare contain genetic mutation 125
The m- fluorescent brightness curve 113 when corresponding of sample to be tested 111.Comparison result matching can confirm the target in sample to be tested 111
The variant sites quantity of gene, or the base Class1 28 of variant sites.
Embodiment one
A kind of to detect and analyse whether the method there are target dna, experimental method principle as shown in Fig. 2, treat respectively
Sample, blank sample are heated.
Preparatory work of experiment：
1st, take sample to be tested 210, and only add in sample to be tested the molecular beacon probe 5 ' of sufficient amount of design-
CY3-TCTACGCCACCAGCTCA-BHQ2-3’。
2nd, take blank sample 200, and add in blank sample the molecular beacon probe 5 ' of sufficient amount of design-
CY3-TCTACGCCACCAGCTCA-BHQ2-3’。
3rd, the warming-up device that can be controlled to heating temperature profile is prepared.
4th, fluorescent brightness detection device is prepared.
Test and record：
Above-mentioned sample 210,200 is heated respectively, heating mode using described in Figure 12 heating temperature curve 1200 (when
Other heating temperature curves can be so used, as long as sample to be tested and blank sample keep that heating temperature curve is consistent to be in contrast test
Can), and sample 210,200 corresponding fluorescent brightness are recorded in real time respectively using fluorescent brightness detection device.Obtain such as Fig. 3
Shown when m- fluorescent brightness curve, wherein, the corresponding unzipping time of sample to be tested 210-fluorescent brightness curve is 310, blank
The corresponding unzipping time of sample 200-fluorescent brightness curve is 300.
Conclusion
The unzipping time of blank sample-fluorescent brightness curve 300 and unzipping time-fluorescence of detected sample DNA is bright
Line 310 of writing music is compared.It can obtain unzipping time-correspondence detection sample 210 of fluorescent brightness curve 310 and not contain target
DNA or contained target dnas quantity is very little or contained target dna variant sites are excessive.
Embodiment two
A kind of to detect and analyse whether the method there are target dna, experimental method principle is as shown in figure 4, treat test sample
Product are heated.
Preparatory work of experiment：
1st, take sample to be tested 400, and only add in sample to be tested the molecular beacon probe 5 ' of sufficient amount of design-
CY3-TCTACGCCACCAGCTCA-BHQ2-3’。
2nd, the warming-up device that can be controlled to heating temperature profile is prepared.
3rd, fluorescent brightness detection device is prepared.
Test and record：
Above-mentioned sample to be tested 400 is heated respectively, heating mode using described in Figure 12 heating temperature curve 1200 (when
Other heating temperature curves can so be used), and 400 corresponding fluorescent brightness of sample to be tested is recorded in real time using fluorescence microscope.
When m- fluorescent brightness curve as shown in Figure 5 is obtained, the corresponding unzipping time of sample to be tested 400-fluorescent brightness curve is 500.
Conclusion
Observation unzipping time-fluorescent brightness curve 500 can obtain, and unzipping time-fluorescent brightness curve 500 corresponds to inspection
Sample 400 contains target dna.
Embodiment three
A kind of method detected and whether analysis DNA is homologous, experimental method principle as shown in fig. 6, treat test sample respectively
Product, standard DNA (any mutation does not occur) sample are heated.
Preparatory work of experiment：
1st, take standard DNA sample 600, and add in standard DNA sample mankind's KRAS genetic fragments single stranded DNA 5 '-
The 5 '-CY3- of molecular beacon probe of GTAGTTGGAGCTGGTGGCGTAGGCAAGAGT-3 ' and sufficient amount of design
TCTACGCCACCAGCTCA-BHQ2-3’；
2nd, take sample to be tested 610, and add in sample to be tested mankind's KRAS genetic fragments single stranded DNA 5 '-
The 5 '-CY3- of molecular beacon probe of GTAGTTGGAGCTGGTGGCGTAGGCAAGAGT-3 ' and sufficient amount of design
TCTACGCCACCAGCTCA-BHQ2-3’；
3rd, take sample to be tested 620, and only add in sample to be tested the molecular beacon probe 5 ' of sufficient amount of design-
CY3-TCTACGCCACCAGCTCA-BHQ2-3’。
4th, the warming-up device that can be controlled to heating temperature profile is prepared.
5th, fluorescent brightness detection device is prepared.
Test and record：
Above-mentioned sample 610,620,600 is heated respectively, heating mode is using the heating temperature curve described in Figure 12
1200 (it is of course possible to use other heating temperature curves, as long as sample to be tested keeps heating bent with standard DNA sample in contrast test
Line is consistent), and sample 610,620,600 corresponding fluorescent brightness are remembered in real time respectively using fluorescent brightness detection device
Record.When m- fluorescent brightness curve as shown in Figure 7 is obtained, wherein, the corresponding unzipping time of sample 610-fluorescent brightness curve
For 710, the corresponding unzipping time of sample 620-fluorescent brightness curve is 720, the corresponding unzipping time-fluorescent brightness of sample 600
Curve is 700.
Conclusion
By the unzipping time of standard DNA sample-fluorescent brightness curve 700 and unzipping time-fluorescence of detected sample DNA
Brightness curve 710,720 is compared.It can obtain, unzipping time-fluorescent brightness curve 710 corresponds to detection sample 610 and contains
Target dna, unzipping time-fluorescent brightness curve 720 correspond to detection sample 620 and do not contain target dna or contained target dna quantity
Very little or contained target dna variant sites are excessive.
Example IV
A kind of method for the bit number of points for detecting and analyzing DNA gene mutations, experimental method principle are divided as shown in Fig. 8
It is other that sample to be tested (determining that producer is mutated) is heated.
Preparatory work of experiment：
1st, the DNA sample 810A of known mutation type is taken, and in the DNA sample 810A of known mutation type
Add the single stranded DNA 5 '-ATCGATTAGGGTGTCCAGCGCGAGCGGTGGGCTAGCTCAT-3 ' and sufficient amount of design
Design molecular beacon probe 5 '-CY5-TAGCCCTCCGCTCCCGCAGGCCACGCTA-BHQ2-3 '；The list of the design
The 5 '-CY5- of molecular beacon probe of chain DNA 5 '-ATCGATTAGGGTGTCCAGCGCGAGCGGTGGGCTAGCTCAT-3 ' and meter
There are 5 gene mutation sites between TAGCCCTCCGCTCCCGCAGGCCACGCTA-BHQ2-3 '.
2nd, take sample to be tested 810B, and add in sample to be tested 810B the single stranded DNA 5 ' of design-
ATCGATTAGGGTGTCCAGCGCGAGCGGTGGGCTAGCTCAT-3 ' and the molecular beacon probe 5 ' of sufficient amount of design-
CY5-TAGCCCTCCGCTCCCGCAGGCCACGCTA-BHQ2-3′；
3rd, the DNA sample 820A of known mutation type is taken, and in the DNA sample 820A of known mutation type
Add the single stranded DNA 5 '-ATCGATTAGGGTGTCCAGCGCGCGCGGTGGGCTAGCTCAT-3 ' and sufficient amount of design
Design molecular beacon probe 5 '-CY5-TAGCCCTCCGCTCCCGCAGGCCACGCTA-BHQ2-3 '；The list of the design
The 5 '-CY5- of molecular beacon probe of chain DNA 5 '-ATCGATTAGGGTGTCCAGCGCGCGCGGTGGGCTAGCTCAT-3 ' and meter
There are 6 gene mutation sites between TAGCCCTCCGCTCCCGCAGGCCACGCTA-BHQ2-3 '.
4th, take sample to be tested 820B, and add in sample to be tested 820B the single stranded DNA 5 ' of design-
ATCGATTAGGGTGTCCAGCGCGCGCGGTGGGCTAGCTCAT-3 ' and the molecular beacon probe 5 ' of sufficient amount of design-
CY5-TAGCCCTCCGCTCCCGCAGGCCACGCTA-BHQ2-3′；
5th, the DNA sample 830A of known mutation type is taken, and in the DNA samples 830A of known mutation type
In add design single stranded DNA 5 '-ATCGATTAGGGTGTCCAGCGCGCGTGGTGGGCTAGCTCAT-3 ' and enough number
The molecular beacon probe 5 '-CY5-TAGCCCTCCGCTCCCGCAGGCCACGCTA-BHQ2-3 ' of the design of amount；The design
The molecular beacon probe 5 ' of single stranded DNA 5 '-ATCGATTAGGGTGTCCAGCGCGCGTGGTGGGCTAGCTCAT-3 ' and meter-
There are 7 gene mutation sites between CY5-TAGCCCTCCGCTCCCGCAGGCCACGCTA-BHQ2-3 '.
6th, take sample to be tested 830B, and add in sample to be tested 830B the single stranded DNA 5 ' of design-
ATCGATTAGGGTGTCCAGCGCGCGCGGTGGGCTAGCTCAT-3 ' and the molecular beacon probe 5 ' of sufficient amount of design-
CY5-TAGCCCTCCGCTCCCGCAGGCCACGCTA-BHQ2-3′。
7th, the warming-up device that can be controlled to heating temperature profile is prepared.
8th, fluorescent brightness detection device is prepared.
Test and record：
Above-mentioned sample 810A, 810B, 820A, 820B, 830A, 830B, 840 are heated respectively, heating mode is adopted
With described in Figure 12 heating temperature curve 1200 (it is of course possible to use other heating temperature curves, as long as in contrast test sample to be tested with
The DNA sample holding heating temperature curve of gene mutation type is consistent), and using fluorescent brightness detection device respectively to sample
810A, 810B, 820A, 820B, 830A, 830B, 840 corresponding fluorescent brightness record in real time.It is m- when obtaining as shown in Figure 9
Fluorescent brightness curve, wherein, the corresponding unzipping times of sample 810A-fluorescent brightness curve is 910A, the corresponding solutions of sample 810B
M- fluorescent brightness curve is 910B during chain, and the corresponding unzipping times of sample 820A-fluorescent brightness curve is 920A, sample 820B
Corresponding unzipping time-fluorescent brightness curve is 920B, and the corresponding unzipping times of sample 830A-fluorescent brightness curve is 930A,
The corresponding unzipping times of sample 830B-fluorescent brightness curve is 930B.
Conclusion
By the unzipping time of the DNA sample of known mutation type-fluorescent brightness curve 910A, 920A, 920A with treating
Unzipping time-fluorescent brightness curve 910B, 920B, 930B of detection sample DNA is compared, and homologous thread highly overlaps, together
When by the numerical value of homologous thread be put into Pearson correlation coefficient formula can obtain homologous thread correlation be 0.999.It can obtain：
The target dna that unzipping time-fluorescent brightness curve 910B is corresponded in detection sample (numbering 810B) has 5 genes
Mutational site；
The target dna that unzipping time-fluorescent brightness curve 920B is corresponded in detection sample (numbering 820B) has 6 genes
Mutational site；
The target dna that unzipping time-fluorescent brightness curve 930B is corresponded in detection sample (numbering 830B) has 7 genes
Mutational site.
Embodiment five
A kind of method of the bit number of points for detecting and analyzing DNA gene mutations and the base type of variation base position, it is real
Test that Method And Principle is as shown in Figure 10, i.e., respectively to sample to be tested (determining that producer is mutated), known mutation type
DNA sample is heated.
Preparatory work of experiment：
1st, the DNA sample 1010A of known mutation type is taken, and in the DNA samples of known mutation type
The single stranded DNA 5 '-GTAGTTGGAGCTGATGGCGTAGGCAAGAGT-3 ' of design and sufficient amount of is added in 1010A)
The molecular beacon probe 5 '-CY3-TCTACGCCACCAGCTCA-BHQ2-3 ' of design；The single stranded DNA 5 ' of the design-
GTAGTTGGAGCTG5 '-the CY3- of molecular beacon probe of TGGCGTAGGCAAGAGT-3 ' and meter
There is 1 gene mutation site between TCTACGCCACCAGCTCA-BHQ2-3 ', become isobase and A (quilts in sequence are changed into from original G
Outlined with square).
2nd, take sample to be tested 1010B, and add in sample to be tested 1010B the single stranded DNA 5 ' of design-
5 '-the CY3- of molecular beacon probe of GTAGTTGGAGCTGATGGCGTAGGCAAGAGT-3 ' and sufficient amount of design
TCTACGCCACCAGCTCA-BHQ2-3′；
3rd, the DNA sample 1020A of known mutation type is taken, and in the DNA samples of known mutation type
The single stranded DNA 5 '-GTAGTTGGAGCTGTTGGCGTAGGCAAGAGT-3 ' of design is added in 1020A and sufficient amount of is set
The molecular beacon probe 5 '-CY3-TCTACGCCACCAGCTCA-BHQ2-3 ' of meter；The single stranded DNA 5 ' of the design-
GTAGTTGGAGCTG5 '-the CY3- of molecular beacon probe of TGGCGTAGGCAAGAGT-3 ' and meter
There is 1 gene mutation site between TCTACGCCACCAGCTCA-BHQ2-3 ', become isobase and T (quilts in sequence are changed into from original G
Outlined with square).
4th, take sample to be tested 1020B, and add in sample to be tested 1020B the single stranded DNA 5 ' of design-
5 '-the CY3- of molecular beacon probe of GTAGTTGGAGCTGTTGGCGTAGGCAAGAGT-3 ' and sufficient amount of design
TCTACGCCACCAGCTCA-BHQ2-3′；
5th, the DNA sample 1030A of known mutation type is taken, and in the DNA samples of known mutation type
The single stranded DNA 5 '-GTAGTTGGAGCTGCTGGCGTAGGCAAGAGT-3 ' of design is added in 1030A and sufficient amount of is set
The molecular beacon probe 5 '-CY3-TCTACGCCACCAGCTCA-BHQ2-3 ' of meter；The single stranded DNA 5 ' of the design-
GTAGTTGGAGCTG5 '-the CY3- of molecular beacon probe of TGGCGTAGGCAAGAGT-3 ' and meter
There is 1 gene mutation site between TCTACGCCACCAGCTCA-BHQ2-3 ', become isobase and C (quilts in sequence are changed into from original G
Outlined with square).
6th, take sample to be tested 1030B, and add in sample to be tested 1030B the single stranded DNA 5 ' of design-
5 '-the CY3- of molecular beacon probe of GTAGTTGGAGCTGCTGGCGTAGGCAAGAGT-3 ' and sufficient amount of design
TCTACGCCACCAGCTCA-BHQ2-3′。
7th, the warming-up device that can be controlled to heating temperature profile is prepared.
8th, fluorescent brightness detection device is prepared.
Test and record：
Above-mentioned sample 1010A, 1010B, 1020A, 1020B, 1030A, 1030B are heated respectively, heating mode is equal
Using the heating temperature curve 1200 described in Figure 12 (it is of course possible to use other heating temperature curves, as long as the sample to be tested in contrast test
It is consistent with the DNA sample holding heating temperature curve of gene mutation type), and using fluorescent brightness detection device respectively to sample
The corresponding fluorescent brightness of 1010A, 1010B, 1020A, 1020B, 1030A, 1030B records in real time.When obtaining as shown in figure 11
M- fluorescent brightness curve, wherein, the corresponding unzipping times of sample 1010A-fluorescent brightness curve is 1110A, 1010B pairs of sample
The unzipping time answered-fluorescent brightness curve is 1110B, and the corresponding unzipping times of sample 1020A-fluorescent brightness curve is 1120A,
The corresponding unzipping times of sample 1020B-fluorescent brightness curve is 1120B, the corresponding unzipping time-fluorescent brightness of sample 1030A
Curve is 1130A, and the corresponding unzipping times of sample 1030B-fluorescent brightness curve is 1130B, and sample 1040 is corresponding when unwinding
M- fluorescent brightness curve is 1140.
Conclusion
By the unzipping time of the DNA sample of known mutation type-fluorescent brightness curve 1110A, 1120A, 1120A
Compared with unzipping time-fluorescent brightness curve 1110B, 1120B, 1130B of detected sample DNA, homologous thread height
Overlap, at the same by the numerical value of homologous thread be put into Pearson correlation coefficient formula can obtain homologous thread correlation be 0.999.Can
To obtain：
The target dna that unzipping time-fluorescent brightness curve 1110B is corresponded in detection sample 1010B has 1 gene mutation position
Point, and become isobase and A is changed into from original G；
The target dna that unzipping time-fluorescent brightness curve 1120B is corresponded in detection sample 1020B has 1 gene mutation
Site, and become isobase and T is changed into from original G；
The target dna that unzipping time-fluorescent brightness curve 1130B is corresponded in detection sample 1030B has 1 gene mutation
Site, and become isobase and C is changed into from original G.
From the above it can be seen that this method can be used for detecting DNA mutation (mutation) and its quantity, the alkali for the base position that makes a variation
Base type or single nucleotide polymorphism genotyping (SNP, single nucleotide polymorphism
genotyping)。
The above is only the preferred embodiment of the present invention, and protection scope of the present invention is not limited merely to above-mentioned implementation
Example, all technical solutions belonged under thinking of the present invention belong to protection scope of the present invention.It should be pointed out that for the art
Those of ordinary skill for, some improvements and modifications without departing from the principles of the present invention, such as change molecular beacon visit
Pin for other can be identified for that with the specific material of marker DNA, by standard DNA, the DNA sample of known mutation type and
Sample to be tested is detected comparison analysis etc., these improvements and modifications also should be regarded as protection scope of the present invention.
Claims (12)
- A kind of 1. method of detection DNA, it is characterised in that it includes the following steps：Obtain unzipping time-fluorescent brightness curve of control group DNA；Obtain unzipping time-fluorescent brightness curve of detection sample DNA；By the unzipping time of control group DNA-fluorescent brightness curve with detection sample DNA unzipping time-fluorescent brightness curve into Row compares, and obtains testing result.
- 2. the method for a kind of detection DNA according to claim 1, it is characterised in that the control group is blank control group.
- 3. the method for a kind of detection DNA according to claim 1, it is characterised in that the control group is standard DNA.
- 4. the method for a kind of detection DNA according to claim 1, it is characterised in that the control group is known mutations class The DNA of type.
- 5. according to the method for detection DNA any one of in claim 1-4 a kind of, it is characterised in that in reaction solution In be put into molecular beacon probe and DNA target, reaction solution is heated, meanwhile, its fluorescence signal is recorded, obtain The unzipping time of DNA-fluorescent brightness curve.
- 6. the method for a kind of detection DNA according to claim 5, it is characterised in that examined obtaining control group DNA and obtaining In the unzipping time of sample DNA-fluorescent brightness curve procedures, the heating temperature curve that both heat process is consistent.
- 7. the method for a kind of detection DNA according to claim 5, it is characterised in that remembered in real time to its fluorescence signal Record.
- A kind of 8. method of detection DNA according to claim 1, it is characterised in that this method be used for DNA mutation detect or DNA homology detects or target dna whether there is detection.
- A kind of 9. method of detection DNA, it is characterised in that by the unzipping time-fluorescent brightness curve for obtaining detection sample DNA Obtain the testing result with or without target dna.
- A kind of 10. method of unzipping time-fluorescent brightness tracing analysis DNA by obtaining DNA.
- 11. the method for a kind of analysis DNA according to claim 10, it is characterised in that molecule is put into reaction solution Beacon probe and DNA target, heat reaction solution, meanwhile, its fluorescence signal is recorded, obtains when unwinding of DNA M- fluorescent brightness curve.
- 12. the method for a kind of analysis DNA according to claim 10, it is characterised in that this method is analyzed for DNA mutation Or DNA homology analysis or target dna whether there is analysis.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711499798.9A CN107937485A (en) | 2017-12-29 | 2017-12-29 | A kind of method detected and analyze DNA |
| PCT/CN2018/088762 WO2019128081A1 (en) | 2017-12-29 | 2018-05-28 | Method for detecting and analyzing dna |
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| WO2019128081A1 (en) * | 2017-12-29 | 2019-07-04 | 澳门大学 | Method for detecting and analyzing dna |
| CN110585754A (en) * | 2019-09-03 | 2019-12-20 | 珠海市迪奇孚瑞生物科技有限公司 | Reagent mixture for drying, preparation of reagent mixture and drying method |
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| WO2019128081A1 (en) * | 2017-12-29 | 2019-07-04 | 澳门大学 | Method for detecting and analyzing dna |
| CN110585754A (en) * | 2019-09-03 | 2019-12-20 | 珠海市迪奇孚瑞生物科技有限公司 | Reagent mixture for drying, preparation of reagent mixture and drying method |
| CN110585754B (en) * | 2019-09-03 | 2020-12-08 | 珠海市迪奇孚瑞生物科技有限公司 | Reagent mixture for drying, preparation of reagent mixture and drying method |
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