CN102191325B - Detection method of vegetal single copy genes - Google Patents
Detection method of vegetal single copy genes Download PDFInfo
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- CN102191325B CN102191325B CN 201110094969 CN201110094969A CN102191325B CN 102191325 B CN102191325 B CN 102191325B CN 201110094969 CN201110094969 CN 201110094969 CN 201110094969 A CN201110094969 A CN 201110094969A CN 102191325 B CN102191325 B CN 102191325B
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Abstract
The invention relates to a detection method of vegetal single copy genes based on fluorescence in situ hybridization of quantum dots. By preparing quantum dot coupled DNA (deoxyribonucleic acid) as a probe, according to the traditional fluorescence in situ hybridization method, the hybridization detection of vegetal single copy genes is carried out on interphase nucleuses, chromosomes, DNA fibers or the like. In the method provided by the invention, a direct marking method is adopted, thus an antibody detection process is omitted; and because the quantum dots have high fluorescence intensity and can resist photo-bleaching, the sensitivity and resolution of hybridization are improved, and the vegetal single copy genes can be quickly and effectively located. The method provided by the invention can be widely used for drawing physical maps and analyzing the composition, structure, rearrangement, evolutionary relationship and the like of genomes.
Description
Technical field
The present invention relates to a kind of detection method of vegetal single copy genes, belong to the molecular cytogenetics field.
Technical background
Fluorescence in situ hybridization technique (Fluorescence in situ hybridization, FISH) cytogenetics and genomics research have been widely used in, can be at special dna sequence dnas in location such as interphasic nucleus, karyomit(e) or dna fibers, the composition of analyzing gene group, structure, rearrangement and evolutionary relationship, especially along with the arrival in gene order-checking epoch, take the drafting of fluorescence in situ hybridization as the physical map on basis, will in gene order-checking and map based cloning research, play important replenishing and booster action.In genome, except comprising a large amount of tumor-necrosis factor glycoproteinss, also have some single-copy sequences.In interphasic nucleus, karyomit(e) or dna fiber, detect accurately and locate single-copy sequence and depend on the specificity of hybridization and the sensitivity of detection.Yet, compare with human genome FISH technology, in Plant Genome Research, the sensitivity of FISH technology and resolving power have been subject to certain restriction.At first, because the existence of plant cell wall, cell walls and cytoplasmic fragment have reduced the chance that probe is combined with target fragment, have increased background, have caused lower signal to noise ratio.Secondly, the Metaphase Chromosome height of plant is concentrated, has also reduced the chance that probe is combined with target fragment, has also reduced the resolving power of hybridization simultaneously.
Quantum dot (quantum dots, QDs) has another name called semiconductor nanocrystal, is a kind of novel fluorescent labeling reagent, is widely used in life science.Compare with traditional organic fluorescent dye, quantum dot has many advantages: (1) excitation wavelength wide ranges and the wavelength of transmitted light narrow range is overlapping little; (2) fluorescence intensity of quantum dot is high, and anti-light floats, and is highly sensitive; (3) can obtain the quantum dot of different colours by size and the composition that changes quantum dot, be applicable to multi-color marking; (4) can carry out coupling, good stability, good biocompatibility with many biomacromolecules.By the retrieval prior art, at present quantum dot is applied in the fluorescence in situ hybridization, mainly contain two kinds of methods: a kind of method is to use biotin labeled probe to hybridize, quantum dot with the streptavidin mark detects (Nucleic Acids Res. 2004 again, 32, e28), the quantum point grain diameter of this method mark is too large, because the existence of plant cell wall, the karyomit(e) height is concentrated, and quantum dot enters relatively difficulty of nucleus, can not be applied to (J Nanobiotechnology in the plant fluorescence in situ hybridization, 2006,4,5); Another kind method is to use quantum dot-labeled oligonucleotide, Chinese patent application numbers 201010503053.7 disclosed " based on the detection method of the spongioblast endosymbiotic bacterium of quantum dot fluorescence in situ hybridization ", the altogether detection of bacterium in having realized in this way.But the dna fragmentation of this method coupling shorter (1-100bp) can only carry out the detection of tumor-necrosis factor glycoproteins, can not carry out the detection of single copy.According to the literature, polyacrylic quantum dot (the Octylamine-modified polyacrylic acid coated CdSe/ZnS quantum dots that octylame is modified, OPA-coated CdSe/ZnS QDs), monodispersity is good, particle diameter is less, stability and optical property good (Bioconjug Chem, 2007,18,323).Therefore, if the long-chain dna molecular of the quantum point coupling of this modification can be applied in the fluorescence in situ hybridization system of plant, sensitivity and the resolving power of hybridization will be improved greatly, and this method is taked the method for direct mark, save the step of antibody test, can realize the rapid detection of single copy gene.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of detection method of vegetal single copy genes, the simple, convenient easy row of present method.
The detection method of vegetal single copy genes provided by the invention, for:
Interphasic nucleus, Metaphase Chromosome or the chromatin fiber of preparation plant; The DNA of preparation quantum point coupling is as probe; According to traditional fluorescence in-situ hybridization method, carry out the hybridization of vegetal single copy genes, then detect; The preparation of described probe specifically comprises the steps:
According to the target gene that will detect, design and synthesize a pair of Oligonucleolide primers, and 5 ' end of one of them primer is connected 5 '-NH
2-(CH
2)
6-TTTTTT, and by amino (NH
2) and quantum dot on carboxyl (COOH) carry out chemical reaction, be coupled to the quantum dot surface;
With coupling Oligonucleolide primers and the another one primer of quantum dot, and the dna profiling that contains the purpose fragment adds in the system of polymerase chain reaction together, obtains the long-chain dna molecular of quantum point coupling by the polymerase chain reaction;
By the product that agarose gel electrophoresis separation of polymeric polymerase chain reaction obtains, obtain the single long-chain dna molecular of single quantum point coupling, with it as probe.
Preferred scheme is:
Quantum dot is polyacrylic quantum dot (the Octylamine-modified polyacrylic acid coated CdSe/ZnS quantum dots that water miscible octylame is modified, OPA-coated CdSe/ZnS QDs), monodispersity is good, particle diameter less (1-20 nm).
Be coupled at the long 200-600bp of DNA on quantum dot surface, because the probe of fluorescence in situ hybridization is better at the 200-600bp crossbreeding effect.
The sex change liquid of fluorescence in situ hybridization comprises 70%(v/v) deionized formamide and 2 * citrate buffer solution (salt-sodium citrate, SSC).
The hybridization solution of fluorescence in situ hybridization is 50%(v/v) deionized formamide (Formamide Deionized, FAD), the single long-chain dna probe of 2 * SSC, 1 mg/ml salmon sperm dna and the single quantum point coupling of 1-2 μ g/m l, do not comprise T 500.
The present invention compared with prior art has following beneficial effect:
The present invention adopts the method for direct mark, saved the process of antibody test, and owing to high, the anti-light of quantum dot fluorescence intensity floats, sensitivity and the resolving power of hybridization have been improved, can fast, effectively carry out detection, the location of vegetal single copy genes, can be widely used in the drafting of physical map, the composition of analyzing gene group, structure, rearrangement and evolutionary relationship etc.
Description of drawings
Fig. 1 for the fragment of the corn alkanoic dehydrogenase gene that uses quantum point coupling as probe, at corn prometaphase chromosomes location corn list copy alkanoic dehydrogenase gene.
Fig. 2 for the fragment of the corn alkanoic dehydrogenase gene that uses quantum point coupling as probe, at corn Metaphase Chromosome location corn list copy alkanoic dehydrogenase gene.
Fig. 3 for the fragment of the corn alkanoic dehydrogenase gene that uses quantum point coupling as probe, location corn list copy alkanoic dehydrogenase gene in the corn interphasic nucleus.
Fig. 4 proves that for using quantum dot as probe quantum dot does not have adsorptivity to maize chromosome, proves the specificity of hybridization.
Fig. 5 proves that for using quantum dot as probe quantum dot does not have adsorptivity to the corn interphasic nucleus, proves the specificity of hybridization.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be appreciated that these embodiment only are used for the present invention being described and being not used in restriction the scope of protection of present invention.Picture among the embodiment is observed by fluorescent microscope (Olympus BX-60), and charge coupled device (charge coupled device, CCD) and Metamorph software carry out image capture and obtains.Scale among the figure (Bar)=10 μ m.
Embodiment 1:
1) interphasic nucleus of preparation corn, Metaphase Chromosome are on slide glass;
2) the polyacrylic quantum dot (Octylamine-modified polyacrylic acid coated CdSe/ZnS quantum dots, OPA-coated CdSe/ZnS QDs) of synthetic octylame modification;
3) according to corn alkanoic dehydrogenase gene, design and synthesize a pair of Oligonucleolide primers, and 5 ' end of one of them primer is connected 5 '-NH
2-(CH
2)
6-TTTTTT, and by amino (NH
2) and quantum dot on carboxyl (COOH) carry out chemical reaction, be coupled to the quantum dot surface;
4) with coupling Oligonucleolide primers and the another one primer of quantum dot, and corn gene group DNA adds in the system of polymerase chain reaction together, obtains the dna molecular of the long 480bp of quantum point coupling by the polymerase chain reaction;
5) product that obtains by agarose gel electrophoresis separation of polymeric polymerase chain reaction obtains the dna molecular of the single long 480bp of single quantum point coupling, with it as probe, and simultaneously with quantum dot as negative control;
6) the sex change liquid of preparation fluorescence in situ hybridization comprises 70%(v/v) FAD, 2 * SSC, slide glass is put in the sex change liquid sex change 2.5min in 70 ℃, then be put in ethanol (v/v ,-20 ℃) each 5min that dewaters of 70%, 95%, 100%, dry;
7) preparation fluorescence in situ hybridization liquid comprises 50%(v/v) FAD, 2 * SSC, the dna probe of the single long 480bp of 1 mg/ml salmon sperm dna and the single quantum point coupling of 1-2 μ g/ml is put in sex change 5min in 75 ℃, places 5min on ice;
8) hybridization solution is dripped on slide glass, covered is hatched 16-24h for 37 ℃;
9) get rid of cover glass, slide glass is placed among 2 * SSC wash-out 10 min under the room temperature, then 37 ℃ of lower wash-out 10 min among 2 * SSC, wash-out 5 min under the room temperature among 2 * SSC, last 1 * phosphoric acid buffer (phosphate buffered solution, PBS) wash-out 5 min under the room temperature in, in the air slide is dried, add 4', 6-diamidino-2-phenylindone (4', 6-diamidino-2-phenylindole, DAPI) the microscopy observation.For using quantum dot as probe, prove that quantum dot does not have adsorptivity to maize chromosome and interphasic nucleus such as Fig. 4, Fig. 5, prove the specificity of hybridization.Fig. 1, Fig. 2, Fig. 3 for the fragment of the corn alkanoic dehydrogenase gene that uses quantum point coupling as probe, at corn prometaphase, Metaphase Chromosome and interphasic nucleus location corn list copy alkanoic dehydrogenase gene.
Embodiment 2:
1) chromatin fiber of preparation corn is on slide glass;
2) the polyacrylic quantum dot (Octylamine-modified polyacrylic acid coated CdSe/ZnS quantum dots, OPA-coated CdSe/ZnS QDs) of synthetic octylame modification;
3) according to somatic embryos of corn generation associated class receptor protein kinase 2 genes, design and synthesize a pair of Oligonucleolide primers, and 5 ' end of one of them primer is connected 5 '-NH
2-(CH
2)
6-TTTTTT, and by amino (NH
2) and quantum dot on carboxyl (COOH) carry out chemical reaction, be coupled to the quantum dot surface;
4) with coupling Oligonucleolide primers and the another one primer of quantum dot, and corn gene group DNA adds in the system of polymerase chain reaction together, obtains the dna molecular of the long 520bp of quantum point coupling by the polymerase chain reaction;
5) product that obtains by agarose gel electrophoresis separation of polymeric polymerase chain reaction obtains the dna molecular of the single long 520bp of single quantum point coupling, with it as probe, and simultaneously with quantum dot as negative control;
6) the sex change liquid of preparation fluorescence in situ hybridization comprises 70%(v/v) FAD, 2 * SSC, slide glass is put in the sex change liquid sex change 2.5min in 70 ℃, then be put in ethanol (v/v ,-20 ℃) each 5min that dewaters of 70%, 95%, 100%, dry;
7) preparation fluorescence in situ hybridization liquid comprises 50%(v/v) FAD, 2 * SSC, the dna probe of the single long 520bp of 1 mg/ml salmon sperm dna and the single quantum point coupling of 1-2 μ g/ml is put in sex change 5min in 75 ℃, places 5min on ice;
8) hybridization solution is dripped on slide glass, covered is hatched 16-24h for 37 ℃;
9) get rid of cover glass, slide glass is placed among 2 * SSC wash-out 10 min, then 37 ℃ of lower wash-out 10 min among 2 * SSC under the room temperature, wash-out 5 min under the room temperature among 2 * SSC, wash-out 5 min under the room temperature in the air dry slide among last 1 * PBS, add the DAPI microscopy and observe.
Embodiment 3:
1) Metaphase Chromosome of preparation parsley is on slide glass;
2) the polyacrylic quantum dot (Octylamine-modified polyacrylic acid coated CdSe/ZnS quantum dots, OPA-coated CdSe/ZnS QDs) of synthetic octylame modification;
3) according to the chalcone synthase genes of parsley, design and synthesize a pair of Oligonucleolide primers, and 5 ' end of one of them primer is connected 5 '-NH
2-(CH
2)
6-TTTTTT, and by amino (NH
2) and quantum dot on carboxyl (COOH) carry out chemical reaction, be coupled to the quantum dot surface;
4) with coupling Oligonucleolide primers and the another one primer of quantum dot, and the parsley genomic dna adds in the system of polymerase chain reaction together, obtains the dna molecular of the long 490bp of quantum point coupling by the polymerase chain reaction;
5) product that obtains by agarose gel electrophoresis separation of polymeric polymerase chain reaction obtains the dna molecular of the single long 490bp of single quantum point coupling, with it as probe, and simultaneously with quantum dot as negative control;
6) the sex change liquid of preparation fluorescence in situ hybridization comprises 70%(v/v) FAD, 2 * SSC, slide glass is put in the sex change liquid sex change 2.5min in 70 ℃, then be put in ethanol (v/v ,-20 ℃) each 5min that dewaters of 70%, 95%, 100%, dry;
7) preparation fluorescence in situ hybridization liquid comprises 50%(v/v) FAD, 2 * SSC, the dna probe of the single long 490bp of 1 mg/ml salmon sperm dna and the single quantum point coupling of 1-2 μ g/ml is put in sex change 5min in 75 ℃, places 5min on ice;
8) hybridization solution is dripped on slide glass, covered is hatched 16-24h for 37 ℃;
9) get rid of cover glass, slide glass is placed among 2 * SSC wash-out 10 min, then 37 ℃ of lower wash-out 10 min among 2 * SSC under the room temperature, wash-out 5 min under the room temperature among 2 * SSC, wash-out 5 min under the room temperature in the air dry slide among last 1 * PBS, add the DAPI microscopy and observe.
Claims (2)
1. the detection method of a vegetal single copy genes is characterized in that: interphasic nucleus, Metaphase Chromosome or the chromatin fiber of preparation plant; The DNA of preparation quantum point coupling is as probe; According to traditional fluorescence in-situ hybridization method, carry out the hybridization of vegetal single copy genes, then detect; The preparation of described probe specifically comprises the steps:
A, according to the target gene that will detect, design and synthesize a pair of Oligonucleolide primers, 5 ' end of one of them primer is connected 5 '-NH
2-(CH
2)
6-TTTTTT, and carry out chemical reaction by the carboxyl on amino and the quantum dot, being coupled to the quantum dot surface, described quantum dot is the polyacrylic quantum dot of water miscible octylame modification;
B, with coupling Oligonucleolide primers and the another one primer of quantum dot, and the dna profiling that contains the purpose fragment adds in the system of polymerase chain reaction together, obtains the long-chain dna molecular of quantum point coupling by the polymerase chain reaction;
C, by the product that agarose gel electrophoresis separation of polymeric polymerase chain reaction obtains, obtain the single long-chain dna molecular of single quantum point coupling, with it as probe.
2. detection method according to claim 1 is characterized in that: the long 200-600bp of DNA that is coupled at the quantum dot surface.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1850988A (en) * | 2006-02-28 | 2006-10-25 | 武汉大学 | Fluorescent quantum dot marking DNA bioprobe, and its preparing method |
| CN101603085A (en) * | 2009-07-07 | 2009-12-16 | 天津工业大学 | A kind of magnetic DNA fluorescent probe and preparation method thereof |
| CN101880733A (en) * | 2010-08-05 | 2010-11-10 | 中国检验检疫科学研究院 | Avian influenza virus typing detection method based on flow microsphere technique |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1850988A (en) * | 2006-02-28 | 2006-10-25 | 武汉大学 | Fluorescent quantum dot marking DNA bioprobe, and its preparing method |
| CN101603085A (en) * | 2009-07-07 | 2009-12-16 | 天津工业大学 | A kind of magnetic DNA fluorescent probe and preparation method thereof |
| CN101880733A (en) * | 2010-08-05 | 2010-11-10 | 中国检验检疫科学研究院 | Avian influenza virus typing detection method based on flow microsphere technique |
Non-Patent Citations (2)
| Title |
|---|
| Quantum dots in biology and medicine;Robert E.Bailey,et al;《Physica E:low-dimensional systems and nanostructures》;20041231;第25卷(第1期);1-12 * |
| Robert E.Bailey,et al.Quantum dots in biology and medicine.《Physica E:low-dimensional systems and nanostructures》.2004,第25卷(第1期),1-12. |
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