CN111009288A - Probe design method of CEBPA gene and application thereof - Google Patents
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Abstract
The invention discloses a probe design method of CEBPA gene and application thereof, aiming at the coding sequence of CEBPA gene, according to the principle of sequence reverse complementation, a probe sequence with the length of 120bp is designed by tiling every 20bp from rs 76249325 locus as the center to two sides; and combining with a cosmic database, and optimizing the probe sequences obtained above to obtain the preferred probe sequences. Meanwhile, a DNA probe library constructed by the probe sequence, a detection reagent for CEBPA gene mutation and a detection method for CEBPA gene mutation are disclosed. The kit has high sensitivity and detection rate for detecting CEBPA gene mutation, can reach the international report level, provides a reliable way for diagnosing diseases of blood tumor patients, and has certain market popularization economic value.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a probe design method of a CEBPA gene and application thereof.
Background
CEBPA gene mutations occur in Acute Myeloid Leukemia (AML) at a rate of about 10-15% (PMID: 21177436). European hematological disease network (ELN) in 2017, National Comprehensive Cancer Network (NCCN) in U.S. in 2019, Chinese medical guideline for acute myelogenous leukemia (non-acute promyelocytic leukemia) in adults (version 2017, J Zhonghua Hematology, 2017,38 (3): 177-182) AML prognosis stratification guideline suggest: CEBPA double mutation AML patients with better prognosis by adopting chemotherapy treatment belong to a low-risk group. In addition, 2016 World Health Organization (WHO) has a new subtype of myeloid tumor associated with germ-line-susceptible genetic variation, of which CEBPA germ-line-susceptible genetic variation belongs. Therefore, the CEBPA double mutation detection has important clinical application significance in accurate medical treatment of blood tumors.
The CEBPA gene is located on chromosome 19q13.11, the gene only has 1 exon, the total length of an open reading frame is 1077bp, and the GC content is as high as 74.7%. The CEBPA gene is mainly divided into three regions, namely a TAD1 region, a TAD2 region and a bZIP region, and the mutation mainly occurs in the TAD1 region close to the N end and the bZIP region at the C end. Currently, the main detection method for CEBPA gene mutation at home and abroad still takes one-generation sanger sequencing as the main, namely, deoxyribonucleic acid (DNA) as a detection object, and adopts a PCR combined one-generation sequencing method for detection (interpretation of China expert consensus (2018) edition, J. clinical hematology, 32 vol. 5 in 2019, 341 & 347), a primer and a detection method for detecting CEBPA gene mutation (an authorization publication number: CN 107841538A), and a PCR primer and a detection method for detecting human gene CEBPA (an authorization publication number: CN 108220411A). The limitation of the first-generation sequencing detection of CEBPA is that the detection sensitivity is not high, and is only about 10-20%. With the development of high-throughput sequencing technology, Ng CWS et al in 2018 disclosed a method for detecting CEBPA mutation by high-throughput sequencing, the method firstly amplifies the CEBPA gene by using a PCR amplification method, and the amplified product is detected by using a Miseq sequencer, and the technology can well detect rs 76249325 single nucleotide polymorphism (c.584-589 dupACCCGC/p.His 195-Pro 196 dup) of the CEBPA gene (PMID: 29180507). However, the document does not disclose CEBPA amplification primer sequences. And because the GC content of the CEBPA gene is high, the technical method can only detect one gene of the CEBPA. With the continuous development of the precise medical treatment of hematological tumors, more and more genes are needed for mutation detection in patients with hematological tumors (J.Zhonghua Hematology, 2017,38 (3): 177-182)). The second generation sequencing is divided into amplicon library construction and probe capture library construction according to the sequencing principle. Because of the high GC content of the CEBPA gene, when the gene is amplified and detected with other genes at the same time, the sequencing depth of the CEBPA gene in the final sequencing result is not very good and single amplification is needed because of the low amplification efficiency caused by the high GC content. The probe capture method has the advantages of better capture efficiency on a high GC content area, and can be placed in a reaction system together with other gene probes for capture and subsequent sequencing. However, no other documents disclose CEBPA gene probe design method and probe sequence, and no probe capture method is utilized to confirm the performance of detecting single nucleotide polymorphism of rs 76249325 of CEBPA gene.
Disclosure of Invention
The invention aims to establish a CEBPA gene probe design method, which takes DNA as a sample source, can effectively capture normal and mutant sequences of a CEPBA gene through the designed probe sequence, and can carry out detection through a high-throughput sequencing method. The probe designed by the invention can well capture the single nucleotide polymorphism rs 76249325 of the CEBPA gene at the same time, and can detect when a patient mutates near rs 76249325. Meanwhile, the probe designed by the invention can be used together with other gene probes, namely the CEBPA gene mutation and other gene mutation conditions can be simultaneously detected through one reaction, and the application value is high.
In order to achieve the purpose, the invention adopts the following technical scheme:
a probe design method of CEBPA gene comprises the following steps:
(1) aiming at the coding sequence of the CEBPA gene, according to the principle of reverse sequence complementation, a probe sequence with the length of 120bp is designed by tiling every 20bp from an rs76245932 locus as a center to two sides;
(2) and (2) optimizing the probe sequence obtained in the step (1) by combining a cosmic database, wherein the optimization principle is as follows: when the cosmic database in the first 6 bases of the probe sequence reports that the cosmic database has more than 6 bases of frame shift mutation, the probe sequence is optimally shifted to two sides of the reported mutation site. And better capture efficiency is ensured when the position has frame shift mutation.
The probe sequence obtained by the probe design method is shown in SEQ ID NO. 1-SEQ ID NO. 55.
A library of DNA probes prepared using the probe sequences described above.
The DNA probe library is applied to the preparation of CEBPA gene mutation detection reagents.
A detection reagent for CEBPA gene mutation, wherein the detection reagent comprises the DNA probe library.
A method of detecting a mutation in the CEBPA gene in a subject comprising the steps of:
(1) extracting the genomic DNA of the subject, and breaking the genomic DNA into a range of 200-220 bp;
(2) preparing a DNA fragment library from the fragmented genomic DNA;
(3) hybridizing the DNA fragment library with the DNA probe library of claim 3 or the DNA probe library comprising claim 3 to capture a gene;
(4) sequencing the target gene captured in the step (3) to obtain sequencing data of the gene;
(5) and (5) comparing the sequencing data of the step (4) with the reference genome, thereby obtaining the mutation condition of the target gene.
Has the advantages that: the invention discloses a probe design method of CEBPA gene and application thereof, and the beneficial effects are mainly embodied in the following aspects:
(1) the social effect is as follows: the CEBPA gene probe sequence designed by the invention can effectively detect CEBPA gene mutation, provides strong evidence for disease diagnosis and risk stratification of blood tumor patients, provides effective technical support for accurate medical development of the blood tumor patients in China, and has certain social effect.
(2) The economic effect is as follows: by utilizing the CEBPA gene probe sequence designed by the invention, CEBPA gene mutation can be detected simultaneously with other genes, the cost for simultaneously detecting multiple gene mutations of a blood tumor patient is reduced, and the CEBPA gene probe sequence has certain market popularization economic value.
(3) The technical effects are as follows: the CEBPA gene probe sequence designed by the invention can ensure the detection rate of CEBPA mutation and reach the international report level.
Drawings
FIG. 1 is a map of the CEBPA gene probe sequence.
FIG. 2 is a diagram of the design concept of CEBPA gene probe sequence.
FIG. 3 is a graph showing the analysis of the detection results in example 1.
FIG. 4 is a graph showing analysis results of example 2.
FIG. 5 is an analysis chart of the detection result of the p.Ala44fs mutation in example 3.
FIG. 6 is a graph showing the analysis of the results of detecting the p.Glu316delinsAspGln mutation in example 3.
FIG. 7 is a graph showing analysis results of example 4.
Detailed Description
The present invention is further described below with reference to specific examples, which are only exemplary and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
The design idea of the probe is as follows: different from the traditional probe design idea of improving the specificity and the capture efficiency of the probe, the invention mainly aims to ensure the capture efficiency of the CEBPA gene probe on the CEBPA gene mutant DNA sequence and ensure the detection rate and the detection sensitivity of the CEBPA gene mutation. It is currently internationally accepted that the specificity and capture efficiency of probes with a probe length of 120bp are relatively optimal. Therefore, the present invention is based on a 120bp DNA probe.
The applicant finds that gene mutation of the CEBPA gene is less around rs 76249325 through searching CEBPA gene mutation documents at earlier stage, and mutation mainly occurs on two sides of rs 76249325 (PMID: 11242107, 14726504 and 15575056), so that the probe is designed by taking single nucleotide of the CEBPA gene rs 76249325 as the center and tiling the probe on two sides every 20bp on average.
At present, it is internationally acknowledged that when a 120bp probe is captured, the capture efficiency of the probe is reduced when a target sequence and a probe sequence generate more than 6 base mismatches. In order to ensure the maximum capture efficiency of the designed probe on CEBPA mutation types, the obtained probe sequence is optimized by combining with a cosmic database. The optimization principle is as follows: when the cosmic database in the first 6 bases of the probe reports that the cosmic database has more than 6 bases of frame shift mutation, the probe sequence is optimally shifted to two sides of the reported mutation site, and better capture efficiency is ensured when the frame shift mutation occurs at the position. The CEBPA gene probe combination profile and the gene probe design concept map are shown in FIG. 1 and FIG. 2, respectively.
By adopting the CEBPA gene probe design idea disclosed by the invention, a CEBPA gene probe sequence can be obtained through one-time probe design, and the rs 76249325 mutation and the CEBPA gene mutation have good detection rates through example verification.
Alternative compositions: it is not excluded that the same detection rate is obtained in the case of encrypted probes and in other cases. However, according to the condition disclosed in the prior art, no method for designing a probe aiming at the mutation detection rate of CEBPA gene rs 7624549325 is disclosed at present, and the CEBPA probe sequence disclosed by the application can be combined with other gene probes to simultaneously detect the CEBPA gene and other gene mutations.
The effect of the probe sequences designed by the method of the present invention on the detection of CEBPA gene mutations is shown in the following examples.
Example 1
And (3) detecting the target DNA: the CEBPA gene contains rs 76249325 heterozygous positive DNA specimen and rs 76249325 negative cell strain K562.
And (3) probe: the invention designs a probe sequence combination shown in SEQ ID NO. 1-SEQ ID NO. 55.
DNA ultrasonic breaking: covaris M220, interrupted to the 200-220bp range.
A library building kit: the kappa hyper Prep kit.
Establishing a library experiment: refer to the Kapa library construction kit instructions.
CEBPA Gene sequencing results coverage: 100% (rs 76249325 positive specimen), 100% (rs 76249325 negative specimen).
CEBPA Gene sequencing results mean sequencing depth: 1928 layer (rs 76249325 positive specimen), 1867 layer (rs 76249325 negative specimen).
And (3) site detection results: data analysis result bam file is analyzed by using IGV software, as shown in FIG. 3, FIG. 3A shows the result of detecting rs 76249325 positive specimen, and FIG. 3B shows the result of detecting rs 76249325 negative specimen.
Example 2
And (3) detecting the target DNA: contains 1% of rs 76249325 variant DNA (rs 76249325 heterozygous variant specimen is mixed with rs 76249325 negative K562 cell strain DNA according to a certain proportion after the concentration is determined to obtain 1% of positive control specimen).
And (3) probe: the invention designs the combination of probe sequence and other 85 gene probe sequences.
DNA ultrasonic breaking: covaris M220, interrupted to the 200-220bp range.
A library building kit: the kappa hyper Prep kit.
Establishing a library experiment: refer to the Kapa library construction kit instructions.
CEBPA sequencing results coverage: 100 percent.
CEBPA sequencing results mean sequencing depth: 2319 and (b) a layer.
And (3) site detection results: data analysis results bam file was analyzed using IGV software as shown in fig. 4.
Example 3
And (3) detecting the target DNA: the double mutation positive samples of CEBPA gene p.Ala44fs and p.Glu316delins AspGln are detected by primary sequencing.
And (3) probe: the invention designs the combination of probe sequence and other 85 gene probe sequences.
DNA ultrasonic breaking: covaris M220, interrupted to the 200-220bp range.
A library building kit: the kappa hyper Prep kit.
Establishing a library experiment: refer to the Kapa library construction kit instructions.
CEBPA sequencing results coverage: 100 percent.
CEBPA sequencing results mean sequencing depth: 1754 and (b) a layer.
And (3) site detection results: data analysis results bam file the results of p.ala44fs mutation were analyzed by IGV software as shown in fig. 5, and the results of p.glu316delins aspgln mutation are shown in fig. 6.
Example 4
And (3) detecting the target DNA: and (3) carrying out follow-up DNA sample after chemotherapy (the initial diagnosis of CEBPA gene p.Ala240fs mutation is positive, and the follow-up DNA sample is negative after the chemotherapy through the first-generation sequencing detection of the p.Ala240fs mutation along with rs 76249325 mutation patients).
And (3) probe: the invention designs the combination of probe sequence and other 85 gene probe sequences.
DNA ultrasonic breaking: covaris M220, interrupted to the 200-220bp range.
A library building kit: the kappa hyper Prep kit.
Establishing a library experiment: refer to the Kapa library construction kit instructions.
CEBPA sequencing results coverage: 100 percent.
CEBPA sequencing results mean sequencing depth: 1995 layer.
And (3) site detection results: data analysis results bam file adopts IGV software to analyze about 2.9% of p.Ala240fs mutation results, and the detection result of CEBPA mutation is consistent with the type of initial diagnosis mutation, as shown in FIG. 7.
The experimental results obtained in the above examples are summarized as follows:
example 1: the probe sequence designed by the application can detect rs 7624579325 mutation;
example 2: the probe sequence designed by the application can detect rs 7624579325 mutation with the proportion of 1%, and can be combined with other gene probes for application.
Example 3: the probe sequence designed by the application can effectively detect the p.Ala44fs mutation of the TAD1 region of the CEBPA gene and the p.Glu316delins AspGln mutation of the bZIP region, and the probe sequence can effectively detect the mutation of other regions of the CEBPA gene.
Example 4: the probe sequence designed by the application can effectively detect CEBPA gene mutation, and the mutation detection sensitivity is higher than that of first-generation sequencing. Meanwhile, when the patient mutates near the rs 7624549325 region, the probe sequence designed by the application can have a certain detection rate.
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<213> Artificial sequence (chemical Synthesis)
<400>23
gggcgcgccc gcgggccccg gcggcgccgt catgcccggg ggagcgcacg ggcccccgcc 60
cggctacggc tgcgcggccg ccggctacct ggacggcagg ctggagcccc tgtacgagcg 120
<210>24
<211>120
<212>DNA
<213> Artificial sequence (chemical Synthesis)
<400>24
gcggcgccgt catgcccggg ggagcgcacg ggcccccgcc cggctacggc tgcgcggccg 60
ccggctacct ggacggcagg ctggagcccc tgtacgagcg cgtcggggcg ccggcgctgc 120
<210>25
<211>120
<212>DNA
<213> Artificial sequence (chemical Synthesis)
<400>25
ggagcgcacg ggcccccgcc cggctacggc tgcgcggccg ccggctacct ggacggcagg 60
ctggagcccc tgtacgagcg cgtcggggcg ccggcgctgc ggccgctggt gatcaagcag 120
<210>26
<211>120
<212>DNA
<213> Artificial sequence (chemical Synthesis)
<400>26
cggctacggc tgcgcggccg ccggctacct ggacggcagg ctggagcccc tgtacgagcg 60
cgtcggggcg ccggcgctgc ggccgctggt gatcaagcag gagccccgcg aggaggatga 120
<210>27
<211>120
<212>DNA
<213> Artificial sequence (chemical Synthesis)
<400>27
ccggctacct ggacggcagg ctggagcccc tgtacgagcg cgtcggggcg ccggcgctgc 60
ggccgctggt gatcaagcag gagccccgcg aggaggatga agccaagcag ctggcgctgg 120
<210>28
<211>120
<212>DNA
<213> Artificial sequence (chemical Synthesis)
<400>28
ctggagcccc tgtacgagcg cgtcggggcg ccggcgctgc ggccgctggt gatcaagcag 60
gagccccgcg aggaggatga agccaagcag ctggcgctgg ccggcctctt cccttaccag 120
<210>29
<211>120
<212>DNA
<213> Artificial sequence (chemical Synthesis)
<400>29
cgtcggggcg ccggcgctgc ggccgctggt gatcaagcag gagccccgcg aggaggatga 60
agccaagcag ctggcgctgg ccggcctctt cccttaccag ccgccgccgc cgccgccgcc 120
<210>30
<211>120
<212>DNA
<213> Artificial sequence (chemical Synthesis)
<400>30
ctcgcacccg cacccgcacc cgccgcccgc gcacctggcc gccccgcacc tgcagttcca 60
gatcgcgcac tgcggccaga ccaccatgca cctgcagccc ggtcacccca cgccgccgcc 120
<210>31
<211>120
<212>DNA
<213> Artificial sequence (chemical Synthesis)
<400>31
cgccgcccgc gcacctggcc gccccgcacc tgcagttcca gatcgcgcac tgcggccaga 60
ccaccatgca cctgcagccc ggtcacccca cgccgccgcc cacgcccgtg cccagcccgc 120
<210>32
<211>120
<212>DNA
<213> Artificial sequence (chemical Synthesis)
<400>32
gccccgcacc tgcagttcca gatcgcgcac tgcggccaga ccaccatgca cctgcagccc 60
ggtcacccca cgccgccgcc cacgcccgtg cccagcccgc accccgcgcc cgcgctcggt 120
<210>33
<211>120
<212>DNA
<213> Artificial sequence (chemical Synthesis)
<400>33
gatcgcgcac tgcggccaga ccaccatgca cctgcagccc ggtcacccca cgccgccgcc 60
cacgcccgtg cccagcccgc accccgcgcc cgcgctcggt gccgccggcc tgccgggccc 120
<210>34
<211>120
<212>DNA
<213> Artificial sequence (chemical Synthesis)
<400>34
ccaccatgca cctgcagccc ggtcacccca cgccgccgcc cacgcccgtg cccagcccgc 60
accccgcgcc cgcgctcggt gccgccggcc tgccgggccc tggcagcgcg ctcaaggggc 120
<210>35
<211>120
<212>DNA
<213> Artificial sequence (chemical Synthesis)
<400>35
ggtcacccca cgccgccgcc cacgcccgtg cccagcccgc accccgcgcc cgcgctcggt 60
gccgccggcc tgccgggccc tggcagcgcg ctcaaggggc tgggcgccgc gcaccccgac 120
<210>36
<211>120
<212>DNA
<213> Artificial sequence (chemical Synthesis)
<400>36
cacgcccgtg cccagcccgc accccgcgcc cgcgctcggt gccgccggcc tgccgggccc 60
tggcagcgcg ctcaaggggc tgggcgccgc gcaccccgac ctccgcgcga gtggcggcag 120
<210>37
<211>120
<212>DNA
<213> Artificial sequence (chemical Synthesis)
<400>37
accccgcgcc cgcgctcggt gccgccggcc tgccgggccc tggcagcgcg ctcaaggggc 60
tgggcgccgc gcaccccgac ctccgcgcga gtggcggcag cggcgcgggc aaggccaaga 120
<210>38
<211>120
<212>DNA
<213> Artificial sequence (chemical Synthesis)
<400>38
gccgccggcc tgccgggccc tggcagcgcg ctcaaggggc tgggcgccgc gcaccccgac 60
ctccgcgcga gtggcggcag cggcgcgggc aaggccaaga agtcggtgga caagaacagc 120
<210>39
<211>120
<212>DNA
<213> Artificial sequence (chemical Synthesis)
<400>39
tggcagcgcg ctcaaggggc tgggcgccgc gcaccccgac ctccgcgcga gtggcggcag 60
cggcgcgggc aaggccaaga agtcggtgga caagaacagc aacgagtacc gggtgcggcg 120
<210>40
<211>120
<212>DNA
<213> Artificial sequence (chemical Synthesis)
<400>40
tgggcgccgc gcaccccgac ctccgcgcga gtggcggcag cggcgcgggc aaggccaaga 60
agtcggtgga caagaacagc aacgagtacc gggtgcggcg cgagcgcaac aacatcgcgg 120
<210>41
<211>120
<212>DNA
<213> Artificial sequence (chemical Synthesis)
<400>41
ctccgcgcga gtggcggcag cggcgcgggc aaggccaaga agtcggtgga caagaacagc 60
aacgagtacc gggtgcggcg cgagcgcaac aacatcgcgg tgcgcaagag ccgcgacaag 120
<210>42
<211>120
<212>DNA
<213> Artificial sequence (chemical Synthesis)
<400>42
cggcgcgggc aaggccaaga agtcggtgga caagaacagc aacgagtacc gggtgcggcg 60
cgagcgcaac aacatcgcgg tgcgcaagag ccgcgacaag gccaagcagc gcaacgtgga 120
<210>43
<211>120
<212>DNA
<213> Artificial sequence (chemical Synthesis)
<400>43
agtcggtgga caagaacagc aacgagtacc gggtgcggcg cgagcgcaac aacatcgcgg 60
tgcgcaagag ccgcgacaag gccaagcagc gcaacgtgga gacgcagcag aaggtgctgg 120
<210>44
<211>120
<212>DNA
<213> Artificial sequence (chemical Synthesis)
<400>44
aacgagtacc gggtgcggcg cgagcgcaac aacatcgcgg tgcgcaagag ccgcgacaag 60
gccaagcagc gcaacgtgga gacgcagcag aaggtgctgg agctgaccag tgacaatgac 120
<210>45
<211>120
<212>DNA
<213> Artificial sequence (chemical Synthesis)
<400>45
cgagcgcaac aacatcgcgg tgcgcaagag ccgcgacaag gccaagcagc gcaacgtgga 60
gacgcagcag aaggtgctgg agctgaccag tgacaatgac cgcctgcgca agcgggtgga 120
<210>46
<211>120
<212>DNA
<213> Artificial sequence (chemical Synthesis)
<400>46
tgcgcaagag ccgcgacaag gccaagcagc gcaacgtgga gacgcagcag aaggtgctgg 60
agctgaccag tgacaatgac cgcctgcgca agcgggtgga acagctgagc cgcgaactgg 120
<210>47
<211>120
<212>DNA
<213> Artificial sequence (chemical Synthesis)
<400>47
gccaagcagc gcaacgtgga gacgcagcag aaggtgctgg agctgaccag tgacaatgac 60
cgcctgcgca agcgggtgga acagctgagc cgcgaactgg acacgctgcg gggcatcttc 120
<210>48
<211>120
<212>DNA
<213> Artificial sequence (chemical Synthesis)
<400>48
gacgcagcag aaggtgctgg agctgaccag tgacaatgac cgcctgcgca agcgggtgga 60
acagctgagc cgcgaactgg acacgctgcg gggcatcttc cgccagctgc cagagagctc 120
<210>49
<211>120
<212>DNA
<213> Artificial sequence (chemical Synthesis)
<400>49
agctgaccag tgacaatgac cgcctgcgca agcgggtgga acagctgagc cgcgaactgg 60
acacgctgcg gggcatcttc cgccagctgc cagagagctc cttggtcaag gccatgggca 120
<210>50
<211>120
<212>DNA
<213> Artificial sequence (chemical Synthesis)
<400>50
cgcctgcgca agcgggtgga acagctgagc cgcgaactgg acacgctgcg gggcatcttc 60
cgccagctgc cagagagctc cttggtcaag gccatgggca actgcgcgtg aggcgcgcgg 120
<210>51
<211>120
<212>DNA
<213> Artificial sequence (chemical Synthesis)
<400>51
acagctgagc cgcgaactgg acacgctgcg gggcatcttc cgccagctgc cagagagctc 60
cttggtcaag gccatgggca actgcgcgtg aggcgcgcgg ctgtgggacc gccctgggcc 120
<210>52
<211>120
<212>DNA
<213> Artificial sequence (chemical Synthesis)
<400>52
acacgctgcg gggcatcttc cgccagctgc cagagagctc cttggtcaag gccatgggca 60
actgcgcgtg aggcgcgcgg ctgtgggacc gccctgggcc agcctccggc ggggacccag 120
<210>53
<211>120
<212>DNA
<213> Artificial sequence (chemical Synthesis)
<400>53
cgccagctgc cagagagctc cttggtcaag gccatgggca actgcgcgtg aggcgcgcgg 60
ctgtgggacc gccctgggcc agcctccggc ggggacccag ggagtggttt ggggtcgccg 120
<210>54
<211>120
<212>DNA
<213> Artificial sequence (chemical Synthesis)
<400>54
cttggtcaag gccatgggca actgcgcgtg aggcgcgcgg ctgtgggacc gccctgggcc 60
agcctccggc ggggacccag ggagtggttt ggggtcgccg gatctcgagg cttgcccgag 120
<210>55
<211>120
<212>DNA
<213> Artificial sequence (chemical Synthesis)
<400>55
actgcgcgtg aggcgcgcgg ctgtgggacc gccctgggcc agcctccggc ggggacccag 60
ggagtggttt ggggtcgccg gatctcgagg cttgcccgag ccgtgcgagc caggactagg 120
Claims (6)
1. A method for designing a probe of a CEBPA gene is characterized by comprising the following steps:
(1) aiming at the coding sequence of the CEBPA gene, according to the principle of reverse sequence complementation, a probe sequence with the length of 120bp is designed by tiling every 20bp from an rs76245932 locus as a center to two sides;
(2) and (2) optimizing the probe sequence obtained in the step (1) by combining a cosmic database, wherein the optimization principle is as follows: when the cosmic database in the first 6 bases of the probe sequence reports that the cosmic database has more than 6 bases of frame shift mutation, the probe sequence is optimally shifted to two sides of the reported mutation site, and better capture efficiency is ensured when the frame shift mutation occurs at the position.
2. The probe sequence obtained by the probe design method of claim 1, wherein the probe sequence is shown in SEQ ID NO. 1-SEQ ID NO. 55.
3. A library of DNA probes prepared using the probe sequence of claim 2.
4. Use of the library of DNA probes of claim 3 in the preparation of a reagent for detecting a mutation in a CEBPA gene.
5. A detection reagent for CEBPA gene mutation, said detection reagent comprises the DNA probe library of claim 3.
6. A method of detecting a mutation in the CEBPA gene in a subject comprising the steps of:
(1) extracting the genomic DNA of the subject, and breaking the genomic DNA into a range of 200-220 bp;
(2) preparing a DNA fragment library from the fragmented genomic DNA;
(3) hybridizing the DNA fragment library with the DNA probe library of claim 3 or the DNA probe library comprising claim 3 to capture a gene;
(4) sequencing the target gene captured in the step (3) to obtain sequencing data of the gene;
(5) and (5) comparing the sequencing data of the step (4) with the reference genome to obtain the mutation condition of the target gene.
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| CN201911190479.9A CN111009288B (en) | 2019-11-28 | 2019-11-28 | Probe design method of CEBPA gene and application thereof |
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Citations (5)
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| CN105779572A (en) * | 2014-12-22 | 2016-07-20 | 深圳华大基因研究院 | Chip and method for capturing target sequences of tumor susceptibility genes, and mutation detection method |
| CN106480205A (en) * | 2016-11-11 | 2017-03-08 | 北京吉因加科技有限公司 | For detecting combined sequence and the probe of various mutations type simultaneously |
| CN106591441A (en) * | 2016-12-02 | 2017-04-26 | 深圳市易基因科技有限公司 | Probes, method and chip for detecting alpha and/or beta-thalassemia mutation based on whole-gene capture sequencing and application of such probes, such method and such chip |
| CN106834507A (en) * | 2017-03-16 | 2017-06-13 | 北京迈博恒业科技有限责任公司 | DMD gene traps probe and its application in DMD detection in Gene Mutation |
| CN109628581A (en) * | 2019-01-30 | 2019-04-16 | 浙江大学 | A kind of diagnostic kit of ob gene mutation and application |
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2019
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105779572A (en) * | 2014-12-22 | 2016-07-20 | 深圳华大基因研究院 | Chip and method for capturing target sequences of tumor susceptibility genes, and mutation detection method |
| CN106480205A (en) * | 2016-11-11 | 2017-03-08 | 北京吉因加科技有限公司 | For detecting combined sequence and the probe of various mutations type simultaneously |
| CN106591441A (en) * | 2016-12-02 | 2017-04-26 | 深圳市易基因科技有限公司 | Probes, method and chip for detecting alpha and/or beta-thalassemia mutation based on whole-gene capture sequencing and application of such probes, such method and such chip |
| CN106834507A (en) * | 2017-03-16 | 2017-06-13 | 北京迈博恒业科技有限责任公司 | DMD gene traps probe and its application in DMD detection in Gene Mutation |
| CN109628581A (en) * | 2019-01-30 | 2019-04-16 | 浙江大学 | A kind of diagnostic kit of ob gene mutation and application |
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