CN111979297A - Method for synthesizing oligonucleotide probe based on multiple PCR - Google Patents
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Abstract
The invention discloses a method for synthesizing an oligonucleotide probe based on multiplex PCR. Based on the cucumber genome sequence information, screening and designing an oligonucleotide library of a cucumber chromosome, and segmenting the oligonucleotide library by a double-joint connection mode. On the basis, the method for synthesizing the oligonucleotide probe based on the multiplex PCR is provided, the chromosome segment required by synthesis is automatically selected according to the experimental needs, and a large amount of oligonucleotide probes required by the experiment are quickly synthesized through the PCR. The method for synthesizing the oligonucleotide probe based on the multiplex PCR can synthesize a large amount of segmented oligonucleotide probes and probes of the whole chromosome rapidly, simply, conveniently and permanently, creates conditions for the popularization and the application of the oligonucleotide probes, and provides a flexible and powerful method for the research of plant chromosomes.
Description
One, the technical field
The invention discloses a method for synthesizing an oligonucleotide probe based on multiple PCR, which is beneficial to developing cytogenetic research of sequenced plant chromosomes and belongs to the technical field of plant biology.
Second, technical background
Fluorescence In Situ Hybridization (FISH) is used to enable a specific chromosome segment, chromosome arm or whole chromosome of a study object to emit Fluorescence of a certain color, so that the chromosome can be visualized under a Fluorescence microscope, namely chromosome painting, and the technology greatly deepens understanding of chromosome structure and change. For example, this technique is used to analyze and study recombination, aberration, and homologous genes of chromosomes (Ried et al, 1998); in addition, comparative studies of genome between closely related species, in which chromosome painting techniques have not been subjected to genome sequencing, provide a new method by which differences in chromosome structure between species can be visually demonstrated, so that we can analyze rearrangement relationships of chromosomes between closely related species (Jiang et al, 2006).
However, plant chromosome painting is difficult to develop over a long period of time due to the presence of a large number of repetitive sequences in the plant cell wall and plant chromosomes. Improvements such as large fragment cloning (BAC, YAC, Fosmid cloning, etc.) probes, single copy probes, etc. have been made by a number of researchers to make this technology applicable to plant chromosome studies (Fransz et al, 2000; Lysak et al, 2003, 2006; Mandakova et al, 2010; Lou et al, 2014). However, the probe preparation process is complicated and labor-intensive (Lysak et al, 2013), and the requirements on chromosome flaking technology are high, so that the application in chromosome research of plants is still limited.
In recent years, the development of oligonucleotide synthesis technology has opened a new way to solve the problem of plant chromosome research (Cuadrado et al, 1998, 2002). Han et al (2015) can conveniently complete the staining of specific chromosome segments or the full length of chromosomes of plants by screening oligonucleotide probes prepared from specific oligonucleotides in the genomes of sequenced plant species. Thus, this method has great potential for the study of sequenced plant chromosomes, and many successful studies have been carried out on some species such as strawberry, potato, tomato, eggplant (Qu et al, 2017; Braz et al, 2017).
However, since the application of oligonucleotide probes is still in the beginning, the conventional oligonucleotide probe synthesis methods are complicated, labor-intensive, and time-consuming, and the reagents required for synthesizing oligonucleotide probes are expensive and large in one-time investment, so that they are used in practical studies less. To this end we propose a technique for multiplex PCR based staining of segmented oligonucleotide probes. The technology screens and synthesizes an oligonucleotide library of cucumber chromosomes based on genome sequence information of cucumber, segments the chromosome library in a double-joint mode in the probe design process, simply, conveniently and quickly synthesizes a certain section, several sections or the whole chromosome oligonucleotide probe required by a large amount of experiments by a multiple PCR method, so that the oligonucleotide probe is more conveniently and flexibly utilized, and the signal intensity can be enhanced by synthesizing the double-stranded oligonucleotide probe. The technology is beneficial to the popularization and application of the oligonucleotide probe in the research of plant chromosomes.
Third, the invention
Technical problem
The invention utilizes cucumber genome information, screens proper oligonucleotides, performs double-joint segmentation design, selects corresponding primers with fluorescent group labels, synthesizes the screened oligonucleotide probes through multiple PCR, can be used for plant chromosome research after purification, and develops a method for flexibly utilizing the oligonucleotide probes only through PCR aiming at the problems that the oligonucleotide probe synthesis technology is complicated and the synthesis library can not be flexibly utilized.
Technical scheme
The invention relates to a method for synthesizing an oligonucleotide probe based on multiplex PCR, which comprises the following steps:
1) the desired oligonucleotide library was screened by the Chorus software for chromosome 4 in cucumber '9930' genome as a reference.
2) All repetitive sequences in the cucumber genome were removed using a RepeatMasker, then the genomic sequence was divided into 48nt oligonucleotides and homopolymers containing more than 6 nucleotides were discarded.
3) Each oligonucleotide was aligned to the reference genome using BLAT and oligonucleotides > 75% similar were selected.
4) Probes associated with a particular chromosome or genomic region are selected from a probe database constructed using Primer3 to retain oligonucleotides dTm > 10 ℃.
5) A total of 93396 oligonucleotide probes covering chromosome 4 (about 23.3Mb) of cucumber were selected, averaging about 3.8 probes per Kb.
6) The oligonucleotide library is segmented by means of ligation of a double linker.
7) After dissolving 300ng of the oligonucleotide library in 300. mu.L of DNA/RNA-free water to prepare a stock solution (-80 ℃ storage) of 1 ng/. mu.L, 2. mu.L of the stock solution was mixed with 26. mu.L of water to prepare a working solution of the oligonucleotide library, which was stored at-20 ℃.
8) Both the front and rear primers used in PCR were modified at the 5' end of the primer using FAM and TAMRA.
9) PCR was performed according to the system described in Table 1.
TABLE 1 PCR System
Oligonucleotide fragments were amplified using the following PCR procedure: 95 ℃ for 3 minutes, 98 ℃ for 20 seconds, 54 ℃ for 15 seconds, 72 ℃ for 30 seconds, 15 cycles starting from step 2, 98 ℃ for 20 seconds, 56 ℃ for 15 seconds, 72 ℃ for 30 seconds, 25 cycles starting from step 5, and finally the temperature is maintained at 4 ℃. The PCR reaction product can be used as a probe directly after purification.
Advantageous effects
Compared with the prior art, the invention has the following advantages and positive effects:
1) compared with the prior art, the method for synthesizing the oligonucleotide probe based on the multiplex PCR fully improves the efficiency of probe synthesis.
2) Oligonucleotide libraries designed by segments in a double-joint mode can be flexibly selected and used according to experimental needs in the synthesis process, and oligonucleotide probe primers covering a certain section, a certain sections or the whole chromosome can be flexibly selected and used, so that the flexibility of the probe in the utilization process is improved.
3) In the process of synthesizing the oligonucleotide probe, one strand of the double-stranded oligonucleotide probe can be provided with green fluorescence, the other strand of the double-stranded oligonucleotide probe can be provided with red fluorescence, the oligonucleotide probes with three colors can be used at one time in the experimental process, and the experimental efficiency is improved.
4) The oligonucleotide probe synthesized by the method can be used as a library to continue synthesizing the available oligonucleotide probe, so that the oligonucleotide probe can be permanently used once being synthesized, and the experiment cost is reduced.
Description of the drawings
FIG. 1: schematic diagram of oligonucleotide probe synthesis by multiplex PCR.
FIG. 2: cucumber chromosome 4 oligonucleotide probe Chr4-T (red) synthesized by PCR was stained on cucumber metaphase and pachytene chromosomes.
FIG. 3: the segmented oligonucleotide probes synthesized by PCR were stained on cucumber pachytene chromosomes. Wherein, the Chr4-1, -3, -5, -7 is a red signal, and the Chr4-2, -4, -6, -8 is a green signal.
FIG. 4: the PCR synthesis of double-stranded-red-green oligonucleotide probes gave rise to a clearly discernible yellow-like signal in FISH.
Fifth, detailed description of the invention
The method for synthesizing the oligonucleotide probe based on the multiplex PCR comprises the following implementation procedures:
1) design of oligonucleotide library: the desired oligonucleotide library was screened by the Chorus software for chromosome 4 in cucumber '9930' genome as a reference. All repetitive sequences in the cucumber genome were removed using a RepeatMasker, then the genomic sequence was divided into 48nt oligonucleotides and homopolymers containing more than 6 nucleotides were discarded. Each oligonucleotide was aligned to the reference genome using BLAT and oligonucleotides > 75% similar were selected. Probes associated with a particular chromosome or genomic region are selected from a probe database constructed using Primer3 to retain oligonucleotides dTm > 10 ℃. A total of 93396 oligonucleotide probes covering chromosome 4 (about 23.3Mb) of cucumber were selected, averaging about 3.8 probes per Kb. In addition, the oligonucleotide library is segmented by means of ligation of the double linker.
2) Synthesis of oligonucleotide probe: after dissolving 300ng of the oligonucleotide library in 300. mu.L of DNA/RNA-free water to prepare a stock solution (-80 ℃ storage) of 1 ng/. mu.L, 2. mu.L of the stock solution was mixed with 26. mu.L of water to prepare a working solution of the oligonucleotide library, which was stored at-20 ℃. PCR was performed according to the above system and procedure, and both the primers used in PCR were modified at the 5' end of the primer using FAM and TAMRA, and the PCR reaction product could be used as a probe directly after purification.
3) Fluorescence in situ hybridization and signal detection: taking 2 μ L of the obtained oligonucleotide probe, preparing probe hybridization solution (containing the oligonucleotide probe, 50% deionized formamide, 2 XSSC, 10% dextran sulfate) to denature in 90 ℃ metal bath for 6 minutes, carrying out ice bath for 10 minutes, carrying out denaturation on a glass slide containing good chromosomes in 70% deionized formamide at 80 ℃ for 90 seconds, dehydrating in 70%, 90% and 100% series of ethanol for 5 minutes respectively, airing, dripping the denatured probe hybridization solution on the denatured glass slide, sealing, and placing in an in-situ hybridization instrument for hybridization at 37 ℃ overnight. Washing, air drying at room temperature, adding DAPI containing anti-fluorescence quencher, sealing with cover glass, and observing hybridization signal with Olympus BX51 fluorescence microscope in dark condition.
Claims (3)
1. A method for synthesizing an oligonucleotide probe based on multiplex PCR, which is characterized by comprising the following steps:
the oligonucleotide probe library is screened according to cucumber genome sequence information, in the probe design process, the oligonucleotide library is segmented by a double-joint method, and a large number of oligonucleotide probes required by chromosome research can be simply, conveniently and rapidly obtained by PCR (polymerase chain reaction) by utilizing different primer combinations.
2. The multiplex PCR-based method for synthesizing oligonucleotide probes according to claim 1, which is carried out by:
1) The desired oligonucleotide library was screened by the Chorus software for chromosome 4 in cucumber '9930' genome as a reference.
2) All repetitive sequences in the cucumber genome were removed using a RepeatMasker, then the genomic sequence was divided into 48nt oligonucleotides and oligomers containing homopolymers of more than 6 nucleotides were discarded.
3) Each oligonucleotide was aligned to the reference genome using BLAT and oligonucleotides > 75% similar were selected.
4) Probes associated with a particular chromosome or genomic region are selected from a probe database constructed using Primer3 to retain oligonucleotides dTm > 10 ℃.
5) A total of 93396 oligonucleotide probes covering chromosome 4 (about 23.3Mb) of cucumber were selected, averaging about 3.8 probes per Kb.
6) The oligonucleotide library is segmented by means of ligation of a double linker.
7) After dissolving 300ng of the oligonucleotide library in 300. mu.L of DNA/RNA-free water to prepare a stock solution (-80 ℃ storage) of 1 ng/. mu.L, 2. mu.L of the stock solution was mixed with 26. mu.L of water to prepare a working solution of the oligonucleotide library, which was stored at-20 ℃.
8) Both the front and rear primers used in PCR were modified at the 5' end of the primer using FAM and TAMRA.
9) PCR was performed according to the system described in Table 1.
TABLE 1PCR System
Oligonucleotide fragments were amplified using the following PCR procedure: 95 ℃ for 3 minutes, 98 ℃ for 20 seconds, 54 ℃ for 15 seconds, 72 ℃ for 30 seconds, 15 cycles starting from step 2, 98 ℃ for 20 seconds, 56 ℃ for 15 seconds, 72 ℃ for 30 seconds, 25 cycles starting from step 5, and finally the temperature is maintained at 4 ℃. The PCR reaction product can be used as a probe directly after purification.
3. Use of the multiplex PCR based oligonucleotide probe synthesis method according to claim 1 for plant chromosome research.
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| CN102191311A (en) * | 2010-03-10 | 2011-09-21 | 常州楚天生物科技有限公司 | Construction and application of general oligonucleotide sequence database |
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