CN107513560B - A kind of quick detection probe of IGH gene breaks of low cost and its preparation method and application - Google Patents
A kind of quick detection probe of IGH gene breaks of low cost and its preparation method and application Download PDFInfo
- Publication number
- CN107513560B CN107513560B CN201710358008.9A CN201710358008A CN107513560B CN 107513560 B CN107513560 B CN 107513560B CN 201710358008 A CN201710358008 A CN 201710358008A CN 107513560 B CN107513560 B CN 107513560B
- Authority
- CN
- China
- Prior art keywords
- probe
- igh
- gene
- sequence
- label
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6811—Selection methods for production or design of target specific oligonucleotides or binding molecules
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6841—In situ hybridisation
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
Abstract
The invention belongs to molecular biology fields, and in particular to a kind of quick detection probe of IGH gene breaks of low cost and preparation method thereof.The preparation method comprises the following steps：IGH gene probes institute overlay area genome non repetitive sequence is downloaded from UCSC Genome Browser, the sequence batch after segmentation is imported into OligoArray softwares 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 IGH gene breaks.The present invention, which prepares the gained quick detection probe of IGH gene breaks, has the advantages that hybridization time short (hybrid experiment can be completed at 15~30 minutes), hybridization signal are bright, signal-to-noise ratio is high, manufacturing 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 IGH gene breaks of low cost
And its preparation method and application.
Background technology
Detection IGH gene breaks are to diagnose most special most sensitive one of the method for acute promyelocytic leukemia (APL),
It is also the most reliable indexs such as the selection of APL therapeutic schemes, efficacy analysis prognostic analysis.Karyotyping at present, PCR methods and FISH methods
It is common several detection modes.FISH has many advantages, such as safety, quick, high sensitivity and shows multiple color simultaneously.
Fluorescence in situ hybridization is a kind of one to grow up in the 1980s radioactive in situ hybridization technical foundation
Kind on-radiation molecular genetic techniques, a kind of new original position for marking substitution labelled with radioisotope with fluorescein and being formed are miscellaneous
Friendship method.The common detection probes of FISH mainly carry out nick-translation by being cloned 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 includes a large amount of repetition sequence
Row are even across COT-1DNA closings but results of hybridization is still with higher background, 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 (be typically concentration and probe concentration 50~
100 times) expensive COT-1DNA blockades to carry out repetitive sequence, and this greatly increases the manufacturing cost of probe.In addition in recent years
Carry out a kind of preparation method of the rapid fluorescence in situ hybridization probe without repetitive sequence and is also established that (1h completes hybridization reality
Test), but such method needs to design a large amount of primers or a large amount of carriers of structure 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；In addition such preparation method is still
So need to rely on human genomic sequence or BAC cloned sequences, and raw material sources have certain limitation, and entirely prepare
Process is cumbersome, and manufacturing cost and uncontrollable factor greatly increase.
Invention content
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 IGH gene breaks of low cost quickly detect
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 IGH gene breaks of low cost, includes the following steps：
(1) the genome non repetitive sequence of IGH gene probes institute overlay area is downloaded from UCSC Genome Browser,
Wherein IGH upstream region of gene spy overlay area is：Chr15 74089693~74634008, IGH downstream of gene probe overlay area
For：Chr17 38059674~38977709；
(2) the genome non repetitive sequence for the IGH 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 sizes；Block batch after segmentation is imported OligoArray softwares to carry out
Probe design, probe screening, then export to the probe filtered out in Microsoft Excel, then respectively at 5 ' ends of every probe
With 3 ' ends plus the sequence label of 18bp, a series of IGH upstream region of gene probe sequences with sequence label are obtained；
(3) the genome non repetitive sequence for the IGH 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 sizes；Block batch after segmentation is imported OligoArray softwares to carry out
Probe design, probe screening, then export to the probe filtered out in Microsoft Excel, then respectively at 5 ' ends of every probe
With 3 ' ends plus the sequence label of 18bp, a series of IGH downstream of gene probe sequences with sequence label are obtained；
(4) use DNA synthesizer to the IGH upstream region of gene probe sequences with sequence label obtained by step (2)
Synthesis is learned, chemically synthesized probe is mixed, IGH upstream region of gene probe libraries are prepared；Similarly, using DNA synthesizer
Chemical synthesis is carried out to the IGH downstream of gene probe sequences with label sets obtained by step (3), by chemically synthesized probe into
Row mixing, is prepared IGH downstream of gene probe libraries；
(5) M13 universal primer and 5 ' ends of the 5 ' ends with green fluorescence group are respectively synthesized and carries Chinese red fluorophor
M13 universal primers, IGH upstream region of gene probe libraries are expanded with the M13 universal primers of green fluorescence group using 5 ' ends
Increase label reaction, IGH downstream of gene probe libraries are expanded using the M13 universal primers of 5 ' fluorophors of the end with Exocarpium Citri Rubrum
Label reaction；
(6) the amplification marked product of IGH upstream region of gene probe libraries obtained by step (5) is purified, obtains fluorescence mark after dilution
The IGH upstream region of gene probe libraries of note, the amplification marked products of IGH downstream of gene probe libraries obtained by step (5) is purified, after dilution
Obtain the IGH downstream of gene probe libraries of fluorescent marker, the IGH upstream region of gene probe library of the fluorescent marker and fluorescent marker
IGH downstream of gene probe libraries constitute the quick detection probe of IGH gene breaks.
In said program, 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 than 40~80%, minimum interval 5bp between probe.
In said program, the base sequence of sequence label described in step (2) and step (3) is as follows：
5 ' end sequence labels be：TGTAAAACGACGGCCAG (M13 upstream sequences)
3 ' end sequence labels be：GGTCATAGCTGTTTCCTG (M13 downstream sequences).
In said program, universal primer sequence is described in step (5)：
M13-F TGTAAAACGACGGCCAGT；
M13-R CAGGAAACAGCTATGACC。
In said program, the PCR reaction systems of step (5) the amplification label reaction are as follows：
In said program, the PCR reaction conditions of step (5) the amplification label reaction are as follows：95℃5min；94 DEG C of 30s,
58 DEG C of 30s, 72 DEG C of 30s carry out 20 cycles altogether；72℃10min.
Including above-mentioned preparation method prepares the kit of the gained quick detection probe of IGH gene breaks.
Mentioned reagent box includes as follows preparing the application in detecting IGH gene break trial products, specific application mode
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 precoolings, it is spare；
(2) preparation of the quick detection probe hybridization mixture of IGH gene breaks：The IGH upstream region of gene of fluorescent marker is visited
Needle library, the IGH 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 IGH gene breaks using 10ul steps (2) and quickly detects
Slide, using rubber glue mounting, is placed in hybridization instrument by probe hybridization mixture using 22 × 22mm coverslip cover plates after mounting
90 DEG C of denaturation 1min, 37 DEG C of 15~30min of hybridization；
(4) post-hybridization washing：It waits throwing off coverslip after the completion of hybridizing, slide is placed in the washing lotion of 60 DEG C of preheatings to wash and is gone
Except unbonded probe；
(5) it redyes and microscopy：10ul anti-cancellation mountant mountings are added dropwise in the slide dried, then under fluorescence microscope
Observe results of hybridization.
In said program, the group of hybridization buffer described in step (2) is divided into：10wt% deionized formamides, 20wt%
Dextran sulfate, 1ugRNaseA, 0.6M sodium chloride, 10mM citrate buffer solutions, 0.5mM CTAB (cetyl trimethyl bromines
Change ammonium).
In said program, washing lotion sodium chloride containing 0.3M, 0.03M sodium citrates and 0.1%Tween- described in step (3)
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 clones completely, and is free of
There is repetitive sequence, does not need expensive COT-1DNA and closed, greatly reduce hybrid context and manufacturing 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 methods is used to expand
And label probe, probe mark rate higher is more uniform, is only held in probe 5 ' and carries out fluorescent marker so not influencing the hybridization of probe
Match reaction；In addition the present invention does not use the nick translation reaction for being easy to be influenced by environment or operation technique and random primer reaction
Carry out label probe, the probe length marked is consistent, increases the stability between batch；
(3) compared with prior art, the present invention coordinates specific hybridization buffer and uniqueness by optimizing probe design
Detection method, the present invention can complete hybrid experiment in 15-30min, this most needs 1h miscellaneous to complete soon 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 with very high specificity and sample recall rate.
Description of the drawings
Fig. 1 is that probe prepares schematic diagram.
Fig. 2 is probe and target gene combination schematic diagram.
Fig. 3 is that chunks.pl programs use code map.
Fig. 4 is IGH gene break detection kits part probe sequence figure after removal repetitive sequence.
Fig. 5 is the testing result figure of IGH gene breaks detection probe of the present invention.
Fig. 6 is IGH gene breaks detection probe of the present invention and Abbott Laboratories' probe difference hybridization time comparative result figure.
Specific implementation mode
For a better understanding of the present invention, with reference to the embodiment content that the present invention is furture elucidated, but the present invention
Content is not limited solely to the following examples.
The preparation of the quick detection probe of embodiment 1IGH gene breaks
The preparation process schematic diagram of the quick detection probe of IGH gene breaks is as shown in Figure 1, specifically include described in the present embodiment
Following steps：
(1) the genome non repetitive sequence of IGH gene probes institute overlay area is downloaded from UCSC Genome Browser,
Wherein IGH upstream region of gene spy overlay area is：Chr15 74089693~74634008, IGH downstream of gene probe overlay area
For：Chr17 38059674~38977709；Acquired sequence saves as fasta formats, repetitive sequence region in genome
Replace with N；
(2) the genome non repetitive sequence for the IGH upstream region of gene probe institute overlay area for obtaining step (1) uses perl
Plug-in card program chunks.pl (chunks.pl programs are shown in Fig. 3 using code map) is divided into the block of 1kb sizes；After segmentation
Block batch imports OligoArray softwares 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 are free of TTTT/GGGG/AAAA/CCCC than 40~80%, minimum interval 5bp between probe；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
Row, obtaining a series of IGH upstream region of gene probe sequences with label, (Fig. 4 show PML gene probes pond, due to sequence mistake
It does not show one by one mostly)；The base sequence of sequence label is as follows：5 ' end sequence labels be：
TGTAAAACGACGGCCAG (M13 upstream sequences), 3 ' end sequence labels are：GGTCATAGCTGTTTCCTG(M13
Downstream sequence)；
(3) the genome non repetitive sequence for the IGH downstream of gene probe institute overlay area for obtaining step (1) uses perl
Plug-in card program chunks.pl (chunks.pl programs are shown in Fig. 3 using code map) is divided into the block of 1kb sizes；After segmentation
Block batch imports OligoArray softwares 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 are free of TTTT/GGGG/AAAA/CCCC than 40~80%, minimum interval 5bp between probe；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
Row obtain a series of IGH downstream of gene probe sequences with label；The same step of base sequence (2) of sequence label；
(4) use DNA synthesizer to the IGH upstream region of gene probe sequences with sequence label obtained by step (2)
Synthesis is learned, chemically synthesized probe is mixed, IGH upstream region of gene probe libraries, the IGH upstream region of gene probe is prepared
Library includes that 1863 IGH upstream region of gene with sequence label are visited；Similarly, using DNA synthesizer to carrying mark obtained by step (3)
The IGH downstream of gene probe sequences for signing sequence carry out chemical synthesis, and chemically synthesized probe is mixed, IGH is prepared
Downstream of gene probe library, the IGH downstream of gene probe library include 1511 IGH downstream of gene probes for carrying sequence label；
(5) 5 ' M13 universal primers of the end with green fluorescence group and Chinese red fluorophor, universal primer are respectively synthesized
Sequence is：M13-F TGTAAAACGACGGCCAGT, M13-R CAGGAAACAGCTATGACC；Green fluorescence is carried using 5 ' ends
The M13 universal primers of group carry out amplification label reaction to IGH upstream region of gene probe library；Use 5 ' fluorescent bases of the end with Exocarpium Citri Rubrum
The M13 universal primers of group carry out amplification label reaction to IGH downstream of gene probe library；The PCR reactions of the amplification label reaction
System is as follows：
The PCR reaction conditions of the amplification label reaction are as follows：95℃5min；94 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 30s, altogether
Carry out 20 cycles；72℃10min；
(6) use amplification marked product of the acetate ethanol sodium method respectively to IGH upstream region of gene probe libraries obtained by step (5) and
The amplification marked product of IGH downstream of gene probe libraries is purified, and is obtained again through dilution after purification：A concentration of 20ng/ul's is glimmering
The IGH downstream of gene probe libraries of the IGH upstream region of gene probe library of signal and the fluorescent marker of a concentration of 20ng/ul.
The detection method of the quick detection probe of embodiment 2IGH gene breaks
The quick detection probe of IGH gene breaks is in the kit for detecting IGH gene breaks described in the present embodiment
Using specifically comprising 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 precoolings, it is spare；
(2) preparation of the quick detection probe hybridization mixture of IGH gene breaks：It is a concentration of that embodiment 1 is prepared into gained
The IGH downstream of gene probes of the IGH upstream region of gene probe library of the fluorescent marker of 20ng/ul, the fluorescent marker of a concentration of 20ng/ul
Library and hybridization buffer are according to 1:1:9 volume ratio mixing；The group of the hybridization buffer is divided into：10wt% deionization formyls
Amine, 20wt% dextran sulfates, 1ugRNaseA, 0.6M sodium chloride, 10mM citrate buffer solutions, 0.5mM CTAB (cetyls
Trimethylammonium bromide)；
(3) probe sample co-variation：Every sample mixes gained IGH gene breaks using 10ul steps (2) and quickly detects
Slide, using rubber glue mounting, is placed in hybridization instrument by probe hybridization mixture using 22 × 22mm coverslip cover plates after mounting
90 DEG C of denaturation 1min, 37 DEG C of hybridization 15min~30min；
(4) post-hybridization washing：It waits throwing off coverslip after the completion of hybridizing, the washing lotion that slide is placed in 60 DEG C of preheatings (contains 0.3M
Sodium chloride, 0.03M sodium citrates and 0.1%Tween-20) in washing removal be not associated with probe；
(5) it redyes and microscopy：10ul anti-cancellation mountant mountings are 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 IGH gene breaks and embodiment 2 prepared using embodiment 1, setting are miscellaneous
The time gradient of friendship is 15min, 30min, 16h；It is carried simultaneously in strict accordance with it using Abbott IGH gene break detection probes
The specification of confession 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 IGH gene breaks 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 IGH gene breaks 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 of the invention prepared, has also illustrated preparation of the present invention
Probe there is high signal-to-noise ratio, very high specificity and sample recall rate.The probe that simultaneously prepared by the present invention is without repetition
Series, and probe length is uniform, probe is with target gene combination schematic diagram 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 be only intended to clearly illustrate made by example, and not 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 domain of the invention.
Claims (8)
1. a kind of preparation method of the quick detection probe of low cost IGH gene breaks, which is characterized in that include the following steps：
（1）The genome non repetitive sequence of IGH gene probes institute overlay area is downloaded from UCSC Genome Browser, wherein
IGH upstream probes overlay area is：Chr15 74089693 ~ 74634008, IGH downstream probe overlay area are：chr17
38059674~38977709；
（2）By step（1）The genome non repetitive sequence of the IGH upstream region of gene probe institute overlay area of acquisition uses perl plug-in units
Program chunks.pl is divided into the block of 1kb sizes；Block batch after segmentation is imported OligoArray softwares 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 obtain a series of IGH upstream region of gene with sequence label and visit plus the sequence label of 18bp
The base sequence of needle sequence, the sequence label is as follows：5 ' end sequence labels be：TGTAAAACGACGGCCAG；3 ' end marks
Signing sequence is：GGTCATAGCTGTTTCCTG；
（3）By step（1）The genome non repetitive sequence of the IGH downstream of gene probe institute overlay area of acquisition uses perl plug-in units
Program chunks.pl is divided into the block of 1kb sizes；Block batch after segmentation is imported OligoArray softwares 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 obtain a series of IGH downstream of gene with sequence label and visit plus the sequence label of 18bp
The base sequence of needle sequence, the sequence label is as follows：5 ' end sequence labels be：TGTAAAACGACGGCCAG；3 ' end marks
Signing sequence is：GGTCATAGCTGTTTCCTG；
（ 4 ）Using DNA synthesizer to step（2）IGH upstream region of gene probe sequence of the gained with sequence label carries out chemistry
Synthesis, chemically synthesized probe is mixed, IGH upstream region of gene probe libraries are prepared；Similarly, it is synthesized using DNA
Instrument is to step（3）IGH downstream of gene probe sequence of the gained with sequence label carries out chemical synthesis, by chemically synthesized probe
It is mixed, IGH downstream of gene probe libraries is prepared；
（5）It is respectively synthesized M13 universal primer and 5 ' end M13s with Chinese red fluorophor of the 5 ' ends with green fluorescence group
Universal primer carries out amplification label using 5 ' M13 universal primers of the end with green fluorescence group to IGH upstream region of gene probe library
Reaction carries out amplification label using the M13 universal primers of 5 ' fluorophors of the end with Chinese red to IGH downstream of gene probe library
Reaction, the sequence of the universal primer are as follows：M13-F TGTAAAACGACGGCCAGT；M13-R
CAGGAAACAGCTATGACC；
（6）By step（5）The amplification marked product purifying of gained IGH upstream region of gene probe libraries obtains fluorescent marker after diluting
IGH upstream region of gene probe libraries, by step（5）The amplification marked product of gained IGH downstream of gene probe libraries purifies, is after dilution
Obtain the IGH downstream of gene probe libraries of fluorescent marker, the IGH upstream region of gene probe library of the fluorescent marker and fluorescent marker
IGH downstream of gene probe libraries constitute the quick detection probe of IGH gene breaks.
2. preparation method according to claim 1, which is characterized in that step（2）And step（3）Described in probe screening item
Part is：85 ~ 99 DEG C of probe length 50bp, TM value, GC is free of TTTT/GGGG/AAAA/CCCC than 40 ~ 80%, between probe between minimum
Every 5bp.
3. preparation method according to claim 1, which is characterized in that step（5）The PCR reactants of the amplification label reaction
System is as follows：
2×PCR Mix 25ul
Template 50ng
M13-F 1ul
M13-R 1ul
H2O complements to 50ul.
4. preparation method according to claim 1, which is characterized in that step（5）The PCR of 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 cycles altogether；72℃10min.
5. one kind preparing the IGH of the gained quick detection probe of IGH gene breaks comprising any preparation method of claim 1 ~ 4
Gene break kit.
6. kit described in claim 5 is being prepared for detecting the application in IGH gene break products.
7. applying according to claim 6, which is characterized in that specific application process 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 precooling, spare；
（2）The preparation of the quick detection probe hybridization mixture of IGH gene breaks：By the IGH upstream region of gene probe library of fluorescent marker,
The IGH downstream of gene probe library and hybridization buffer of fluorescent marker are according to 1:1:9 volume ratio mixing；
（3）Probe sample co-variation：Every sample uses 10ul steps（2）The mixing gained quick detection probe of IGH gene breaks
Slide, using rubber glue mounting, 90 DEG C is placed in hybridization instrument after mounting by hybridization mixture using 22 × 22mm coverslip cover plates
It is denaturalized 1min, 37 DEG C of 15 ~ 30min of hybridization；
（4）Post-hybridization washing：It waits throwing off coverslip after the completion of hybridizing, slide, which is placed in washing in the washing lotion of 60 DEG C of preheatings, to be removed not
Bonding probes；
（5）It redyes and microscopy：10ul anti-cancellation mountant mountings are added dropwise in the slide dried, then in fluorescence microscopy microscopic observation
Results of hybridization.
8. applying according to claim 7, which is characterized in that step（2）Described in the group of hybridization buffer be divided into：10wt%
Deionized formamide, 20wt% dextran sulfates, 1ugRNaseA, 0.6M sodium chloride, 10mM citrate buffer solutions.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710358008.9A CN107513560B (en) | 2017-05-19 | 2017-05-19 | A kind of quick detection probe of IGH gene breaks of low cost and its preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710358008.9A CN107513560B (en) | 2017-05-19 | 2017-05-19 | A kind of quick detection probe of IGH gene breaks of low cost and its preparation method and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107513560A CN107513560A (en) | 2017-12-26 |
| CN107513560B true CN107513560B (en) | 2018-09-18 |
Family
ID=60721774
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710358008.9A Active CN107513560B (en) | 2017-05-19 | 2017-05-19 | A kind of quick detection probe of IGH gene breaks of low cost and its preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107513560B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN110195106A (en) * | 2019-05-10 | 2019-09-03 | 广州安必平医药科技股份有限公司 | For detecting probe groups, kit and its application of PD-L1 gene unconventionality |
| CN112795649A (en) * | 2021-01-05 | 2021-05-14 | 武汉友芝友医疗科技股份有限公司 | Probe set for detecting HER2 gene amplification level and application thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100124741A1 (en) * | 2008-11-18 | 2010-05-20 | Quest Disgnostics Investments Incorporated | METHODS FOR DETECTING IgH/BCL-1 CHROMOSOMAL TRANSLOCATION |
| CN102747156B (en) * | 2012-07-13 | 2015-01-14 | 中国医学科学院肿瘤医院 | Application of Bcl-2 (B cell lymphoma)/IgH (immunoglobulin H) gene rearrangement used as B cell lymphoma bone marrow infiltration marks |
| CN104560978B (en) * | 2015-01-20 | 2017-04-05 | 中国人民解放军第三军医大学 | The multiple PCR primer and method of people's BCR heavy chain libraries are built based on high-flux sequence |
-
2017
- 2017-05-19 CN CN201710358008.9A patent/CN107513560B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN107513560A (en) | 2017-12-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Shi et al. | A review: Fabrications, detections and applications of peptide nucleic acids (PNAs) microarray | |
| CN106834492B (en) | A kind of BCR/ABL fusions quick detection probe of low cost and its preparation method and application | |
| CN107513560B (en) | A kind of quick detection probe of IGH gene breaks of low cost and its preparation method and application | |
| CN110129415B (en) | NGS library-building molecular joint and preparation method and application thereof | |
| CN107227346B (en) | A kind of quick detection probe of CBFB gene break of low cost and its preparation method and application | |
| EP3971282A1 (en) | Array and method for detecting spatial information of nucleic acids | |
| CN107619870B (en) | Molecular marker capable of indicating and identifying length of sheep wool and specific primer pair and application thereof | |
| CN110004225B (en) | Tumor chemotherapeutic drug individualized gene detection kit, primers and method | |
| CN106566876A (en) | Oligonucleotide probe and acquisition method thereof | |
| CN106282413B (en) | Probe combinations, kit and the method for the high-risk strain Genotyping detection of HPV | |
| CN105505755A (en) | Space transcriptome database building and sequencing method and device adopted for same | |
| CN108517567A (en) | Connector, primer sets, kit and the banking process in library are built for cfDNA | |
| CN107904283A (en) | A kind of instant multiprobe hybridized slides and its preparation method and application | |
| CN106929576B (en) | A kind of PML/RARA fusions quick detection probe of low cost and its preparation method and application | |
| CN106701936A (en) | Breast cancer susceptibility gene BRCA1 and BRCA2 detection kit and method | |
| CN106834493B (en) | A kind of AML1-ETO fusions quick detection probe of low cost and its preparation method and application | |
| CN106399589A (en) | Method, kit and probe assembly for virus genotyping detection | |
| CN108642208A (en) | A kind of Cinnamomum and its general SSR molecular marker of relative genus plant and its development approach and application | |
| CN112592962B (en) | Detection method suitable for high-throughput transcriptome spatial position information and application thereof | |
| CN102140509B (en) | Gene mutation detection method based on nucleic acid amplification on solid carrier | |
| TW201321520A (en) | Method and system for virus detection | |
| CN109439751B (en) | It is a kind of for detecting the fluorescence in situ hybridization probe kit of KIAA1549-BRAF fusion | |
| CN109234400B (en) | It is a kind of for detecting the fluorescence in situ hybridization probe and its preparation method and application of KIAA1549-BRAF fusion | |
| CN110241211B (en) | Molecular combination marker for diagnosing colorectal cancer and detection kit | |
| CN116144811B (en) | Multiplex primer set, method and kit for detecting cerebrospinal fluid pathogen |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |