CN108441539B - Method for detecting end of forest chromosome by using oligonucleotide sequence - Google Patents
Method for detecting end of forest chromosome by using oligonucleotide sequence Download PDFInfo
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Abstract
The invention provides a method for detecting the end of a forest chromosome by using an oligonucleotide sequence. The method comprises (AGGGTTT)3Oligonucleotide sequence probe and kit comprising the probe, said (AGGGTTT)3The base sequence of the oligonucleotide probe is as follows: 5'-AGGGTTTAGGGTTTAGGGTTT-3', for identifying and marking the end of a forest dye; the kit comprises: a probe; enzymolysis liquid; 20 XSSC buffer; 2 XSSC buffer; 70% of FA solution; 50% DS solution; salmon sperm DNA; the method comprises obtaining a forest chromosome slide specimen; denaturation; fluorescence in situ hybridization; detecting a signal; and recovering the glass slide. The method can repeatedly use the slide, the probe preparation is easy and quick, the sensitivity is high, the probe only aims at the oligonucleotide sequence at the end part of the chromosome, the root tip slide is easy, the metaphase chromosome is conveniently obtained, the time of the whole experimental process is short, and the operation is simple and convenient.
Description
Technical Field
The invention belongs to the application field of fluorescent in situ hybridization probes, and particularly relates to a method for detecting the end part of a forest chromosome by using an oligonucleotide sequence.
Background
At present, the method for detecting the forest chromosome is mostly simple microscopic observation, photographs are carried out and analyzed, the forest chromosome is small compared with herbaceous plants, the number, the size and the form of the chromosome can not be clearly distinguished in many times, and huge errors of experiments are caused.
Fluorescence In Situ Hybridization (FISH) is a new in situ hybridization method formed by replacing isotope labeling with fluorescent labeling, a non-radioactive molecular cytogenetic technology developed on the basis of radioactive in situ hybridization technology. The FISH can observe the forest chromosome more intuitively, is efficient and quick, but becomes a difficult point in the forest chromosome when the chromosome is subjected to fluorescence labeling. Besides lacking a simple and rapid method for carrying out fluorescent labeling on chromosomes, the method also has the problems that the quantity of target DNA required in the labeling process is large, labeling errors can be caused, repeated labeling is caused, and the number of chromosomes is incomplete and unclear.
The invention establishes a stable and convenient fluorescence in situ hybridization reaction system by designing and synthesizing an oligonucleotide probe for detecting the end part of the forest chromosome, overcomes a plurality of problems of the existing forest chromosome detection, and provides a new method for detecting the forest plant chromosome.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for detecting the chromosome end of a forest tree by using an oligonucleotide sequence. The technical scheme of the invention is as follows:
a method for detecting the ends of the chromosomes of a forest tree by using an oligonucleotide sequence (AGGGTTT)3Oligonucleotide sequence probe and kit comprising the probe, said (AGGGTTT)3The base sequence of the oligonucleotide probe is as follows: 5'-AGGGTTTAGGGTTTAGGGTTT-3', for identifying and marking the end of a forest dye; the kit comprises: (1) the probe; (2) enzymolysis liquid; (3)20 XSSC buffer; (4)2 XSSC buffer; (5) 70% of FA solution; (6) 50% DS solution; (7) salmon sperm DNA; the method comprises the following steps:
1) obtaining a forest chromosome glass slide specimen: shearing a 1.0-1.5 cm newly-born root tip tissue of a forest seed, sequentially treating with an ice-water mixture for 18-24 h and glacial acetic acid for 5min, cleaning with double-distilled water, cutting 1-2 mm of the root tip meristem, and placing in an enzymolysis solution for enzymolysis at 37 ℃ for 45-60 min;
removing enzymolysis liquid, sequentially cleaning root tip meristem with double distilled water, 70%, 90% and 100% alcohol, adding glacial acetic acid to prepare root tip tissue suspension, dripping the suspension on a glass slide, drying in the air, performing microscopic examination, marking the glass slide with a chromosome clearly observed, and storing in a-20 ℃ storage box;
2) denaturation: a1.5 mL centrifuge tube ready for sterilization was added 2. mu.L of salmon sperm DNA, 0.5. mu.L of probe, followed by 10. mu.L of 100% FA, 2. mu.L of 20 XSSC, and 4. mu.L of 50% DS per tube. Mixing, centrifuging instantly, denaturing in boiling water bath for 10min, immediately placing on ice for 20min to prevent renaturation;
3) fluorescence in situ hybridization: treating the glass slide in the step 1) with 2 XSSC buffer solution twice, each time for 5min, then sequentially carrying out gradient treatment with 70% alcohol, 90% alcohol and 100% alcohol for 5min, drying at room temperature, then dripping 70% FA solution, covering with a cover glass, and treating at 80 ℃ for 2 min;
then, gradient treatment is carried out by using 70% alcohol, 90% alcohol and 100% alcohol in sequence, each time is 5min, 10 mu L of the denatured mixed liquid obtained in the step 2) is dripped after air drying, the glass slide is placed in a thermostat at 37 ℃ for 1.5-2 h under shading, and is washed by using 2 XSSC buffer solution for 3min and double distilled water for 3min in sequence, and finally, the glass slide is dried at normal temperature;
4) signal detection: adding DAPI into the dried glass slide, detecting by using a fluorescence microscope, and collecting a detection image;
5) and (3) recovering the glass slide: the glass slides are placed in 2 XSSC buffer solution, water bath is carried out at 60 ℃ for 10min, 70%, 90% and 100% alcohol are respectively used for 10min, then the glass slides are placed under a fluorescent lamp for 24h, and the glass slides are collected together and placed in a storage box at 20 ℃ below zero for the next use.
Further, the probe is labeled with FAM at the 5' end of its sequence.
Further, the preparation method of the enzymolysis liquid comprises the following steps: every 10mLddH2O, 2g cellulase and 1g pectinase were added.
Further, the preparation method of the 20 XSSC buffer solution comprises the following steps: ddH per 1000mL2O add 88.23g lemonSodium salt and 175.32g of sodium chloride.
Further, the preparation method of the 2 XSSC buffer solution comprises the following steps: 20 XSSC buffer was diluted 10 fold.
Further, the 70% FA solution was composed of deionized Formamide (FA) and 2 × SSC buffer at a volume ratio of 7: 3.
Further, the preparation method of the 50% DS solution comprises the following steps: ddH every 500. mu.L20, adding 0.5g of dextran sulfate, shaking overnight at 37 ℃, and instantly centrifuging and uniformly mixing after completely melting.
Further, the processing method of the salmon sperm DNA comprises the following steps: the salmon sperm DNA (10mg/mL) dissolved at room temperature is autoclaved for 10min, and the DNA is denatured by boiling water for 10min and stored at-20 ℃.
The invention has the beneficial effects that: the invention utilizes a probe for detecting the end part of the forest chromosome on the basis of the fluorescence in situ hybridization technology, and the probe mainly identifies the oligonucleotide sequence of the end part of the forest chromosome, thereby helping to establish a more stable and convenient chromosome fluorescence in situ hybridization reaction system. The method is applied to detecting the end part of the forest chromosome, and the reagent in the adopted kit has simpler composition, is convenient to prepare, and has the advantages of simple and convenient operation, easy understanding and good repeatability. The method can repeatedly use the slide, the probe preparation is easy and quick, the sensitivity is high, the probe only aims at the oligonucleotide sequence at the end part of the chromosome, the root tip slide is easy, the metaphase chromosome is conveniently obtained, the time of the whole experimental process is short, and the operation is simple and convenient.
Drawings
FIG. 1 shows the present invention (AGGGTTT)3Signal site distribution images of probe-labeled Croton tiglium chromosomes; (AGGGTTT)3The probe was labeled with FAM at the 5' end, and a hybridization signal was shown as a green signal on a scale of 3 μm.
FIG. 2 shows the present invention (AGGGTTT)3Signal site distribution image of probe-labeled Erythrina crista-galli chromosome; (AGGGTTT)3The probe was labeled with FAM at the 5' end, and a hybridization signal was shown as a green signal on a scale of 3 μm.
FIG. 3 shows the present invention (AGGGTTT)3A signal site distribution image of the probe-labeled litseaoviensis chromosome; (AGGGTTT)3The probe was labeled with FAM at the 5' end, and a hybridization signal was shown as a green signal on a scale of 3 μm.
FIG. 4 shows the present invention (AGGGTTT)3Signal site distribution image of probe-labeled litseeelongata chromosome; (AGGGTTT)3The probe was labeled with FAM at the 5' end, and a hybridization signal was shown as a green signal on a scale of 3 μm.
FIG. 5 shows the present invention (AGGGTTT)3Signal site distribution images of probe-labeled Podocarpusacryphyllus chromosomes; (AGGGTTT)3The probe was labeled with FAM at the 5' end, as shown by the green signal on a scale of 5 μm.
FIG. 6 shows the present invention (AGGGTTT)3Signal site distribution images of probe-labeled Quercusaquifolioides chromosomes; (AGGGTTT)3The probe was labeled with FAM at the 5' end, as shown by the green signal on a scale of 5 μm.
FIG. 7 shows the present invention (AGGGTTT)3Probe-labeled Signal site distribution image of Robiniapseudoacacia chromosome, (AGGGTTT)3The probe was labeled with FAM at the 5' end, as shown by the green signal on a 3 μm scale.
Detailed Description
The probe used in the practice of the present invention was synthesized by Beijing Biochemical company, and the synthesized probe was diluted to a concentration of 100M with 1 XTE and stored at-20 ℃ and the working concentration of the probe was further diluted to 20M and also stored at-20 ℃.
The wood materials used in the practice of the present invention are shown in table 1.
TABLE 1 Wood species materials used in the practice of the invention
The present invention will now be described in further detail with reference to the following figures and specific examples, which are intended to be illustrative, but not limiting, of the invention.
The embodiments of the present invention provide a method for detecting ends of forest chromosome by using oligo sequence (AGGGTTT)3Oligonucleotide sequence probe and kit comprising the probe, said (AGGGTTT)3The base sequence of the oligonucleotide probe is as follows: 5'-AGGGTTTAGGGTTTAGGGTTT-3', used for identifying and marking the ends of the forest chromosome, and marking the 5 ' end of the sequence by FAM; the kit comprises: (1) the probe; (2) enzymolysis liquid; the preparation method comprises the following steps: every 10mLddH2Adding 2g of cellulase and 1g of pectinase into the O; (3)20 XSSC buffer; the preparation method comprises the following steps: ddH per 1000mL2O88.23 g of sodium citrate and 175.32g of sodium chloride are added; (4)2 XSSC buffer; the preparation method comprises the following steps: diluting 20 XSSC buffer solution by 10 times; (5) 70% of FA solution; consists of deionized Formamide (FA) and 2 XSSC buffer solution according to the volume ratio of 7: 3; (6) 50% DS solution; the preparation method comprises the following steps: every 500. mu.LddH20, adding 0.5g of dextran sulfate, shaking overnight at 37 ℃, and instantly centrifuging and uniformly mixing after completely melting; (7) salmon sperm DNA; the method for treating the salmon sperm DNA comprises the following steps: the salmon sperm DNA (10mg/mL) dissolved at room temperature is autoclaved for 10min, and the DNA is denatured by boiling water for 10min and stored at-20 ℃. The method comprises the following steps:
1) forest chromosome slide specimen: shearing a 1.0-1.5 cm newly-born root tip tissue of a forest seed, sequentially treating with an ice-water mixture for 18-24 h and glacial acetic acid for 5min, cleaning with double-distilled water, cutting 1-2 mm of the root tip meristem, and placing in an enzymolysis solution for enzymolysis at 37 ℃ for 45-60 min;
removing enzymolysis liquid, sequentially cleaning root tip meristem with double distilled water, 70%, 90% and 100% alcohol, adding glacial acetic acid to prepare root tip tissue suspension, dripping the suspension on a glass slide, drying in the air, performing microscopic examination, marking the glass slide with a chromosome clearly observed, and storing in a-20 ℃ storage box;
2) denaturation: a1.5 mL centrifuge tube ready for sterilization was added 2. mu.L of salmon sperm DNA, 0.5. mu.L of probe, followed by 10. mu.L of 100% FA, 2. mu.L of 20 XSSC, and 4. mu.L of 50% DS. Mixing, centrifuging instantly, denaturing in boiling water bath for 10min, immediately placing on ice for 20min to prevent renaturation;
3) fluorescence in situ hybridization: treating the glass slide in the step 1) with 2 XSSC buffer solution twice, each time for 5min, then sequentially carrying out gradient treatment with 70% alcohol, 90% alcohol and 100% alcohol for 5min, drying at room temperature, then dripping 70% FA solution, covering with a cover glass, and treating at 80 ℃ for 2 min;
then, gradient treatment is carried out by using 70% alcohol, 90% alcohol and 100% alcohol in sequence, each time is 5min, 10 mu L of the denatured mixed liquid obtained in the step 2) is dripped after air drying, the glass slide is placed in a thermostat at 37 ℃ for 1.5-2 h under shading, and is washed by using 2 XSSC buffer solution for 3min and double distilled water for 2min in sequence, and finally, the glass slide is dried at normal temperature;
4) signal detection: adding DAPI into the dried glass slide, detecting by using a fluorescence microscope, and collecting a detection image;
5) and (3) recovering the glass slide: the glass slides are placed in 2 XSSC buffer solution, water bath is carried out at 60 ℃ for 10min, 70%, 90% and 100% alcohol are respectively used for 10min, then the glass slides are placed under a fluorescent lamp for 24h, and the glass slides are collected together and placed in a storage box at 20 ℃ below zero for the next use.
Example 1
This example uses (AGGGTTT)3The probe and the kit thereof mark the Croton tiglium chromosome by the method, and a green hybridization signal can be observed by a fluorescence microscope, wherein a scale in the figure is 3 mu m.
Example 2
This example uses (AGGGTTT)3The probe and the kit thereof mark the Erythrina crista-galli chromosome by the above method, and a green hybridization signal can be observed by a fluorescence microscope, and the scale in the figure is 3 μm.
Example 3
This example uses (AGGGTTT)3The probes and the kit thereof were labeled with the Litseabaviansis chromosome by the above method, and a green hybridization signal was observed by a fluorescence microscope, with a scale of 3 μm in the figure.
Example 4
This example uses (AGGGTTT)3The probes and the kit thereof mark the Litseaelangata chromosome by the method, and green can be observed by a fluorescence microscopeColor hybridization signal, scale in the figure is 3 μm.
Example 5
This example uses (AGGGTTT)3The probes and the kit thereof mark Podocarpusacryphyllus chromosomes by the method, and a green hybridization signal can be observed by a fluorescence microscope, wherein a scale in the figure is 3 mu m.
Example 6
This example uses (AGGGTTT)3The probes and the kit thereof mark the chromosomes of Quercusaquifolioides by the method, and a green hybridization signal can be observed by a fluorescence microscope, wherein a scale in the figure is 3 mu m.
Example 7
This example uses (AGGGTTT)3The probe and the kit thereof mark the Robiniapseudoacacia chromosome by the above method, and a green hybridization signal can be observed by a fluorescence microscope, and the scale in the figure is 3 μm.
The method of the specific embodiment of the invention can be used for repeatedly preparing the slide, the probe is easy and quick to prepare and high in sensitivity, the probe only aims at the oligonucleotide sequence at the end part of the forest chromosome, the slide of the root tip is easy, the metaphase chromosome is convenient to obtain, the time of the whole experimental process is short, and the operation is simple and convenient.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Sequence listing
<110> Sichuan university of agriculture
<120> a method for detecting ends of chromosomes of forest trees by using oligonucleotide sequences
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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agggtttagg gtttagggtt t 21
Claims (8)
1. A method for detecting the ends of the chromosomes of a forest tree by using an oligonucleotide sequence, which is characterized in that the method comprises (AGGGTTT)3Oligonucleotide sequence probe and kit comprising the probe, said (AGGGTTT)3The base sequence of the oligonucleotide probe is as follows: 5'-AGGGTTTAGGGTTTAGGGTTT-3', for identifying and marking the end of a forest dye; the kit comprises: (1) the probe; (2) enzymolysis liquid; (3)20 XSSC buffer; (4)2 XSSC buffer; (5) 70% of FA solution; (6) 50% DS solution; (7) salmon sperm DNA; the forest isCroton tiglium、Erythrina crista-galli、Litseabaviensis、Litsea elongata、Podocarpusmacrophyllus、Quercusaquifolioides、RobiniapseudoacaciaOne of (1); the method comprises the following steps:
1) obtaining a forest chromosome glass slide specimen: shearing a 1.0-1.5 cm newly-born root tip tissue of a forest seed, sequentially treating with an ice-water mixture for 18-24 h and glacial acetic acid for 5min, cleaning with double-distilled water, cutting 1-2 mm of the root tip meristem, and placing in an enzymolysis solution for enzymolysis at 37 ℃ for 45-60 min;
removing enzymolysis liquid, sequentially cleaning root tip meristem with double distilled water, 70%, 90% and 100% alcohol, adding glacial acetic acid to prepare root tip tissue suspension, dripping the suspension on a glass slide, drying in the air, performing microscopic examination, marking the glass slide with a chromosome clearly observed, and storing in a-20 ℃ storage box;
2) denaturation: preparing a sterilized 1.5mL centrifuge tube, adding 2 muL salmon sperm DNA and 0.5 muL probe into each tube, then adding 10 muL 100% FA, 2 muL 20 XSSC and 4 muL 50% DS, mixing uniformly, centrifuging instantly, denaturing in a boiling water bath for 10min, immediately placing on ice for 20min, and preventing the renaturation;
3) fluorescence in situ hybridization: treating the glass slide in the step 1) with 2 XSSC buffer solution twice, each time for 5min, then sequentially carrying out gradient treatment with 70% alcohol, 90% alcohol and 100% alcohol for 5min, drying at room temperature, then dripping 70% FA solution, covering with a cover glass, and treating at 80 ℃ for 2 min;
then, gradient treatment is carried out by using 70% alcohol, 90% alcohol and 100% alcohol in sequence, each time is 5min, 10 mu L of the denatured mixed liquid obtained in the step 2) is dripped after air drying, the glass slide is placed in a thermostat at 37 ℃ for 1.5-2 h under shading, and is washed by using 2 XSSC buffer solution for 3min and double distilled water for 3min in sequence, and finally, the glass slide is dried at normal temperature;
4) signal detection: adding DAPI into the dried glass slide, detecting by using a fluorescence microscope, and collecting a detection image;
5) and (3) recovering the glass slide: the glass slides are placed in 2 XSSC buffer solution, water bath is carried out at 60 ℃ for 10min, 70%, 90% and 100% alcohol are respectively used for 10min, then the glass slides are placed under a fluorescent lamp for 24h, and the glass slides are collected together and placed in a storage box at 20 ℃ below zero for the next use.
2. The method for detecting the ends of the chromosomes of the forest trees by using the oligonucleotide sequences as claimed in claim 1, wherein the probes are labeled with FAM at the 5' ends of the sequences.
3. The method for detecting the chromosome end of the forest trees by using the oligonucleotide sequence as claimed in claim 1, wherein the preparation method of the enzymatic hydrolysate is as follows: every 10mLddH2O, 2g cellulase and 1g pectinase were added.
4. The method for detecting the ends of the chromosomes of forest trees by using the oligonucleotide as claimed in claim 1, wherein the 20 XSSC buffer is prepared by the following steps: ddH per 1000mL2O88.23 g of sodium citrate and 175.32g of sodium chloride were added.
5. The method for detecting the ends of the chromosomes of forest trees by using the oligonucleotide as claimed in claim 4, wherein the 2 XSSC buffer is prepared by the following steps: 20 XSSC buffer was diluted 10 fold.
6. The method for detecting the chromosome end of forest trees by using the oligonucleotide sequence as claimed in claim 1 or 4, wherein the 70% FA solution is composed of deionized formamide and 2 XSSC buffer at a volume ratio of 7: 3.
7. The method for detecting the chromosome end of the forest trees by using the oligonucleotide sequence as claimed in claim 1, wherein the 50% DS solution is prepared by the following steps: ddH every 500. mu.L2And adding 0.5g of dextran sulfate into the O, shaking overnight at 37 ℃, and instantly centrifuging and uniformly mixing after completely melting.
8. The method for detecting the chromosome end of the forest trees by using the oligonucleotide as claimed in claim 1, wherein the salmon sperm DNA is processed by the following steps: autoclaving 10mg/mL salmon sperm DNA dissolved at room temperature for 10min, boiling in boiling water to denature the DNA for 10min, and storing at-20 deg.C.
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