CN107227346B - A kind of quick detection probe of CBFB gene break of low cost and its preparation method and application - Google Patents
A kind of quick detection probe of CBFB gene break of low cost and its preparation method and application Download PDFInfo
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Abstract
The invention belongs to molecular biology fields, and in particular to a kind of quick detection probe of CBFB gene break of low cost and preparation method thereof.The preparation method comprises the following steps：CBFB gene probe institute overlay area genome non repetitive sequence is downloaded from UCSC Genome Browser, the sequence batch after segmentation is imported into OligoArray software again and carries out probe design, then it is directly synthesized after the probe after design being added sequence label, it reuses the Tag primer with fluorophor probe is expanded and marked, obtains the quick detection probe of CBFB gene break.The present invention, which prepares the gained quick detection probe of CBFB gene break, has the advantages that hybridization time short (can be in 15~30 minutes completion hybrid experiments), hybridization signal are bright, signal-to-noise ratio is high, preparation cost is low etc..
Description
Technical field
The invention belongs to molecular biology fields, and in particular to a kind of quick detection probe of CBFB gene break of low cost
And its preparation method and application.
Background technique
Detection CBFB gene break be diagnose acute promyelocytic leukemia (APL) most special most sensitive method it
The most reliable indexs such as the selection of one and APL therapeutic scheme, efficacy analysis prognostic analysis.Karyotyping at present, PCR method and
FISH method is common several detection modes.FISH have safety, quickly, high sensitivity and to show multiple color simultaneously etc. excellent
Point.
Fluorescence in situ hybridization is a kind of one to grow up in radioactive in situ hybridization technical foundation the 1980s
Kind on-radiation molecular genetic techniques mark a kind of new original position for replacing labelled with radioisotope and being formed miscellaneous with fluorescein
Friendship method.The common detection probe of FISH mainly carries out nick-translation by cloning to specific human genome BAC at present
Or random priming label, then hybridization in situ experiment is used for after COT-1DNA is closed, but the method to include a large amount of repetition sequence
Column are even across COT-1DNA closing but results of hybridization background still with higher, and the probe of such method label usually needs
Want 12-16h (overnight) complete that hybrid experiment is time-consuming and laborious and such side need it is a large amount of (usually the 50 of concentration and probe concentration~
100 times) expensive COT-1DNA blockades to carry out repetitive sequence, and this greatly increases the preparation cost of probe.Furthermore in recent years
A kind of preparation method of rapid fluorescence in situ hybridization probe without repetitive sequence is also established (1h completion hybridization reality
Test), but such method needs to design a large amount of primers or a large amount of carriers of building to realize the preparation of probe, and the preparation of probe
Process still uses the conventional methods such as nick translation or random primer, causes unstable between batch；Furthermore such preparation method is still
So need to rely on human genomic sequence or BAC cloned sequence, and raw material sources have certain limitation, and entirely prepare
Process is cumbersome, and preparation cost and uncontrollable factor greatly increase.
Summary of the invention
The present invention is in view of the deficiencies of the prior art, and it is an object of the present invention to provide a kind of CBFB gene break of low cost quickly detects
Probe and its preparation method and application.
For achieving the above object, the technical solution adopted by the present invention is：
A kind of preparation method of the quick detection probe of CBFB gene break of low cost, includes the following steps：
(1) the genome non repetitive sequence of CBFB gene probe institute overlay area is downloaded from UCSC Genome Browser,
Wherein CBFB upstream probe overlay area is：chr16:66885331-67101057, CBFB downstream probe overlay area are：
chr16:67122220-67337946；
(2) the genome non repetitive sequence for the CBFB upstream region of gene probe institute overlay area for obtaining step (1) uses
Perl plug-in card program chunks.pl is divided into the block of 1kb size；Block batch after segmentation is imported into OligoArray software
Probe design, probe screening are carried out, is then exported to the probe filtered out in Microsoft Excel, then respectively in every probe
5 ' ends and 3 ' ends add the sequence label of 18bp, obtain a series of CBFB upstream region of gene probe sequences with sequence label；
(3) the genome non repetitive sequence for the CBFB downstream of gene probe institute overlay area for obtaining step (1) uses
Perl plug-in card program chunks.pl is divided into the block of 1kb size；Block batch after segmentation is imported into OligoArray software
Probe design, probe screening are carried out, is then exported to the probe filtered out in Microsoft Excel, then respectively in every probe
5 ' ends and 3 ' ends add the sequence label of 18bp, obtain a series of CBFB downstream of gene probe sequences with sequence label；
(4) using DNA synthesizer to the CBFB upstream region of gene probe sequence for having sequence label obtained by step (2)
Synthesis is learned, chemically synthesized probe is mixed, CBFB upstream region of gene probe library is prepared；Similarly, using DNA synthesizer
Chemical synthesis is carried out to the CBFB downstream of gene probe sequence obtained by step (3) with label sets, by chemically synthesized probe
It is mixed, CBFB downstream of gene probe library is prepared；
(5) M13 universal primer and 5 ' ends of the 5 ' ends with green fluorescence group are respectively synthesized with Chinese red fluorophor
M13 universal primer, CBFB upstream region of gene probe library is expanded with the M13 universal primer of green fluorescence group using 5 ' ends
Increase label reaction, the M13 universal primer using 5 ' fluorophors of the end with Exocarpium Citri Rubrum expands CBFB downstream of gene probe library
Increase label reaction；
(6) the amplification marked product of CBFB upstream region of gene probe library obtained by step (5) is purified, obtains fluorescence mark after dilution
The CBFB upstream region of gene probe library of note, the amplification marked product of step (5) gained CBFB downstream of gene probe library is purified, dilutes
The CBFB downstream of gene probe library of fluorescent marker, the CBFB upstream region of gene probe library and fluorescent marker of the fluorescent marker are obtained afterwards
CBFB downstream of gene probe library constitute the quick detection probe of CBFB gene break.
In above scheme, the condition of the screening of probe described in step (2) and step (3) is：Probe length 50bp, TM value 85
~99 DEG C, GC is free of TTTT/GGGG/AAAA/CCCC, minimum interval 5bp between probe than 40~80%.
In above scheme, the base sequence of sequence label described in step (2) and step (3) is as follows：
5 ' end sequence labels be：TGTAAAACGACGGCCAG (M13 upstream sequence)
3 ' end sequence labels be：GGTCATAGCTGTTTCCTG (M13 downstream sequence).
In above scheme, universal primer sequence described in step (5) is：
M13-F TGTAAAACGACGGCCAGT；
M13-R CAGGAAACAGCTATGACC。
In above scheme, the PCR reaction system of step (5) the amplification label reaction is as follows：
In above scheme, the PCR reaction condition of step (5) the amplification label reaction is as follows：95℃5min；94 DEG C of 30s,
58 DEG C of 30s, 72 DEG C of 30s carry out 20 circulations altogether；72℃10min.
The kit of the gained quick detection probe of CBFB gene break is prepared comprising above-mentioned preparation method.
Application of the mentioned reagent box in preparation detection CBFB gene break trial product, specific application mode includes as follows
Step：
(1) sample process：Peripheral blood cells drop piece sample is put into the container for filling 2 × SSC, the high fire heating of micro-wave oven
3min is handled until liquid boiling, then again in low fire continue to heat 10min, slide is placed in immediately after the completion of processing-
It is dehydrated and dries in the graded ethanol of 20 DEG C of pre-coolings, it is spare；
(2) preparation of the quick detection probe hybridization mixture of CBFB gene break：By the CBFB upstream region of gene of fluorescent marker
Probe library, the CBFB downstream of gene probe library of fluorescent marker and hybridization buffer are according to 1:1:9 volume ratio mixing；
(3) probe sample co-variation：Every sample mixes gained CBFB gene break using 10ul step (2) and quickly detects
Slide, using rubber glue mounting, is placed in hybridization instrument by probe hybridization mixture using 22 × 22mm coverslip cover plate after mounting
90 DEG C of denaturation 1min, 37 DEG C of 15~30min of hybridization；
(4) post-hybridization washing：Wait throw off coverslip after the completion of hybridizing, slide is placed in the washing lotion of 60 DEG C of preheatings to wash and be gone
Except unbonded probe；
(5) it redyes and microscopy：10ul anti-cancellation mountant mounting is added dropwise in the slide dried, then under fluorescence microscope
Observe results of hybridization.
In above scheme, the group of hybridization buffer described in step (2) is divided into：10wt% deionized formamide, 20wt%
Dextran sulfate, 1ugRNaseA, 0.6M sodium chloride, 10mM citrate buffer solution, 0.5mM CTAB (cetyl trimethyl bromine
Change ammonium).
In above scheme, washing lotion sodium chloride containing 0.3M described in step (3), 0.03M sodium citrate and 0.1%Tween-
20。
Beneficial effects of the present invention are as follows：
(1) FISH is cloned compared to traditional BAC, preparation method of the present invention does not depend on BAC clone completely, and is free of
There is repetitive sequence, does not need expensive COT-1DNA and closed, greatly reduce hybrid context and preparation cost.
(2) compared to the preparation method for the rapid fluorescence in situ hybridization probe without repetitive sequence established in recent years, originally
It invents in the preparation method, the screening of probe relies primarily on program completion, and more stable PCR method is used to expand
And label probe, probe mark rate is higher more uniform, only holds in probe 5 ' and carries out fluorescent marker so not influencing the hybridization of probe
Match reaction；Furthermore the present invention does not use the nick translation reaction and random primer reaction for being easy to be influenced by environment or operation technique
Carry out label probe, the probe length marked is consistent, increases the stability between batch；
(3) compared with prior art, the present invention cooperates specific hybridization buffer and uniqueness by optimization probe design
Detection method, the present invention can complete hybrid experiment in 15-30min, this most needs 1h miscellaneous to complete fastly than in the prior art
The time for handing over experiment spent is compared, and reduces 50% or more.
(4) probe of the present invention has high signal-to-noise ratio, and has very high specificity and sample recall rate.
Detailed description of the invention
Fig. 1 is that probe prepares schematic diagram.
Fig. 2 is probe and target gene combination schematic diagram.
Fig. 3 is that chunks.pl program uses code map.
Fig. 4 is CBFB gene break detection kit part probe sequence figure after removal repetitive sequence.
Fig. 5 is the testing result figure of CBFB gene break detection probe of the present invention.
Fig. 6 is CBFB gene break detection probe of the present invention and Abbott Laboratories' probe difference hybridization time comparative result figure.
Specific embodiment
For a better understanding of the present invention, below with reference to the embodiment content that the present invention is furture elucidated, but it is of the invention
Content is not limited solely to the following examples.
The preparation of the quick detection probe of embodiment 1CBFB gene break
The preparation process schematic diagram of the quick detection probe of CBFB gene break described in the present embodiment is as shown in Figure 1, specific packet
Include following steps：
(1) the genome non repetitive sequence of CBFB gene probe institute overlay area is downloaded from UCSC Genome Browser,
Wherein CBFB upstream probe overlay area is：chr16:66885331-67101057, CBFB downstream probe overlay area are：
chr16:67122220-67337946；Acquired sequence saves as fasta format, and repetitive sequence region is replaced in genome
For N；
(2) the genome non repetitive sequence for the CBFB upstream region of gene probe institute overlay area for obtaining step (1) uses
Perl plug-in card program chunks.pl (chunks.pl program is shown in Fig. 3 using code map) is divided into the block of 1kb size；It will segmentation
Block batch afterwards imports OligoArray software and carries out probe design, probe screening, and the condition of probe screening is：Probe length
85~99 DEG C of 50bp, TM value, GC is free of TTTT/GGGG/AAAA/CCCC, minimum interval 5bp between probe than 40~80%；Then
The probe filtered out is exported in Microsoft Excel, then adds the label sequence of 18bp at 5 ' ends of every probe and 3 ' ends respectively
Column, obtaining a series of CBFB upstream region of gene probe sequences with label, (Fig. 4 show CBFB gene probe pond, due to sequence
It does not show one by one excessively)；The base sequence of sequence label is as follows：
5 ' end sequence labels be：TGTAAAACGACGGCCAG (M13 upstream sequence),
3 ' end sequence labels be：GGTCATAGCTGTTTCCTG (M13 downstream sequence)；
(3) the genome non repetitive sequence for the CBFB downstream of gene probe institute overlay area for obtaining step (1) uses
Perl plug-in card program chunks.pl (chunks.pl program is shown in Fig. 3 using code map) is divided into the block of 1kb size；It will segmentation
Block batch afterwards imports OligoArray software and carries out probe design, probe screening, and the condition of probe screening is：Probe length
85~99 DEG C of 50bp, TM value, GC is free of TTTTT/GGGG/AAAAA/CCCC, minimum interval 5bp between probe than 40~80%；So
The probe filtered out is exported in Microsoft Excel afterwards, then adds the label of 18bp at 5 ' ends of every probe and 3 ' ends respectively
Sequence obtains a series of CBFB downstream of gene probe sequences with label；The same step of the base sequence of sequence label (2)；
(4) using DNA synthesizer to the CBFB upstream region of gene probe sequence for having sequence label obtained by step (2)
Synthesis is learned, chemically synthesized probe is mixed, CBFB upstream region of gene probe library is prepared, the CBFB upstream region of gene is visited
Needle library includes that 1109 CBFB upstream region of gene with sequence label are visited；Similarly, using DNA synthesizer to band obtained by step (3)
There is the CBFB downstream of gene probe sequence of sequence label to carry out chemical synthesis, chemically synthesized probe is mixed, is prepared into
To CBFB downstream of gene probe library, the CBFB downstream of gene probe library includes under 1238 CBFB genes with sequence label
Swim probe；
(5) the M13 universal primer that 5 ' ends have green fluorescence group and Chinese red fluorophor, universal primer are respectively synthesized
Sequence is：M13-F TGTAAAACGACGGCCAGT, M13-R CAGGAAACAGCTATGACC；Green fluorescence is had using 5 ' ends
The M13 universal primer of group carries out amplification label reaction to CBFB upstream region of gene probe library；The fluorescence of Exocarpium Citri Rubrum is had using 5 ' ends
The M13 universal primer of group carries out amplification label reaction to CBFB downstream of gene probe library；The PCR of the amplification label reaction is anti-
Answer system as follows：
The PCR reaction condition of the amplification label reaction is as follows：95℃5min；94 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 30s, altogether
Carry out 20 circulations；72℃10min；
(6) using acetate ethanol sodium method respectively to the amplification marked product of CBFB upstream region of gene probe library obtained by step (5)
It is purified with the amplification marked product of CBFB downstream of gene probe library, is obtained again through dilution after purification：Concentration is 20ng/ul's
The CBFB upstream region of gene probe library and concentration of fluorescent marker are the CBFB downstream of gene probe library of the fluorescent marker of 20ng/ul.
The detection method of the 2 quick detection probe of CBFB gene break of embodiment
The quick detection probe of CBFB gene break is in the kit for detecting CBFB gene break described in the present embodiment
Application, specifically comprise the following steps：
(1) sample process：Peripheral blood cells drop piece sample is put into the container for filling 2 × SSC, the high fire heating of micro-wave oven
3min is handled until liquid boiling, then again in low fire continue to heat 10min, slide is placed in immediately after the completion of processing-
It is dehydrated and dries in the graded ethanol of 20 DEG C of pre-coolings, it is spare；
(2) preparation of the quick detection probe hybridization mixture of CBFB gene break：Embodiment 1, which is prepared gained concentration, is
The CBFB downstream of gene spy of the CBFB upstream region of gene probe library of the fluorescent marker of 20ng/ul, the fluorescent marker that concentration is 20ng/ul
Needle library and hybridization buffer are according to 1:1:9 volume ratio mixing；The group of the hybridization buffer is divided into：10wt% deionization formyl
Amine, 20wt% dextran sulfate, 1ugRNaseA, 0.6M sodium chloride, 10mM citrate buffer solution, 0.5mM CTAB (cetyl
Trimethylammonium bromide)；
(3) probe sample co-variation：Every sample mixes gained CBFB gene break using 10ul step (2) and quickly detects
Slide, using rubber glue mounting, is placed in hybridization instrument by probe hybridization mixture using 22 × 22mm coverslip cover plate after mounting
90 DEG C of denaturation 1min, 37 DEG C of hybridization 15min~30min；
(4) post-hybridization washing：Wait throw off coverslip after the completion of hybridizing, slide is placed in the washing lotion of 60 DEG C of preheatings (containing 0.3M
Sodium chloride, 0.03M sodium citrate and 0.1%Tween-20) in washing remove unbonded probe；
(5) it redyes and microscopy：10ul anti-cancellation mountant mounting is added dropwise in the slide dried, then under fluorescence microscope
Observe results of hybridization.
Points for attention：Step (2)~step (4) need to be protected from light operation, and testing result is as shown in Figure 5.
Detection method described in the quick detection probe of CBFB gene break and embodiment 2 prepared using embodiment 1 is arranged miscellaneous
The time gradient of friendship is 15min, 30min, 16h；Simultaneously using Abbott CBFB gene break detection probe in strict accordance with it
The specification of offer carries out sample process and crossover operation, same setting hybridization 15min, 30min, 16h time gradient, as right
According to.Testing result is as shown in fig. 6, as can be seen from Figure 6：The quick detection probe of CBFB gene break prepared by the present invention is in hybridization
Between just have a preferable hybridization signal intensities when being 15min, hybridization time all has strong hybridization signals when being 30min or more；
Abbott of Abbott CBFB gene break detection probe is only visible clear hybridization signal occur in 16 hours in hybridization time,
It has absolutely proved the probe hybridization feature used that the time is short and hybridization signal is strong prepared by the present invention, has also illustrated preparation of the present invention
Probe have high signal-to-noise ratio, it is very high specificity and sample recall rate.Probe prepared by the present invention is without repetition simultaneously
Series, and probe length is uniform, probe and target gene combination schematic diagram are as shown in Fig. 2, from figure 2, it is seen that the probe and target base
Because the binding specificity of non-duplicate sequence of regions is very high.
Obviously, above-described embodiment is only intended to clearly illustrate made example, and is not the limitation to embodiment.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or
It changes.There is no necessity and possibility to exhaust all the enbodiments.And the obvious variation or change therefore amplified
It moves within still in the protection scope of the invention.
Claims (7)
1. a kind of preparation method of the quick detection probe of low cost CBFB gene break, which is characterized in that include the following steps：
(1) from the genome non repetitive sequence of UCSC Genome Browser downloading CBFB gene probe institute overlay area, wherein
CBFB upstream probe overlay area is：chr16:66885331-67101057, CBFB downstream probe overlay area are：chr16:
67122220-67337946；
(2) the genome non repetitive sequence for the CBFB upstream region of gene probe institute overlay area for obtaining step (1) is inserted using perl
Part program chunks.pl is divided into the block of 1kb size；Block batch after segmentation is imported OligoArray software to visit
Needle design, probe screening, then export to the probe filtered out in Microsoft Excel, then add respectively at 5 ' ends of every probe
The sequence label of upper 17bp, 3 ' ends add the sequence label of 18bp, obtain a series of CBFB upstream region of gene with sequence label
Probe sequence；The condition of probe screening is：85~99 DEG C of value of probe length 50bp, TM, GC is free of than 40~80%
TTTT/GGGG/AAAA/CCCC, minimum interval 5bp between probe；The base sequence of the sequence label is as follows：
5 ' end sequence labels be：TGTAAAACGACGGCCAG,
3 ' end sequence labels be：GGTCATAGCTGTTTCCTG；
(3) the genome non repetitive sequence for the CBFB downstream of gene probe institute overlay area for obtaining step (1) is inserted using perl
Part program chunks.pl is divided into the block of 1kb size；Block batch after segmentation is imported OligoArray software to visit
Needle design, probe screening, then export to the probe filtered out in Microsoft Excel, then add respectively at 5 ' ends of every probe
The sequence label of upper 17bp, 3 ' ends add the sequence label of 18bp, obtain a series of CBFB downstream of gene with sequence label
Probe sequence；The condition of probe screening is：85~99 DEG C of value of probe length 50bp, TM, GC is free of than 40~80%
TTTT/GGGG/AAAA/CCCC, minimum interval 5bp between probe；The base sequence of the sequence label is as follows：
5 ' end sequence labels be：TGTAAAACGACGGCCAG,
3 ' end sequence labels be：GGTCATAGCTGTTTCCTG；
(4) chemical conjunction is carried out to the CBFB upstream region of gene probe sequence obtained by step (2) with sequence label using DNA synthesizer
At chemically synthesized probe is mixed, CBFB upstream region of gene probe library is prepared；Similarly, using DNA synthesizer to step
Suddenly the CBFB downstream of gene probe sequence obtained by (3) with sequence label carries out chemical synthesis, and chemically synthesized probe is carried out
Mixing, is prepared CBFB downstream of gene probe library；
(5) it is respectively synthesized the M13 that M13 universal primer and 5 ' ends of the 5 ' ends with green fluorescence group have Chinese red fluorophor
Universal primer carries out amplification mark to CBFB upstream region of gene probe library with the M13 universal primer of green fluorescence group using 5 ' ends
Note reaction carries out amplification mark to CBFB downstream of gene probe library with the M13 universal primer of Chinese red fluorophor using 5 ' ends
Note reaction；The sequence of the universal primer is as follows：
M13-F TGTAAAACGACGGCCAGT、
M13-R CAGGAAACAGCTATGACC；
(6) fluorescent marker will be obtained after the amplification marked product purifying of CBFB upstream region of gene probe library obtained by step (5), dilution
CBFB upstream region of gene probe library, after the amplification marked product purifying of CBFB downstream of gene probe library obtained by step (5), dilution
Obtain the CBFB downstream of gene probe library of fluorescent marker, the CBFB upstream region of gene probe library and fluorescent marker of the fluorescent marker
CBFB downstream of gene probe library constitute the quick detection probe of CBFB gene break.
2. preparation method according to claim 1, which is characterized in that the PCR reactant of step (5) the amplification label reaction
It is as follows：
3. preparation method according to claim 1, which is characterized in that the PCR of step (5) the amplification label reaction reacts item
Part is as follows：95℃5min；94 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 30s carry out 20 circulations altogether；72℃10min.
4. a kind of prepare the gained quick detection probe of CBFB gene break comprising any preparation method of claims 1 to 3
CBFB gene break kit.
5. kit described in claim 4 is in preparation for detecting the application in CBFB gene break product.
6. applying according to claim 5, which is characterized in that specific detection method includes the following steps：
(1) sample process：Peripheral blood cells drop piece sample is put into the container for filling 2 × SSC, the high fire heat treatment of micro-wave oven
3min until liquid boiling, then again in low fire continue to heat 10min, slide is placed in -20 DEG C immediately after the completion of processing
Dehydration is dried in the graded ethanol of pre-cooling, spare；
(2) preparation of the quick detection probe hybridization mixture of CBFB gene break：By the CBFB upstream region of gene probe of fluorescent marker
Library, the CBFB downstream of gene probe library of fluorescent marker and hybridization buffer are according to 1:1:9 volume ratio mixing；
(3) probe sample co-variation：Every sample mixes the gained quick detection probe of CBFB gene break using 10ul step (2)
Slide, using rubber glue mounting, is placed in hybridization instrument 90 DEG C after mounting using 22 × 22mm coverslip cover plate by hybridization mixture
It is denaturalized 1min, 37 DEG C of 15~30min of hybridization；
(4) post-hybridization washing：Wait throw off coverslip after the completion of hybridizing, slide is placed in washing in the washing lotion of 60 DEG C of preheatings and is removed not
Bonding probes；
(5) it redyes and microscopy：10ul anti-cancellation mountant mounting is added dropwise in the slide dried, then in fluorescence microscopy microscopic observation
Results of hybridization.
7. applying according to claim 6, which is characterized in that the group of hybridization buffer described in step (2) is divided into：10wt%
Deionized formamide, 20wt% dextran sulfate, 1ugRNaseA, 0.6M sodium chloride, 10mM citrate buffer solution.
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| CN109439751B (en) * | 2018-11-13 | 2019-09-20 | 武汉康录生物技术股份有限公司 | It is a kind of for detecting the fluorescence in situ hybridization probe kit of KIAA1549-BRAF fusion |
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