CN107385042B - Multi-PCR primer and method for detecting gene fusion by combining anchoring nest type multi-PCR with high-throughput sequencing - Google Patents
Multi-PCR primer and method for detecting gene fusion by combining anchoring nest type multi-PCR with high-throughput sequencing Download PDFInfo
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Abstract
The invention provides an anchoring nest type multiple PCR combined high-throughput sequencing detection gene fusion multiple PCR primer, which comprises a multiple PCR specific primer group 1 and a multiple PCR specific primer group 2, wherein the multiple PCR specific primer group 1 is a primer group consisting of nucleotide sequences shown in SEQ ID NO. 1-SEQ ID NO. 32, and the multiple PCR specific primer group 2 is a primer group consisting of nucleotide sequences shown in SEQ ID NO. 33-SEQ ID NO. 64. The invention also provides a multiplex PCR primer and a method for detecting gene fusion by combining anchoring nested multiplex PCR with high-throughput sequencing, the detection method has higher universality, and the detection method can accurately and sensitively detect the fusion gene under the condition that the structure of the fusion gene is only partially known or unknown.
Description
Technical Field
The invention relates to the field of detection, in particular to a multiplex PCR primer and a method for detecting gene fusion by combining anchoring nested multiplex PCR with high-throughput sequencing
Background
Studies have shown that gene fusion is one of the causes of certain diseases (e.g., cancer) in mammals (e.g., humans).
In the case of mixed lineage leukemia gene (MLL), MLL fusion proteins can cause abnormal epigenetic modification of downstream genes, leading to activation of downstream oncogenes, and thus induce leukemia.
Fusion proteins produced ectopically from MLL function as oncogenes, and MLL forms a fusion gene with the following partner genes in addition to the most common MLL-MLLT3(acute myelogenous leukemia (AML)) and MLL-MLLT2(AF 4): MLLT1(ENL), MLLT10(AF10), MLLT4(AF6), ELL and the like.
Among many MLL fusion genes, for acute myeloid leukemia of the liver myeloloid leukemia (AML), about 18% of AML are caused by the rearrangement (MLL-r) of MLLT3 gene (also called AF9) with MLL; at present, the MLLT3 gene is the second most of more than 50 genes fused with MLL, and the fusion gene research of MLLT3-MLL shows that the fusion gene is closely related to the occurrence and the development of tumors.
At present, most of the research on fusion genes adopts an RT-PCR method, and the small part of the research adopts an immunohistochemical FISH method for detection.
The RT-PCR method is to design primers according to the type and fusion mode of known MLL fusion gene, obtain cDNA by reverse transcription of RNA, and then detect by PCR amplification of target fragment. The limitations of this approach: 1) the gene to which the MLL gene is fused must be known; 2) the way in which the two genes are fused must be known; 3) fusion of different genes with MLL cannot or is difficult to detect simultaneously. Therefore, different primers must be designed for different genes fused with MLL according to different fusion modes for detection, and unknown MLL fusion genes and unknown fusion modes cannot be detected, which directly results in difficult detection of MLL fusion genes and low detection rate.
The principle of the non-radioactive in situ hybridization FISH method is to design a nucleic acid probe which is homologous and complementary with the detected MLL-MLLT3, and the nucleic acid probe and the DNA of the MLL-MLLT3 fusion gene can form a hybrid of the nucleic acid probe and the DNA through denaturation, annealing and renaturation. A reporter molecule (such as biotin and digoxigenin) is used to label a certain nucleotide of a nucleic acid probe, and the immunochemical reaction between the reporter molecule and a fluorescein-labeled specific ligand (such as avidin which is specifically combined with biotin) can be used to perform qualitative and relative positioning analysis on the DNA to be detected under a microscope by using a fluorescent detection system. However, this approach has limitations: 1) the gene to which the MLL gene is fused must be known; 2) the chromosome on which the gene fused to MLL is located must be located at a short distance from MLL; 3) fusion of different genes with MLL cannot be detected simultaneously. Therefore, this method requires designing different probes for different genes fused with the MLL gene, and cannot detect an unknown MLL fusion gene, which affects the detection rate of the MLL gene.
In view of the fact that expression products of various fusion genes (such as MLL fusion gene) play an important role in the development of diseases such as cancer, there is an urgent need in the art to develop more general fusion gene detection methods.
In addition, the present invention is also eagerly desired to develop a method for detecting a fusion gene (e.g., MLL fusion gene) by an accurate and sensitive detection method with partial knowledge or no knowledge of the structure of the fusion gene.
Disclosure of Invention
The invention aims to provide a multiplex PCR primer for detecting gene fusion by combining anchoring nested multiplex PCR with high-throughput sequencing, and also provides a multiplex PCR primer and a method for detecting gene fusion by combining anchoring nested multiplex PCR with high-throughput sequencing.
In order to achieve the purpose, the invention adopts the technical scheme that: a multiplex PCR primer for detecting gene fusion by anchoring nested multiplex PCR combined high-throughput sequencing comprises a multiplex PCR specific primer group 1 and a multiplex PCR specific primer group 2, wherein the multiplex PCR specific primer group 1 is a primer group consisting of nucleotide sequences shown in SEQ ID NO. 1-SEQ ID NO. 32, and the multiplex PCR specific primer group 2 is a primer group consisting of nucleotide sequences shown in SEQ ID NO. 33-SEQ ID NO. 64.
The invention provides a kit for detecting gene fusion by combining anchoring nested multiplex PCR with high-throughput sequencing, which comprises the multiplex PCR primer according to claim 1, and further comprises a linker sequence, an anchoring primer 1 and an anchoring primer 2;
the adapter sequence comprises a long sequence of the adapter sequence and a short sequence of the adapter sequence, wherein the long sequence of the adapter sequence consists of a complementary sequence of an upstream sequencing primer, a tag sequence and a complementary sequence of the short sequence of the adapter sequence which are connected in sequence;
the anchor primer 2 consists of a part of the sequence of the sequencing sequence;
the anchor primer 1 is composed of a part of the sequence of the anchor primer 2.
Preferably, the tag sequence is a DNA sequence of 10 bases in length. The tag is used for the statistics of the molecular weight on the original template, theoretically, the tag sequence with N bases can have 4NA linker, and thus a 10 base tag sequence, is sufficient for statistics of sample molecular weight. The position of the tag sequence on the long sequence is 5' to the long sequence except for the segment of the sequence complementary to the short sequence.
Preferably, the long sequence of the linker sequence is represented by SEQ ID NO. 67, and the short sequence of the linker sequence is represented by SEQ ID NO. 68.
Preferably, the sequence of the anchor primer 1 is shown as SEQ ID NO. 65, and the sequence of the anchor primer 2 is shown as SEQ ID NO. 66.
Preferably, a sequence complementary to a downstream sequencing primer for distinguishing the tagged sequence of the sample is also included.
The invention provides a method for detecting gene fusion by adopting the multiple PCR primers.
The invention provides application of the multiplex PCR primer in preparation of a kit for detecting gene fusion.
The invention provides a method for detecting gene fusion by adopting the kit, which comprises the following steps:
(1) taking total RNA of a detected sample as a template, performing reverse transcription by using a random primer after high-temperature breaking to obtain a first chain of cDNA, and then synthesizing a second chain of cDNA;
(2) performing end repair and joint sequence connection on the double-stranded cDNA;
(3) carrying out a first round of multiplex PCR reaction on the product connected with the adaptor sequence, wherein an upstream primer is the anchored primer 1, and a downstream primer is a multiplex PCR gene specific primer group 1;
(4) performing a second round of multiplex PCR reaction on the product obtained from the first round of multiplex PCR reaction to obtain a target region library, wherein the upstream primer is an anchor primer 2, and the downstream primer is a combination of a multiplex PCR primer group 2 and a downstream sequencing primer complementary sequence with a tag sequence for distinguishing a sample;
(5) whether the gene is fused or not and the fusion mode are determined by a sequencing method.
The method provided by the invention is a non-diagnostic method for in vitro detecting the presence or absence of a gene fusion in a polynucleotide in a sample, wherein the fused gene is generally formed by fusing a first gene and a second gene, wherein the linker sequence comprises a long sequence and a short sequence which are complementary, the long sequence is complementary to the short sequence near the junction, and the remainder of the long sequence comprises an unknown tag sequence and an upstream sequencing primer complementary sequence;
at least a portion of the anchor primer 1 is identical to the upstream sequencing primer complement sequence, and the multiplex PCR primer set 1 comprises a plurality of sequences that specifically bind to the target region;
the gene-specific primer group 1 is positioned at the far end of the first gene and the second gene fused with the first gene, namely the gene-specific primer group 1, the first gene (or part thereof) and the second gene fused with the first gene are sequentially arranged in the amplification direction of the gene-specific primer group 1;
at least a part of the anchor primer 2 is identical to the sequence of the sequencing primer and is longer than the anchor primer 1 sequence of the first round of multiplex PCR, the multiplex PCR primer set 2 comprises a plurality of sequences, the 5 '-end of each sequence has at least a part of the sequence complementary to the downstream sequencing primer, and the 3' -end of each sequence specifically binds to the target region, and finally, a target region library is obtained.
The gene specific primer 2 is positioned at the far end of the first gene and the gene fused with the first gene, and is not more than the gene specific primer 1, and the gene specific primer 1, the gene specific primer 2, the first gene (or part of the first gene) and the second gene fused with the first gene are sequentially arranged from the amplification direction of the gene specific primer 1;
as a further improvement of the present invention, the tag sequence is a sequence of 5 to 10 bases, and the tag sequence is different between samples. The tag sequences are set to distinguish between different samples.
As a further improvement of the present invention, the DNA after the end repair has overhanging A bases at both ends and forms a sticky end junction with the overhanging T bases of the linker sequence.
In a further improved aspect of the present invention, the long sequence of the linker sequence has the T base at the 3 '-end and an unknown tag sequence at the 5' -end, the unknown tag sequence is a 10-base sequence, the unknown tag sequence is used for statistics of expression levels of fusion genes, and the short sequence is complementary to the long sequence near the T base.
The invention has the beneficial effects that:
the detection method of the present invention is more versatile, and can accurately and sensitively detect a fusion gene (e.g., MLL fusion gene) with only partial knowledge or no knowledge of the structure of the fusion gene.
Drawings
FIG. 1 is a schematic diagram of the schematic flow chart of the method for detecting gene fusion by anchoring nested multiplex PCR in the embodiment of the present invention;
FIG. 2 is a graph showing the results of Agilent 2100 detection after purification of the final amplified product in the example of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the following detailed description and accompanying drawings.
The method for detecting gene fusion by anchoring nested multiplex PCR of one embodiment of the invention comprises the following steps:
(1) taking total RNA of a detected sample as a template, performing reverse transcription by using a random primer after high-temperature breaking to obtain a first chain of cDNA, and then synthesizing a second chain of cDNA;
(2) subjecting the double-stranded cDNA to end repair and ligation of adaptor sequences, wherein said adaptor sequences comprise a long sequence of adaptor sequences complementary to a short sequence of adaptor sequences, said long sequence being complementary to said short sequence near the junction end, and the remainder of said long sequence comprising an unknown tag sequence and an upstream sequencing primer complementary sequence;
(3) performing a first round of multiplex PCR reaction on the products connected to the linker sequence, wherein the upstream primer is an anchor primer 1, the downstream primer is a multiplex PCR specific primer set 1, at least a portion of the anchor primer 1 is identical to the sequence required for the sequencing, and the multiplex PCR specific primer set 1 comprises a plurality of sequences specifically binding to the target region;
(4) performing a second round of multiplex PCR reaction on the product obtained from the first round of multiplex PCR reaction, wherein the upstream primer is the anchor primer 2, the downstream primer is a multiplex PCR specific primer set 2, the multiplex PCR primer set 2 comprises a plurality of sequences, the 5 '-end of each sequence has a complementary sequence of a downstream sequencing primer, and the 3' -end of each sequence is specifically combined on a target region, and finally obtaining a target region library;
(5) whether the gene is fused or not and the fusion mode are determined by a sequencing method.
In the above scheme, the end repair mainly refers to the end compensation of DNA, and the like, and may also include the end-A addition reaction, and the like. The DNA may be ligated to the linker sequence by blunt-end ligation or cohesive-end ligation, for example, by directly blunt-end ligating the DNA with blunt ends to the blunt-end linker sequence, or by cohesive-end ligating the DNA with overhanging A bases after the end-plus-A reaction to the cohesive-end linker sequence with overhanging T bases. In one embodiment of the present invention, the ligation of DNA to linker sequences is achieved by sticky-end ligation based on the ease of manipulation of the end-plus-A reaction and the high efficiency of sticky-end ligation.
In the above scheme, the linker sequence includes complementary long and short sequences, which are relatively speaking and do not have absolute length criteria. Since the adaptor sequence is double-stranded, with one strand being longer than the other, it is referred to as a long sequence and a short sequence, respectively, where the short sequence is generally complementary to the long sequence at one end, i.e., the end of the adaptor sequence that is ligated to the DNA, and in fact is blunt-ended or sticky-ended.
In the above embodiment, the other part of the long sequence comprises an unknown tag sequence and the sequence complementary to the upstream sequencing primer, which means that the long sequence comprises a sequence other than the one complementary to the short sequence, and the sequence comprises an unknown tag sequence. The tag sequence is used to count the expression level of RNA. The tag sequence has a length of 10 bases, and theoretically, the tag sequence having N bases may have 4NA linker, and thus a 10 base tag sequence, is sufficient for quantification of sample expression. The position of the tag sequence on the long sequence is 5' to the long sequence except for the segment of the sequence complementary to the short sequence.
In the above scheme, at least a part of the anchor primer 1 is identical to the complementary sequence of the upstream sequencing primer, which means that a part of the sequence of the anchor primer 1 can be completely identical to the sequence of the upstream sequencing primer, and only then the tag sequences between different samples can be completely retained in the multiplex PCR reaction, thereby performing statistics on the expression amount of RNA. In general, anchor primer 1 is only partially identical to the sequence complementary to the forward sequencing primer, and anchor primer 2 should completely cover the sequence complementary to the forward sequencing primer.
In the above scheme, the multiplex PCR primer set 1 includes a plurality of sequences that specifically bind to the target region, i.e., the multiplex PCR primer set 1 includes a set of primer sequences rather than a single primer sequence, which is why the PCR in the present invention is referred to as multiplex PCR. The number of primer sequences in the multiplex PCR primer 1 can be determined according to the number of target regions to be captured, and generally, as long as 2 or more (e.g., 2, 3, 5, 10, 20, 100, or 1000 or more) primer sequences are included in the multiplex PCR primer set 1.
In the above-mentioned embodiment, in performing the second round of multiplex PCR reaction, the above-mentioned anchor primer 2 is used as the upstream primer, and the multiplex PCR primer set 2 is used as the downstream primer, and the multiplex PCR primer set 2 is also a set of primer sequences consisting of a plurality of PCR primers instead of one primer sequence, and the set of multiplex PCR primers is characterized in that the 5 '-end has a downstream sequencing primer complementary sequence, and the 3' -end specifically binds to the target region, so that the target region library is finally obtained by the second round of multiplex PCR reaction. Wherein, the sequencing primer sequence is a sequencing primer sequence which is universal for each sequencing platform. The resulting library can be sequenced directly on a specific sequencing platform (e.g., Hiseq, proton, or CG platform). In a preferred embodiment of the invention, the sequence of multiplex PCR primer set 2 that specifically binds to the target region is located distal to the first gene and the gene to which it is fused, and does not exceed gene-specific primer set 1. One of the purposes of the second round of multiplex PCR reaction is to amplify the target region and simultaneously introduce a downstream sequencing primer complementary sequence, so that the subsequent direct sequencing is facilitated.
In a preferred embodiment of the present invention, as shown in FIG. 1, the DNA after end repair has an overhanging A base at both ends, the long sequence of the linker sequence has an overhanging T base at the 3 '-end and an unknown tag sequence at the 5' -end, the overhanging A base forms a sticky end junction with the overhanging T base, the short sequence of the linker sequence is complementary to the long sequence near the T base, and a portion of the anchor primer is identical to a sequence required for sequencing. Thus, in the first round of multiplex PCR reaction, first, the multiplex PCR primer set 1 specifically binds to the target region, a complementary strand is generated by extension, then the anchor primer 1 binds to the complementary strand, another complementary strand is generated by extension, and thereafter, exponential PCR amplification is performed; in the second round of multiplex PCR reaction, the complementary sequences of the anchor primer 2, the multiplex PCR primer set 2 and the labeled downstream sequencing primer are respectively used as an upstream primer and a downstream primer for exponential PCR amplification.
The "upstream primer" and the "downstream primer" are merely relative terms, and are intended to distinguish between two primers in different directions in a PCR reaction, and the present invention is not particularly limited. Meanwhile, the "multiplex PCR primer set 1" and the "multiplex PCR primer set 2" may also be referred to as a "multiplex PCR 1 st primer set" and a "multiplex PCR 2 nd primer set", respectively, and wherein "1" and "2" are only for the purpose of distinguishing objects and have no technical meaning.
The features, effects and advantages of the present invention will be described in more detail by the following examples, which should be understood as being exemplary only, and used for illustrating the feasibility of the technical solution of the present invention, and not limiting the protection scope of the present invention.
Example 1: an RNA sample of an Acute myelogenous leukemia cell line THP1 of Acute Myeloloid Leukemia (AML) is extracted.
The AML cell line of THP1 comprises MLL-MLLT3 fusion gene.
The method comprises the following steps: THP1 cell sample RNA (100ng) was cleaved at 94 ℃ for 10 minutes using the Eichi Library Kit (TrueLib mRNA Library Prep Kit for Illumina, NGS00-2013), and then single-strand cDNA reverse reaction was performed using the fragmented RNA as a template, at 25 ℃, 15 minutes → 42 ℃, 15 minutes → 70 ℃ and 10 minutes. After the first strand was reversed, a two-strand cDNA synthesis reaction was carried out at 16 ℃ for 60 minutes
After the reversal, the cDNA was purified using VAHTS DNA Clean Beads (purchased from N411-03, Nyvowed Biotech, Inc., Nanjing).
Step two: the cDNA purified from the magnetic beads was end-repaired with A block using NEBNext ultrafast end-repair/dA tail block (# E7442L) in a reaction sequence of 20 ℃, 30 min → 65 ℃, 30 min.
Step three: the ligation of adapters of specific sequences was performed by cohesive ligation using NEBNext ultrafast ligation module (# E7445L) at 20 ℃ for 30 min, and the ligation products were subjected to subsequent nested multiplex PCR amplification.
Step four: aiming at the connection product, the first round of multiplex PCR amplification is carried out by adopting an upstream primer as an anchor primer 1 and adopting a downstream primer as a multiplex PCR primer group 1. The amplification system and conditions are shown in tables 1 and 2:
table 1: amplification system
Table 2: reaction conditions are as follows:
step five: and performing second round of multiplex PCR on the magnetic bead purified one-round PCR amplification product, wherein the upstream primer of the amplification primer is an anchor primer II, the downstream primer is a combination of a multiplex PCR specific primer group II and a downstream sequencing primer complementary sequence (Index primer) for distinguishing the tagged sequence of the sample. The amplification system and conditions are shown in tables 3 and 4:
table 3: amplification system
Table 4: reaction conditions
The sequences of the linker and the primer used in the above steps are shown in Table 5:
table 5: sequence of
After the final amplified product is purified by magnetic beads, Agilent 2100 detection is carried out, and the detection result is shown in FIG. 2.
Results
The MLL fusion gene is detected by using the method, and the detection result of the THP1 cell line containing the MLL-MLLT3 fusion gene is MLL-MLLT3 fusion gene. The sequencing results are shown in table 6:
table 6: sequencing results
Discussion of the related Art
The most commonly used methods for detecting fusion genes at present are RT-PCR and FISH methods.
The establishment of the anchoring nested multiplex PCR combined sequencing detection method is based on the characteristics of fusion genes NTRK1, ALK, RET, ROS1, JAK2, NTRK3, BRAF, ABL1, RAF1, MLLT3, KRAS, FGFR3, ROS1 and NTRK2, namely all fusion genes of the method comprise and begin at the exon positions. In the case where the structure of the fusion gene is partially known or unknown, the above fusion gene can be detected accurately and sensitively.
Designing specific primers by using the known gene sequence behind the exon, walking to the upstream of the fusion gene by two rounds of anchoring nested PCR, obtaining a product spanning the fusion point of the fusion gene, and determining the gene fused with the gene and the fusion mode by a sequencing method.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Sequence listing
<110> Guangzhou Yongno health science and technology, Inc., Guangzhou Yongno Biotechnology, Inc
<120> multiple PCR primers and method for detecting gene fusion by anchoring nest type multiple PCR combined high-throughput sequencing
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atctgggcca ccttccgaa 19
<210> 32
<211> 20
<212> DNA
<213> Artificial sequence
<400> 32
gcaacgaaaa ggttggggtc 20
<210> 33
<211> 39
<212> DNA
<213> Artificial sequence
<400> 33
agacgtgtgc tcttccgatc taccagtggt gcatctcca 39
<210> 34
<211> 41
<212> DNA
<213> Artificial sequence
<400> 34
agacgtgtgc tcttccgatc ttcgctgtga cttcgaattg c 41
<210> 35
<211> 39
<212> DNA
<213> Artificial sequence
<400> 35
agacgtgtgc tcttccgatc tatggcttgc agctcctgg 39
<210> 36
<211> 41
<212> DNA
<213> Artificial sequence
<400> 36
agacgtgtgc tcttccgatc tccacagaga ggatcagcga g 41
<210> 37
<211> 40
<212> DNA
<213> Artificial sequence
<400> 37
agacgtgtgc tcttccgatc tctgactgca caggacaggg 40
<210> 38
<211> 43
<212> DNA
<213> Artificial sequence
<400> 38
agacgtgtgc tcttccgatc tgggaatgcc tggtttattt ggg 43
<210> 39
<211> 43
<212> DNA
<213> Artificial sequence
<400> 39
agacgtgtgc tcttccgatc ttgacacgaa agacaaagct tcc 43
<210> 40
<211> 41
<212> DNA
<213> Artificial sequence
<400> 40
agacgtgtgc tcttccgatc tttctcgctt cagcacgatg t 41
<210> 41
<211> 41
<212> DNA
<213> Artificial sequence
<400> 41
agacgtgtgc tcttccgatc ttcatcactc gagtcccgtc t 41
<210> 42
<211> 39
<212> DNA
<213> Artificial sequence
<400> 42
agacgtgtgc tcttccgatc tcaccgccgc tttccaaag 39
<210> 43
<211> 42
<212> DNA
<213> Artificial sequence
<400> 43
agacgtgtgc tcttccgatc tacaccattc gttctgaaga ct 42
<210> 44
<211> 41
<212> DNA
<213> Artificial sequence
<400> 44
agacgtgtgc tcttccgatc ttgcattcag gtggtaccca t 41
<210> 45
<211> 41
<212> DNA
<213> Artificial sequence
<400> 45
agacgtgtgc tcttccgatc taggagacgg actggtgaga a 41
<210> 46
<211> 41
<212> DNA
<213> Artificial sequence
<400> 46
agacgtgtgc tcttccgatc tccaccagct ccaactacca c 41
<210> 47
<211> 41
<212> DNA
<213> Artificial sequence
<400> 47
agacgtgtgc tcttccgatc ttctgggatg gtgtgaagct g 41
<210> 48
<211> 41
<212> DNA
<213> Artificial sequence
<400> 48
agacgtgtgc tcttccgatc tgtccttacc gtgacgtcca c 41
<210> 49
<211> 41
<212> DNA
<213> Artificial sequence
<400> 49
agacgtgtgc tcttccgatc tcactgtcac cccttccttg g 41
<210> 50
<211> 50
<212> DNA
<213> Artificial sequence
<400> 50
agacgtgtgc tcttccgatc ttcaaacagt gtttatattc aatgacattt 50
<210> 51
<211> 41
<212> DNA
<213> Artificial sequence
<400> 51
agacgtgtgc tcttccgatc tgtggtgtcc ccgatgtcat t 41
<210> 52
<211> 42
<212> DNA
<213> Artificial sequence
<400> 52
agacgtgtgc tcttccgatc tcctgtggta ttgggtggtg tt 42
<210> 53
<211> 42
<212> DNA
<213> Artificial sequence
<400> 53
agacgtgtgc tcttccgatc ttggcacata cattcccatg aa 42
<210> 54
<211> 41
<212> DNA
<213> Artificial sequence
<400> 54
agacgtgtgc tcttccgatc tgggtctcca gatgtgctgt t 41
<210> 55
<211> 41
<212> DNA
<213> Artificial sequence
<400> 55
agacgtgtgc tcttccgatc tctcaccact gatgacagcc a 41
<210> 56
<211> 43
<212> DNA
<213> Artificial sequence
<400> 56
agacgtgtgc tcttccgatc tagcttgaat ctctctgtcc acg 43
<210> 57
<211> 45
<212> DNA
<213> Artificial sequence
<400> 57
agacgtgtgc tcttccgatc ttggtaacat gtcattttct gttaa 45
<210> 58
<211> 41
<212> DNA
<213> Artificial sequence
<400> 58
agacgtgtgc tcttccgatc tagcagacaa acctgtggtt g 41
<210> 59
<211> 41
<212> DNA
<213> Artificial sequence
<400> 59
agacgtgtgc tcttccgatc tcgagggaag gcaggaagat t 41
<210> 60
<211> 39
<212> DNA
<213> Artificial sequence
<400> 60
agacgtgtgc tcttccgatc tacagctcgt cgcacagtg 39
<210> 61
<211> 42
<212> DNA
<213> Artificial sequence
<400> 61
agacgtgtgc tcttccgatc tgaaccaagt tcttccgagg ga 42
<210> 62
<211> 41
<212> DNA
<213> Artificial sequence
<400> 62
agacgtgtgc tcttccgatc ttagcctaag acccggagct t 41
<210> 63
<211> 41
<212> DNA
<213> Artificial sequence
<400> 63
agacgtgtgc tcttccgatc tggctggcag agtcatcatc a 41
<210> 64
<211> 41
<212> DNA
<213> Artificial sequence
<400> 64
agacgtgtgc tcttccgatc taagtcagat gctactggcc g 41
<210> 65
<211> 35
<212> DNA
<213> Artificial sequence
<400> 65
aatgatacgg cgaccaccga gatctacact ctttc 35
<210> 66
<211> 45
<212> DNA
<213> Artificial sequence
<400> 66
aatgatacgg cgaccaccga gatctacact ctttccctac acgac 45
<210> 67
<211> 87
<212> DNA
<213> Artificial sequence
<220>
<221> misc_feature
<222> (59)..(68)
<223> n is a, c, g, or t
<400> 67
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctnn 60
nnnnnnnnct gtctcttata cacatct 87
<210> 68
<211> 18
<212> DNA
<213> Artificial sequence
<400> 68
gatgtgtata agagacag 18
Claims (9)
1. An anchoring nest type multiple PCR combined high-throughput sequencing detection gene fusion multiple PCR primer,
the primer set is characterized by comprising a multiple PCR specific primer set 1 and a multiple PCR specific primer set 2, wherein the multiple PCR specific primer set 1 is a primer set consisting of nucleotide sequences shown in SEQ ID NO. 1-SEQ ID NO. 32, and the multiple PCR specific primer set 2 is a primer set consisting of nucleotide sequences shown in SEQ ID NO. 33-SEQ ID NO. 64.
2. A kit for detecting gene fusion by combining anchoring nested multiplex PCR and high-throughput sequencing, which is characterized by comprising the multiplex PCR primer according to claim 1, and further comprising a linker sequence, an anchor primer 1 and an anchor primer 2;
the adapter sequence comprises a long sequence of the adapter sequence and a short sequence of the adapter sequence, wherein the long sequence of the adapter sequence consists of a complementary sequence of an upstream sequencing primer, a tag sequence and a complementary sequence of the short sequence of the adapter sequence which are connected in sequence;
the anchor primer 2 consists of a part of the long sequence of the linker sequence;
the anchor primer 1 is composed of a part of the sequence of the anchor primer 2.
3. The kit of claim 2, wherein the tag sequence is a DNA sequence of 10 bases in length.
4. The kit according to claim 2 or 3, wherein the long sequence of the linker sequence is represented by SEQ ID NO 67 and the short sequence of the linker sequence is represented by SEQ ID NO 68.
5. The kit according to claim 2, wherein the sequence of the anchor primer 1 is shown as SEQ ID NO. 65, and the sequence of the anchor primer 2 is shown as SEQ ID NO. 66.
6. The kit of claim 2, further comprising a sequence complementary to a downstream sequencing primer for distinguishing tagged sequences of a sample.
7. A method for detecting gene fusion using the multiplex PCR primers of claim 1, said method being of non-diagnostic use.
8. Use of the multiplex PCR primer of claim 1 for the preparation of a kit for detecting gene fusion.
9. A method for detecting gene fusion using a kit according to any one of claims 2 to 6, wherein the method is for non-diagnostic use and comprises the steps of:
(1) taking total RNA of a detected sample as a template, performing reverse transcription by using a random primer after high-temperature breaking to obtain a first chain of cDNA, and then synthesizing a second chain of cDNA;
(2) performing end repair and joint sequence connection on the double-stranded cDNA;
(3) carrying out a first round of multiplex PCR reaction on the product connected with the adaptor sequence, wherein an upstream primer is the anchored primer 1, and a downstream primer is a multiplex PCR gene specific primer group 1;
(4) performing a second round of multiplex PCR reaction on the product obtained from the first round of multiplex PCR reaction to obtain a target region library, wherein the upstream primer is an anchor primer 2, and the downstream primer is a combination of a multiplex PCR primer group and a downstream sequencing primer complementary sequence with a tag sequence for distinguishing a sample;
(5) whether the gene is fused or not and the fusion mode are determined by a sequencing method.
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| CN109929920A (en) * | 2017-12-19 | 2019-06-25 | 李劲风 | For detecting the multiple PCR method, kit and composition of Gene Fusion |
| WO2019129239A1 (en) * | 2017-12-29 | 2019-07-04 | Act Genomics Co., Ltd. | Method and system for sequence alignment and variant calling |
| CN110628891B (en) * | 2018-06-25 | 2024-01-09 | 深圳华大智造科技股份有限公司 | Method for screening embryo genetic abnormality |
| CN110669823B (en) * | 2018-07-03 | 2022-05-24 | 中国医学科学院肿瘤医院 | ctDNA library construction and sequencing data analysis method for simultaneously detecting multiple liver cancer common mutations |
| WO2020036926A1 (en) * | 2018-08-17 | 2020-02-20 | Cellecta, Inc. | Multiplex preparation of barcoded gene specific dna fragments |
| CN110820051B (en) * | 2018-12-28 | 2023-04-28 | 广州表观生物科技有限公司 | High-sensitivity fusion gene detection method and application thereof |
| WO2020191521A1 (en) * | 2019-03-22 | 2020-10-01 | 深圳华大智造科技有限公司 | Nucleotide sequence, and method for constructing rna target area sequencing library and application thereof |
| CN111321208B (en) * | 2020-02-14 | 2023-10-03 | 上海厦维医学检验实验室有限公司 | Library construction method based on high-throughput sequencing |
| CN112280864B (en) * | 2020-11-13 | 2021-06-15 | 苏州科贝生物技术有限公司 | Thyroid polygene joint detection kit |
| CN116377076A (en) * | 2023-04-07 | 2023-07-04 | 安徽安龙基因科技有限公司 | Primer combination, method and kit for detecting skip mutation of fusion gene and/or MET gene of solid tumor |
| CN117343989A (en) * | 2023-12-06 | 2024-01-05 | 广州迈景基因医学科技有限公司 | Targeting library construction method for detecting gene fusion |
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| CN105838793A (en) * | 2016-04-22 | 2016-08-10 | 上海荻硕贝肯生物科技有限公司 | Primers, kit and method for qualitatively detecting leukaemia fusion genes |
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| EP3283645B1 (en) * | 2015-04-17 | 2019-03-27 | Roche Diagnostics GmbH | Multiplex pcr to detect gene fusions |
| CN106192018B (en) * | 2015-05-07 | 2020-03-24 | 深圳华大智造科技有限公司 | Method and kit for enriching DNA target region by anchoring nested multiplex PCR |
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Denomination of invention: A multiplex PCR primer and method for anchored nested multiplex PCR combined with high-throughput sequencing to detect gene fusion Effective date of registration: 20230423 Granted publication date: 20210101 Pledgee: Bank of China Limited by Share Ltd. Guangzhou Haizhu branch Pledgor: GUANGZHOU FOREVERGEN MEDICAL LABORATORY Co.,Ltd.|GUANGZHOU FOREVERGEN HEALTH TECHNOLOGY Co.,Ltd. Registration number: Y2023980038850 |
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