CN113512580A - Primer and method for detecting all exons of Kalman Syndrome (KS) related gene - Google Patents
Primer and method for detecting all exons of Kalman Syndrome (KS) related gene Download PDFInfo
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Abstract
The invention discloses a method, primers and a kit for detecting the sequencing of the whole exons of related genes of Kalman Syndrome (KS), which comprise the detection of the whole exons of amplified KAL1, FGFR1, PROKR2, PROK2, CHD7 and FGF 8; coating the forward and reverse primers to be detected in the micropores of a 96-well plate, and storing at normal temperature or 4 ℃; during detection, PCR amplification can be rapidly carried out only by adding the PCR reaction solution mixed with the genome DNA of the sample to be detected into a 96-well plate; and then obtaining the exon sequence of the gene by a Sanger sequencing technology. The method, the primer and the kit for detection have the advantages of easy storage, simplicity, convenience, rapidness, high accuracy and the like, and have an auxiliary effect on early diagnosis and early intervention of the Karman syndrome.
Description
Technical Field
The invention belongs to the field of molecular diagnosis and detection, and particularly relates to detection of Kalman Syndrome (KS) related genes.
Background
Kalman Syndrome (KS) is a hypogonadotropic hypogonadism with hyposmia or hyposmia. Is a disease with clinical and genetic heterogeneity. KS can be familial or sporadic, and its inheritance pattern is three: x-linked recessive inheritance, autosomal dominant inheritance, and autosomal recessive inheritance.
The pathogenesis of KS is not well understood. It is presently believed that GnRH neurons, which may originate in the olfactory tract, fail to normally migrate for various reasons, localize in the hypothalamus and result in complete or partial loss of the ability to synthesize and secrete GnRH, resulting in hypothalamic-pituitary-gonadal shaft dysfunction and failure to initiate puberty, manifested as delayed puberty development. With the intensive research on KS genetics, a plurality of genes related to KS pathogenesis, such as KAL1 gene, fibroblast growth factor receptor 1 gene (FGFRI), fibroblast growth factor 8 gene (FGF8), prokineticin 2 gene receptor (PROKR2) and prokineticin 2 gene (PROK2) are discovered in succession, and the functions of the genes are probably closely related to the normal migration of GnRH neurons, the development of olfactory bulbs and the projection process of the axon of the GnRH neurons to the median bulge. However, only 30% of Kallmann's syndrome cases were associated with the above-mentioned genes, suggesting that other KS-onset-associated genes have not yet been found.
Disclosure of Invention
The invention aims to provide a primer for detecting sequencing of all exons of KaL1, FGFR1, PROKR2, PROK2, CHD7 and FGF8 related genes of Kalman Syndrome (KS). The method comprises the following steps: primers for amplifying all exons of KAL1, FGFR1, PROKR2, PROK2, CHD7 and FGF8, and the base sequences thereof are shown in Table 1.
Further, the 5 'end of the upstream primer described in Table 1 further comprises a M13-F sequence, and the 5' end of the downstream primer further comprises a M13-R sequence.
Further, the kit also comprises a sequencing primer, the base sequence of which is
M13-F:TGTAAAACGACGGCCAGT
M13-R:AACAGCTATGACCATG。
The invention also provides a method for detecting the sequencing condition of the whole exons of KaL1, FGFR1, PROKR2, PROK2, CHD7 and FGF8 related genes of Kalman Syndrome (KS), which comprises the following steps:
(1) coating the upstream and downstream primers in the micropores of a 96-well plate, and storing at normal temperature or 4 ℃ for later use;
(2) extracting the genome DNA of a sample to be detected;
(3) preparing PCR reaction liquid containing the genomic DNA extracted in the step (2), and adding the PCR reaction liquid into a 96-well plate for PCR amplification;
(4) sequencing the amplification product in step (3);
(5) judging the sequencing result, and determining the mutation condition of each gene;
wherein, the base sequence of the amplification primer in the PCR reaction solution in the step (1) is shown in the 5 'end of the upstream primer in Table 1 and also comprises an M13-F sequence, and the 5' end of the downstream primer also comprises an M13-R sequence.
Further, the sequencing primer base sequence in the step (4) further comprises a sequencing primer, and the base sequence is
M13-F:TGTAAAACGACGGCCAGT
M13-R:AACAGCTATGACCATG。
The invention also provides a kit for detecting the sequencing of the whole exons of the KaL1, FGFR1, PROKR2, PROK2, CHD7 and FGF8 related genes of Kalman Syndrome (KS), which comprises
(1) A 96-well plate coated with upstream and downstream primers;
(2) peripheral blood DNA extraction reagent;
(3) detecting a system PCR amplification reaction solution;
(4) sequencing system reagents;
wherein the base sequence of the amplification primer in the PCR amplification reaction solution is shown in the 5 'end of the upstream primer in Table 1 and also comprises an M13-F sequence, and the 5' end of the downstream primer also comprises an M13-R sequence.
Further, the sequencing primer base sequence in the step (4) further comprises a sequencing primer, and the base sequence is
M13-F:TGTAAAACGACGGCCAGT
M13-R:AACAGCTATGACCATG。
Has the advantages that: the invention designs primers for sequencing all exons of genes KAL1, FGFR1, PROKR2, PROK2, CHD7 and FGF8 related to amplification Kalman Syndrome (KS), coats the positive primers and the reverse primers of the genes to be detected in micropores of a 96-well plate, and stores the primers at normal temperature or 4 ℃. During detection, PCR amplification can be rapidly carried out by adding the PCR reaction solution mixed with the genome DNA of the sample to be detected into a 96-well plate, and then a gene exon sequence is obtained by a Sanger sequencing technology. The invention is designed aiming at the condition that a plurality of primers exist, can simplify the experimental steps, reduce the operation errors and improve the detection efficiency. The 96-well plate coated with the primers can be stably stored at normal temperature through determination, and is simpler and more convenient to store. Coating the positive and reverse primers of the gene to be detected in the micropores of a 96-well plate, and storing at normal temperature or 4 ℃. During detection, PCR amplification can be rapidly carried out by adding the PCR reaction solution mixed with the genome DNA of the sample to be detected into a 96-well plate, and then a gene exon sequence is obtained by a Sanger sequencing technology. Can be used for rapidly detecting the whole exon sequences of KAL1, FGFR1, PROKR2, PROK2, CHD7 and FGF8 of KS patients and analyzing the mutation condition of the patients.
Drawings
FIG. 196 well plate coating primer profiles. C, CDH 7; k is KAL 1; FR, FGFR 1; PR is PROKR 2; p is PROK 2; con denotes blank control
FIG. 2 shows the results of electrophoresis in 96-well plate with primer coating. M is Marker DL 2000, Con: blank control.
FIG. 3 is an agarose gel electrophoresis image of the amplification product of sample 1. M is Marker DL 2000, Con: blank control.
FIG. 4 sample 1PROKR2 gene positive sequencing result screenshot.
Detailed Description
Example 1
The invention will be further elucidated with reference to the specific embodiments and the accompanying drawings. It should be noted that the conventional conditions and methods not described in the examples are generally employed by those skilled in the art according to the routine procedures: such as OsOb and Kingston, fourth edition, or following the manufacturer's suggested procedures and conditions.
A primer for detecting the sequencing of the whole exons of KAL1, FGFR1, PROKR2, PROK2, CHD7 and FGF8 related genes of Kalman Syndrome (KS), is designed as a specific amplification primer aiming at the whole exon sequences of KAL1, FGFR1, PROKR2, PROK2, CHD7 and FGF8, and the base sequence of the specific amplification primer is shown in Table 1. In a preferred design, the upstream primer described in Table 1 further comprises a M13-F sequence at the 5 'end and a M13-R sequence at the 5' end of the downstream primer.
Table 1 shows the base sequences of PCR amplification primers and sequencing primers for each gene. F is an upstream primer, and R is a downstream primer.
M13-F and M13-R are sequencing primers.
A kit for detecting complete exon sequencing of KaL1, FGFR1, PROKR2, PROK2, CHD7 and FGF8 related genes of Kalman Syndrome (KS) comprises
(1) A 96-well plate coated with upstream and downstream primers;
(2) peripheral blood DNA extraction reagent;
(3) detecting a system PCR amplification reaction solution;
(4) sequencing system reagents;
the blood DNA extraction reagent can be purchased from commercial reagents such as Tiangen DNA extraction kit and the like.
The PCR amplification reaction solution of the detection system comprises: 2 times PCR Buffer; 2mM dNTPs; KOD FX DNA Polymerase (1U/. mu.l); and (3) testing the genomic DNA of the sample.
The sequencing system reagent comprises: sequencing purification solution (ExoI:0.6U, CIP:1.2U), EDTA (125mmol), absolute ethanol, 75% ethanol, HIDI (highly deionized formamide), sequencing primers: m13 upstream and downstream primers (3.2 μ M).
Example 2
(1) Coating 96-well plate: 1 mu L of upstream and downstream primers (10 mu m) for sequencing of KAL1, FGFR1, PROKR2, PROK2, CHD7 and FGF8 gene full exons are added into a 96-well plate micropore for 80 wells in total. Another well was blank and 1. mu.L ddH was added2O, drying at 37 ℃ for 1h, sealing by a sealing film, and storing at normal temperature or 4 ℃ for later use.
(2) Extraction of genomic DNA from blood: 1) mu.l of blood was taken and added to 900. mu.l of erythrocyte lysate, mixed by inversion, left at room temperature for 5 minutes, and mixed by inversion several times in the meantime. Centrifuge at 12,000 rpm for 1min, aspirate the supernatant, leave the leukocyte pellet, add 200. mu.l of buffer GA, and shake until thoroughly mixed. 2) Add 20. mu.l proteinase K solution and mix well. 3) Add 200. mu.l buffer GB, mix well by inversion, stand at 70 ℃ for 10 minutes, clear the solution, centrifuge briefly to remove beads on the inner wall of the tube cap. 4) Add 200. mu.l of absolute ethanol, mix well with shaking for 15 seconds, at which time a flocculent precipitate may appear, and centrifuge briefly to remove water droplets on the inner wall of the tube cover. 5) Adding the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3 (the adsorption column is put into a collecting pipe), centrifuging at 12,000 rpm for 30 s, pouring off waste liquid, and putting the adsorption column CB3 back into the collecting pipe. 6) Add 500. mu.l buffer GD (check whether absolute ethanol has been added before use) to adsorption column CB3, centrifuge at 12,000 rpm for 30 seconds, dump the waste and place adsorption column CB3 in the collection tube. 7) To the adsorption column CB3, 700. mu.l of a rinsing solution PW (previously used, whether or not absolute ethyl alcohol has been added) was added, and the mixture was centrifuged at 12,000 rpm for 30 seconds, and the waste liquid was discarded, and the adsorption column CB3 was put into a collection tube. 8) To the adsorption column CB3, 500. mu.l of a rinsing solution PW was added, and the mixture was centrifuged at 12,000 rpm for 30 seconds, and then the waste liquid was discarded. 9) The adsorption column CB3 was returned to the collection tube, centrifuged at 12,000 rpm for 2 minutes, and the waste liquid was discarded. The adsorption column CB3 was left at room temperature for several minutes to completely dry the residual rinse solution in the adsorption material. 10) Transferring the adsorption column CB3 into a clean centrifuge tube, suspending and dripping 100 mu l of elution buffer TE into the middle part of the adsorption membrane, standing for 2-5 minutes at room temperature, centrifuging for 2 minutes at 12,000 rpm, and collecting the solution into the centrifuge tube.
(3) Reagent preparation: preparing X mu l of PCR reaction solution of a detection system according to 1 part of a person:
20. mu.l of reaction solution X81
(3) Sample adding: adding 20 mul of reaction solution into the 96-well plate coated with the primers and the blank micropores, and sealing by a sealing film.
(4) Amplification: the detection is carried out on a conventional PCR instrument, and available instruments include ABI veriti (Applied Biosystems, USA) and the like. The reaction conditions were as follows:
the preparation method of the PCR amplification system reagent comprises the following steps:
(5) electrophoresis: electrophoresis on 1.5% agarose gel at 110V for 35min, and observation on a gel imaging system.
As shown in FIG. 3, the electropherogram of the product obtained after 1 blood sample was amplified with the primers. Analysis of an electrophoretogram shows that the invention has effective amplification, clear bands, good specificity and correct product size.
(6) Sanger sequencing:
take 9. mu.l of PCR product and 2. mu.l of purification system. Purification was performed according to the following procedure:
mu.l of the purified product was mixed with the upper and lower sequencing primers, respectively, according to the following system:
sequencing reaction program:
and (3) a precipitation link:
adding 2 mu l of 125mmol EDTA into the product after the sequencing reaction, and standing for 5 min; adding 15 mu L of absolute ethyl alcohol, and mixing uniformly by vortex; centrifuging at 3700rpm for 30 min; inverting, centrifuging for 15sec, adding 50ml 70% ethanol, and mixing by vortex; centrifuging at 3700rpm for 15 min; inverting and centrifuging for 15sec, and placing on a metal bath at 95 ℃; after addition of 10. mu.l CBL, denaturation was carried out for 5min and finally sequencing was carried out on a sequencer (ABI3730) at-20 ℃ for 2 min.
(7) And (5) judging a result: the sequencing results were aligned with KAL1(NG _007088.2), FGFR 1(NG _007729.1), PROKR2(NG _008132.2), PROK2(NG _008275.1), CHD7 (NG _007009.1) and FGF8(NG _007151.1) negative reference sequences, respectively, and the results were reported according to the actual mutation status.
Example 3
The 7 primer pairs of PROKR2 and PROK2 were coated in eight tubes as described in example 2 and a blank was set. The coated eight-connected tubes are randomly divided into two groups, one group is stored at normal temperature, the other group is stored at 4 ℃, the electrophoresis results of normal temperature storage and 4 ℃ storage are not different after detection is carried out for 0, 7, 14, 21 and 28 days according to the method in the embodiment 2, and the stability and the specificity of the primers can be ensured after normal temperature storage. The electrophoresis results are shown in FIG. 2.
Example 4
5 clinical peripheral blood samples were taken, and the genome was extracted, reagents were prepared and tested as described in example 2. The PCR product can be amplified by the sample, the electrophoresis band is clear, the specificity is good, and the size of the product is correct. After sequencing of sample 1, two synonymous mutations of PROKR2 gene are found, namely c.465C > CT; p.L155L and c.585G > C; p.T195T. No mutations were found in any of the other gene exons. The result of agarose gel electrophoresis of sample 1 is shown in FIG. 3; the mutation sequencing results are shown in FIG. 4.
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gtgaagggac gaagtgtagc c 21
<210> 25
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
tcagcagcct taatgggtaa t 21
<210> 26
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
caagttgaca gcaccaaatg a 21
<210> 27
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
tgcttggtga aaagtggaat ag 22
<210> 28
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
gaccaggtct aggattctac catct 25
<210> 29
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
taaatctggt ccagcctgtg a 21
<210> 30
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
cctccaactg gtattccctg a 21
<210> 31
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
ggcaggacat agatcaagca a 21
<210> 32
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
acccaaaggc tttagatgtt c 21
<210> 33
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
ttcctcctaa aggcgaaact g 21
<210> 34
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
ttagaccttc ccaagtcacc a 21
<210> 35
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
tagaatgggt acgcttagtg g 21
<210> 36
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
aacgggacta tcaagtcagc a 21
<210> 37
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
atgagtagga gtagaacaat gggtg 25
<210> 38
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
cctttatgat gatgacctgg aa 22
<210> 39
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
attatgcaac cttcatgcac tg 22
<210> 40
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
tttctggtgc ttcaatcgtc a 21
<210> 41
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
gagtttggtg ttctggttcc t 21
<210> 42
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
gcccctttat catagattcc acttt 25
<210> 43
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
aaggagcaag tatgttgtcg c 21
<210> 44
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
agaggtgcag tttagaagaa gg 22
<210> 45
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
ggtggggaga cagaaacatt a 21
<210> 46
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
acccttcatc ctcctcatcc a 21
<210> 47
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
agcatttgtt tagtctgcaa acctc 25
<210> 48
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
aggtgagggc aaataccagt g 21
<210> 49
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
agtgtctggc ataagtggag g 21
<210> 73
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 73
ccttcactgc actgtagagc ct 22
<210> 51
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
<210> 52
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
tctatcttac gtagtgagtc ctact 25
<210> 53
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
ttagaagcag tgaactgagg gg 22
<210> 54
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
gcacaaacag catggtggag a 21
<210> 55
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
cataaacccc atatttccct g 21
<210> 56
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
gtccctcagt tgaccctcca a 21
<210> 57
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
cttggcagga tgatggatga a 21
<210> 58
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
caagcacgaa ggacaaatac tg 22
<210> 59
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
taaccttgct gatacctccc c 21
<210> 60
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
ggtagacgcc aagagtcctt t 21
<210> 61
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
gctgtgattt tgccagtgat g 21
<210> 62
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
tgaaccctgc caatagatgt g 21
<210> 63
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 63
ttaaaagtag cactgggcag at 22
<210> 64
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 64
ttctgggacc ttacctgtgg c 21
<210> 65
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 65
ttactctttc ctacccaccc c 21
<210> 66
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 66
aagcaggaca gtccctacca c 21
<210> 67
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 67
aaaatgaggg cactgagatg c 21
<210> 68
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 68
gttggagcga gcctttcttt g 21
<210> 69
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 69
tcaaaggtag tgaccaccaa a 21
<210> 70
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 70
tcggctcaat tatggaggag a 21
<210> 71
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 71
caagccagat gatggtaggt a 21
<210> 72
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 72
gtgcatccaa gaaggataac tc 22
<210> 73
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 73
gtcagtcgga cggattatca c 21
<210> 74
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 74
agaaagcaac gcatctcaca a 21
<210> 96
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 96
gacaacagtg cccaatacca t 21
<210> 76
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 76
gggtaagtaa cctaaaagcc acat 24
<210> 77
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 77
aggaccttct gccagagcaa t 21
<210> 78
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 78
gcaaactcag ttaaggctta gag 23
<210> 79
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 79
gttttggctc actgcaactc t 21
<210> 80
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 80
gctttcatac aatgctgctg a 21
<210> 81
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 81
ccagcctcat tttctgactt t 21
<210> 82
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 82
gggatttcta tgttgtaagg ga 22
<210> 83
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 83
<210> 84
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 84
gggctaagaa atcgcatagt g 21
<210> 85
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 85
agaatcctgt ctgcccgaat g 21
<210> 86
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 86
caggtgagct gttaagtagg ct 22
<210> 87
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 87
acattgagat caagttgtct tcgac 25
<210> 88
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 88
cttgactaga agcagtagtg gtgt 24
<210> 89
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 89
ggaaacacca ctactgcttc tag 23
<210> 90
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 90
ctgcagtctt atccaggctc t 21
<210> 91
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 91
cagcattggc aggacttcag a 21
<210> 92
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 92
gcactttctt ccattagtgc c 21
<210> 93
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 93
ttgaactttc cggctcagtc 20
<210> 94
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 94
<210> 95
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 95
gtgtagcttt ctaatggatc a 21
<210> 96
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 96
<210> 97
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 97
<210> 98
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 98
tgaccccacg taagcatagt c 21
<210> 99
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 99
tcagacttca tgtgtcttta atgga 25
<210> 100
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 100
<210> 101
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 101
gttcttcctc aacttttact tca 23
<210> 102
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 102
cagacactac ctccaggatg a 21
<210> 103
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 103
gatccaacta acatgtcgga at 22
<210> 104
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 104
<210> 105
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 105
<210> 106
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 106
ccctctgtgg gaataacaat c 21
<210> 107
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 107
ttgcaatgaa gatgagagac g 21
<210> 108
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 108
<210> 109
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 109
<210> 110
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 110
tggcttgaca tttacttctt caaa 24
<210> 111
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 111
<210> 112
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 112
cttccatcaa gtcattactc c 21
<210> 113
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 113
atccttgttg gatggaatat g 21
<210> 114
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 114
<210> 115
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 115
acaccttctc cagtcgccta 20
<210> 116
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 116
agcagtagat accaatgaca ca 22
<210> 117
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 117
cggtgagcat gctcttttat g 21
<210> 118
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 118
tttctctatg tccacaagac ctg 23
<210> 119
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 119
<210> 120
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 120
tctggaagtg tgcatgtctc 20
<210> 121
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 121
<210> 122
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 122
<210> 123
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 123
<210> 124
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 124
tcaccttcct ctgaaactgg c 21
<210> 125
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 125
cgtgttcatc tggaactgc 19
<210> 126
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 126
aatcaggact tcctaactcg g 21
<210> 127
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 127
<210> 128
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 128
<210> 129
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 129
aggtttacaa cccatcactg g 21
<210> 130
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 130
<210> 131
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 131
aagaagatgt cagggagcat t 21
<210> 132
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 132
<210> 133
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 133
<210> 134
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 134
<210> 139
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 139
<210> 135
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 135
<210> 136
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 136
aacaagtttg gatgaagtgg g 21
<210> 137
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 137
<210> 138
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 138
<210> 140
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 140
<210> 141
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 141
<210> 142
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 142
<210> 143
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 143
<210> 144
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 144
<210> 145
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 145
actgaccctg aaagagcaga aggt 24
<210> 146
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 146
agcctgtcag agcctaaatg agga 24
<210> 147
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 147
<210> 148
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 148
<210> 149
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 149
<210> 150
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 150
acacagatgt catttcccat gc 22
<210> 151
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 151
<210> 152
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 152
<210> 153
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 153
<210> 154
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 154
<210> 155
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 155
gtatcttgct ccgccagttc 20
<210> 156
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 156
aggacagacc caactctatg g 21
<210> 157
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 157
<210> 158
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 158
ttgaggaagc aagagcattt c 21
<210> 159
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 159
gcgagttgtg agggattaga ga 22
<210> 160
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 160
<210> 161
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 161
<210> 162
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 162
Claims (5)
1. A primer for detecting whole exon sequencing of a Kalman Syndrome (KS) related gene is characterized by comprising: primers for amplifying all exons of KAL1, FGFR1, PROKR2, PROK2, CHD7 and FGF8 have the base sequences shown in Table 1.
2. The primer of claim 1, wherein the upstream primer further comprises a M13-F sequence at the 5 'end and the downstream primer further comprises a M13-R sequence at the 5' end of table 1.
3. The primer of claim 2, further comprising a sequencing primer having a base sequence of
M13-F:TGTAAAACGACGGCCAGT
M13-R:AACAGCTATGACCATG。
4. A method for detecting sequencing of all exons of a Kalman Syndrome (KS) related gene KAL1, FGFR1, PROKR2, PROK2, CHD7 and FGF8, comprising the steps of:
(1) coating the upstream and downstream primers in the micropores of a 96-well plate, and storing at normal temperature or 4 ℃ for later use;
(2) extracting the genome DNA of a sample to be detected;
(3) preparing PCR reaction liquid containing the genomic DNA extracted in the step (2), and adding the PCR reaction liquid into a 96-well plate for PCR amplification;
(4) sequencing the amplification product in step (3);
(5) judging the sequencing result, and determining the mutation condition of each gene;
wherein the base sequence of the amplification primer in the PCR amplification of step (2) comprises the primer of claim 2;
wherein the sequencing primer base sequence in the sequencing in the step (4) is
M13-F:TGTAAAACGACGGCCAGT
M13-R:AACAGCTATGACCATG。
5. The method of claim 3, wherein the method for coating a 96-well plate comprises adding upstream and downstream primers for sequencing all exons of KAL1, FGFR1, PROKR2, PROK2, CHD7 and FGF8 genes into micropores of the 96-well plate, setting one hole as a blank, drying at 37 ℃ for 1h, sealing with a sealing film, and storing at normal temperature or 4 ℃ for later use.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110609499.6A CN113512580A (en) | 2021-06-01 | 2021-06-01 | Primer and method for detecting all exons of Kalman Syndrome (KS) related gene |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110609499.6A CN113512580A (en) | 2021-06-01 | 2021-06-01 | Primer and method for detecting all exons of Kalman Syndrome (KS) related gene |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113512580A true CN113512580A (en) | 2021-10-19 |
Family
ID=78065279
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110609499.6A Pending CN113512580A (en) | 2021-06-01 | 2021-06-01 | Primer and method for detecting all exons of Kalman Syndrome (KS) related gene |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113512580A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107058531A (en) * | 2017-04-01 | 2017-08-18 | 杭州艾迪康医学检验中心有限公司 | A kind of kit and method for detecting Niemann-Pick disease SMPD1 gene mutations |
| CN107841554A (en) * | 2017-11-24 | 2018-03-27 | 南京艾迪康医学检验所有限公司 | Detect the primer, kit and method of Hageman factor (F12) gene mutation |
-
2021
- 2021-06-01 CN CN202110609499.6A patent/CN113512580A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107058531A (en) * | 2017-04-01 | 2017-08-18 | 杭州艾迪康医学检验中心有限公司 | A kind of kit and method for detecting Niemann-Pick disease SMPD1 gene mutations |
| CN107841554A (en) * | 2017-11-24 | 2018-03-27 | 南京艾迪康医学检验所有限公司 | Detect the primer, kit and method of Hageman factor (F12) gene mutation |
Non-Patent Citations (2)
| Title |
|---|
| J.-P. HARDELIN等: "The Complex Genetics of Kallmann Syndrome: KAL1, FGFR1, FGF8, PROKR2, PROK2, et al.", SEX DEV, no. 2, pages 181 * |
| JA HYE KIM等: "Targeted Gene Panel Sequencing for Molecular Diagnosis of Kallmann Syndrome and Normosmic Idiopathic Hypogonadotropic Hypogonadism", BIBLIOGRAPHY, pages 1 - 12 * |
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