CN111979297B

CN111979297B - Method for synthesizing oligonucleotide probe based on multiplex PCR

Info

Publication number: CN111979297B
Application number: CN201910432072.6A
Authority: CN
Inventors: 娄群峰; 毕云飞; 赵勤政
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2019-05-22
Filing date: 2019-05-22
Publication date: 2023-10-27
Anticipated expiration: 2039-05-22
Also published as: CN111979297A

Abstract

The invention discloses a method for synthesizing an oligonucleotide probe based on multiplex PCR. Screening an oligonucleotide library for designing cucumber chromosomes based on the genomic sequence information of the cucumbers, and segmenting the oligonucleotide library by a double-joint connection mode. On the basis, a synthesis method of oligonucleotide probes based on multiplex PCR is provided, chromosome segments required by synthesis are selected independently according to experimental requirements, and a large number of oligonucleotide probes required by the experiment are rapidly synthesized through PCR. The method for synthesizing the oligonucleotide probe based on the multiplex PCR can synthesize a large amount of segmented oligonucleotide probes and whole chromosome probes rapidly, simply, conveniently and permanently, creates conditions for popularization and application of the oligonucleotide probes, and provides a flexible and powerful method for research on plant chromosomes.

Description

Method for synthesizing oligonucleotide probe based on multiplex PCR

1. Technical field

The invention discloses a method for synthesizing an oligonucleotide probe based on multiplex PCR, which is favorable for developing cytogenetic research of sequenced plant chromosomes and belongs to the technical field of plant biology.

2. Background of the art

The technique of using fluorescence in situ hybridization (Fluorescence in situ hybridization, FISH) to fluoresce a certain color for a particular chromosome segment, chromosome arm, or whole chromosome of a subject, so that the chromosome can be visualized under a fluorescence microscope is called chromosome staining, which greatly enhances our understanding of chromosome structure and changes. For example, this technique is used to analyze and study chromosomal recombination, aberration, and homologous genes (Ried et al, 1998); in addition, comparative studies of the genome between closely related species, where the chromosome staining technique has not undergone genome sequencing, provide a new approach by which the differences in chromosome structure between species can be visualized, so that we can analyze the rearrangement relationship of chromosomes between closely related species (Jiang et al 2006).

However, plant chromosome painting has been difficult to develop for a long time due to the presence of numerous repetitive sequences in plant cell walls and plant chromosomes. Numerous researchers have made improvements such as large fragment cloning (BAC, YAC, fosmid cloning, etc.) probes, single copy probes, etc. to make this technology applicable to plant chromosome studies (frankz et al, 2000; lysak et al, 2003, 2006;Mandakova et al, 2010; lou et al, 2014). However, due to the cumbersome and labor-intensive process of probe preparation (Lysak et al, 2013), the requirements on chromosome flaking technology are high, and 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 problems of plant chromosome studies (cuadrao et al, 1998, 2002). Han et al (2015) have readily accomplished the staining of plant specific chromosome segments or the full length of the chromosome by screening oligonucleotide probes made of specific oligonucleotides in the genome of sequenced plant species. Thus, this method has great potential for chromosomal studies of sequenced plants, and many successful studies have been conducted on some species such as strawberry, potato, tomato, eggplant (Qu et al, 2017; braz et al, 2017).

However, since the application of the oligonucleotide probe is still in the initial stage, the existing oligonucleotide probe synthesis method is complex, labor-intensive and time-consuming, and in addition, the reagent required for synthesizing the oligonucleotide probe is expensive and the investment at one time is large, so that the method is less in practical research. For this purpose we propose a technique for multiplex PCR based staining of segmented oligonucleotide probes. The technology screens an oligonucleotide library synthesized with cucumber chromosomes based on genome sequence information of the cucumber, segments the chromosome library in a double-joint mode in the probe design process, synthesizes a plurality of oligonucleotide probes of a certain section, a certain section or the whole chromosome required by experiments simply, conveniently and rapidly through a multiple PCR method, ensures that the oligonucleotide probes are more simply, conveniently and flexibly utilized, and can also strengthen the strength of signals through synthesizing the double-chain oligonucleotide probes. The technology is favorable for popularization and application of the oligonucleotide probe in plant chromosome research.

3. Summary of the invention

Technical problem

The invention utilizes cucumber genome information to screen proper oligonucleotides and performs double-joint segment design, selects corresponding primers with fluorescent group marks, synthesizes the screened oligonucleotide probes through multiplex PCR, and can be used for plant chromosome research after purification, and the method is developed for solving the problems that the oligonucleotide probe synthesis technology is complex and the synthesis library is not flexibly utilized due to fixation.

Technical proposal

The method for synthesizing the oligonucleotide probe based on multiplex PCR comprises the following steps of:

1) Screening the required oligonucleotide library by chord software with reference to cucumber '9930' genome against cucumber chromosome 4.

2) The repeat sequence was removed from the cucumber genome using a repeat mask, the genomic sequence was then split into 48nt oligonucleotides and homopolymers containing more than 6 nucleotides were discarded.

3) Each oligonucleotide was aligned to a reference genome using blast, and oligonucleotides with > 75% similarity were selected.

4) The Primer3 was used to retain the dTm > 10℃oligonucleotides to construct a database of probes from which probes associated with a particular chromosomal or genomic region were selected.

5) A total of 93396 oligonucleotide probes covering chromosome 4 (about 23.3 Mb) of cucumber were selected, with an average of about 3.8 probes per Kb.

6) The oligonucleotide library was fragmented by means of a double-linker ligation.

7) A working solution of the oligonucleotide library was prepared by dissolving 300ng of the oligonucleotide library in 300. Mu.L of DNA/RNA free water to prepare 1 ng/. Mu.L of stock solution (-80 ℃ C. For storage), and then mixing 2. Mu.L of stock solution with 26. Mu.L of water, and storing in-20 ℃.

8) The 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 from step 2, 98℃for 20 seconds, 56℃for 15 seconds, 72℃for 30 seconds, 25 cycles 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 multiplex PCR fully improves the efficiency of probe synthesis.

2) The oligonucleotide library which is designed in a sectional way by using a double-joint mode can flexibly select oligonucleotide probe primers which cover a certain section, a certain section or a whole chromosome according to experimental requirements in the synthesis process, so that the flexibility in the probe utilization process is improved.

3) In the process of synthesizing the oligonucleotide probe, one chain belt of the double-chain oligonucleotide probe has green fluorescence, the other chain belt has red fluorescence, and the oligonucleotide probes with three colors can be utilized at one time in the experimental process, so that the experimental efficiency is improved.

4) The oligonucleotide probe synthesized by the method can be used as a library to continuously synthesize available oligonucleotide probes, so that the oligonucleotide probes can be permanently used once synthesized, and the experimental cost is reduced.

4. Description of the drawings

Fig. 1: schematic representation of the principle of multiplex PCR synthesis of oligonucleotide probes.

Fig. 2: the PCR synthesized cucumber chromosome 4 oligonucleotide probe Chr4-T (red) is used for the painting of the middle-stage and the thick-line stage chromosomes of the cucumber.

Fig. 3: the PCR synthesized segmented oligonucleotide probe is applied to the chromosome of the cucumber in the pachytene period. Wherein Chr4-1, -3, -5, -7 is a red signal and Chr4-2, -4, -6, -8 is a green signal.

Fig. 4: the synthesis of double-stranded red-green oligonucleotide probes by PCR gave a clearly discernible yellow-like signal in FISH.

5. Detailed description of the preferred embodiments

The implementation procedure of the method for synthesizing oligonucleotide probes based on multiplex PCR of the invention comprises:

1) Design of oligonucleotide library: screening the required oligonucleotide library by chord software with reference to cucumber '9930' genome against cucumber chromosome 4. The repeat sequence was removed from the cucumber genome using a repeat mask, the genomic sequence was then split into 48nt oligonucleotides and homopolymers containing more than 6 nucleotides were discarded. Each oligonucleotide was aligned to a reference genome using blast, and oligonucleotides with > 75% similarity were selected. The Primer3 was used to retain the dTm > 10℃oligonucleotides to construct a database of probes from which probes associated with a particular chromosomal or genomic region were selected. A total of 93396 oligonucleotide probes covering chromosome 4 (about 23.3 Mb) of cucumber were selected, with an average of about 3.8 probes per Kb. In addition, the oligonucleotide library is segmented by means of a double-linker ligation.

2) Synthesis of oligonucleotide probes: a working solution of the oligonucleotide library was prepared by dissolving 300ng of the oligonucleotide library in 300. Mu.L of DNA/RNA free water to prepare 1 ng/. Mu.L of stock solution (-80 ℃ C. For storage), and then mixing 2. Mu.L of stock solution with 26. Mu.L of water, and storing in-20 ℃. PCR was performed according to the above system and procedure, and primers before and after use in PCR were modified at the 5' end of the primer using FAM and TAMRA, and the PCR reaction product was directly used as a probe after purification.

3) Fluorescence in situ hybridization and signal detection: 2 mu L of the obtained oligonucleotide probe is taken to prepare a probe hybridization solution (containing the oligonucleotide probe, 50% deionized formamide, 2 XSSC and 10% dextran sulfate), the probe hybridization solution is denatured in a metal bath at 90 ℃ for 6 minutes, the glass slide containing good chromosomes is denatured in 70% deionized formamide at 80 ℃ for 10 minutes, dehydrated in 70%, 90% and 100% series of glacial ethanol for 5 minutes respectively, dried in the air, and the denatured probe hybridization solution is dripped on the denatured glass slide, sealed and placed in an in situ hybridization instrument for hybridization at 37 ℃ for overnight. After developing, air-dried at room temperature, DAPI containing anti-fluorescence quenching agent is added, and hybridization signals are observed by an Olympus BX51 fluorescence microscope under the condition of light shielding after cover glass sealing.

Claims

1. The application of the method for synthesizing double-stranded oligonucleotide probes based on multiplex PCR in cucumber chromosome research is characterized by comprising the following steps:

the oligonucleotide probe library is screened according to the genome sequence information of cucumber, in the process of probe design, the oligonucleotide library is segmented by a method of respectively adding different segment specific primer joints at the 5 'end and the 3' end of each segment oligonucleotide and adding fluorescent marked integral primer joints at the outer side, and a large number of oligonucleotide probes required by chromosome research can be simply, conveniently and quickly obtained by using different primer combinations through PCR;

the method for synthesizing the oligonucleotide probe based on multiplex PCR comprises the following steps:

1) Screening a required oligonucleotide library by chord software by taking cucumber '9930' genome as a reference aiming at cucumber chromosome 4;

2) Removing all repetitive sequences in the cucumber genome using a repeater mask, then dividing the genomic sequence into 48nt oligonucleotides, and discarding oligomers containing homopolymers of more than 6 nucleotides;

3) Each oligonucleotide was aligned to a reference genome using blast, selecting oligonucleotides with > 75% similarity;

4) Preserving the oligonucleotides of dTm > 10 ℃ using Primer3 to construct a probe database from which probes associated with a particular chromosomal or genomic region are selected;

5) Screening out 93396 oligonucleotide probes covering cucumber chromosome 4 with a chromosome size of 23.3 Mb;

6) Segmenting the oligonucleotide library by a connection mode that different segment specific primer joints are respectively added to the 5 'end and the 3' end of each segment oligonucleotide and then fluorescent marked integral primer joints are added to the outer side;

7) 300ng of the oligonucleotide library was dissolved in 300. Mu.L of DNA/RNA free water to prepare 1 ng/. Mu.L of stock solution, stored at-80℃and then 2. Mu.L of 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) The primers before and after the PCR are modified at the 5' end of the primer by FAM and TAMRA;

the PCR system is as follows:

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 are performed from the step 2, 98 ℃ for 20 seconds, 56 ℃ for 15 seconds, 72 ℃ for 30 seconds, 25 cycles are performed from the step 5, and finally the temperature is kept at 4 ℃; the PCR reaction product can be used as a probe directly after purification.