CN114875148A

CN114875148A - Familial multiple lipoma detection kit and application of primer group

Info

Publication number: CN114875148A
Application number: CN202210532328.2A
Authority: CN
Inventors: 贺曦; 刘彦伶; 熊寅
Original assignee: Xiangya Hospital of Central South University
Current assignee: Xiangya Hospital of Central South University
Priority date: 2022-05-11
Filing date: 2022-05-11
Publication date: 2022-08-09

Abstract

The invention relates to the field of biotechnology and medicine, and particularly discloses a familial multiple lipoma detection kit, which comprises a forward amplification primer shown as SEQ ID No.33, a reverse amplification primer shown as SEQ ID No.34 and a sequencing primer shown as SEQ ID No. 126; the detection kit is used for detecting a pathogenic gene of familial multiple lipoma, the pathogenic gene is a mutant ABCA13 gene, the difference between the mutant ABCA13 gene and a wild ABCA13 gene comprises 1 mutation, the mutation is that the 2908 th base C of the exon 17 in the coding region of the wild ABCA13 gene is mutated into T, and the nucleotide sequence of the mutant ABCA13 gene is shown as SEQ ID NO. 139. The kit has the characteristics of convenient detection, high sensitivity and accurate result. The invention also discloses application of the primer group in a familial multiple lipoma disease causing gene detection product, and the detection product can be used for detecting base mutation of a wild ABCA13 gene at any site.

Description

Familial multiple lipoma detection kit and application of primer group

Technical Field

The invention relates to the field of biotechnology and medicine, in particular to a familial multiple lipoma detection kit and application of a primer group.

Background

Familial Multiple Lipomatosis (FML) is a rare lipomatous disease of subcutaneous fat (RAD) (OMIM 151900) with an estimated prevalence of 1/50,000, characterized by well-enveloped subcutaneous nodules in the limbs and trunk, soft texture, consisting mainly of mature adipose tissue, enveloped by connective tissue envelopes, of varying sizes, small pea, large fist, or even larger, occurring at any age, unequal household penetrance, and most common in young, old, or obese. Lipomas are generally not painful or tender, but during growth they may cause a slight itching or burning sensation when formed. If lipomas occur in stressed areas, such as the back of the legs, tenderness is caused. The blood fat of patients is generally normal, the deposition of fat is generally asymptomatic and asymmetric, the large number of focuses of some patients can influence the beauty and daily life, lipoma near joints and limbs can cause dysfunction of the joints, the limbs and the like sometimes, and the phenomena that the sensitivity of insulin is lower than a normal level and the metabolic clearance of the insulin is lower than a normal level appear in some patients in FML family. At present, the cause of familial multiple lipomatosis is not clear, the treatment mode is limited, the surgical operation mode is mainly used for removing, but scars are generated after the removal, and the recurrence risk exists. Therefore, it is important to explore the pathogenesis of FML and provide new possibilities for its treatment.

Familial multiple lipomatosis is usually inherited in an autosomal dominant manner. However, the genetic mechanism of FML is rarely studied, and only two pathogenic genes, PALB2 and HMGA2, are reported at present. Both genes were identified in the caucasian population and there were no reports of association of these genes with FML in the east asian population. Therefore, we need to locate more FML pathogenic genes, so as to reveal the pathogenic mechanism of FML, and provide guidance for the prevention, treatment and diagnosis of FML, and prenatal diagnosis.

The existing detection products for the familial multiple lipomatosis have the problems of long detection period, low detection accuracy, more primer sequencing times, more sequencing times and the like in the detection process, and need to be improved urgently.

Disclosure of Invention

The invention aims to provide a familial multiple lipoma detection kit, which is used for detecting a mutant pathogenic gene shown as SEQ ID No.139 and has the characteristics of convenience in detection, high sensitivity, accurate result and more economy.

The second technical problem solved by the invention is to provide the application of the primer group in the detection product of the familial multiple lipoma pathogenic gene, the primer group can be used for the base mutation of the wild ABCA13 gene at any site, and the primer group has the characteristics of convenient detection, high sensitivity, accurate result and more economy.

The technical problem solved by the invention is realized by adopting the following technical scheme:

a familial multiple lipoma detection kit comprises a forward amplification primer shown as SEQ ID No.33, a reverse amplification primer shown as SEQ ID No.34 and a sequencing primer shown as SEQ ID No. 126.

Further, the detection kit is used for detecting a pathogenic gene of familial multiple lipoma, the pathogenic gene is a mutant ABCA13 gene, the difference between the mutant ABCA13 gene and a wild ABCA13 gene comprises 1 mutation, the 2908 th base C of the 17 th exon in the coding region of the wild ABCA13 gene is mutated into T, and the nucleotide sequence of the mutant ABCA13 gene is shown as SEQ ID NO. 139.

Further, the detection kit also comprises a DNA amplification enzyme, a buffer solution and a dNTP mixture for PCR, wherein the DNA amplification enzyme is Taq DNA polymerase, and the concentration of the Taq DNA polymerase is 1U/muL; the buffer solution is MgCl2 solution, and the concentration of the MgCl2 solution is 10 mM; the concentration of the dNTP mixture is 10 mM.

Furthermore, in the detection kit, the volume ratio of the primer group, the DNA amplification enzyme, the buffer solution and the water is 2:1:2: 4.

Further, the volume ratio of the added DNA template to the buffer was 1:1, and the concentration of the DNA template was 50 ng/. mu.L at the time of detection.

An application of a primer group in a detection product of a familial multiple lipoma pathogenic gene,

the primer group comprises 62 primer pairs and 14 sequencing primers;

the forward amplification primer of the primer pair is shown as SEQ ID No. (2N-1), the reverse amplification primer is shown as SEQ ID No.2N, and N is 1-62;

the sequencing primer is shown as SEQ ID No. M, wherein M is 125-138.

Further, the detection product is used for detecting a pathogenic gene of the familial multiple lipoma, the pathogenic gene is a base mutation of a wild-type ABCA13 gene at any position, and the nucleotide sequence of the wild-type ABCA13 gene is shown as SEQ ID No. 140.

One of the important points of the invention lies in the design of primers in the detection kit, the primers contained in the detection kit do not contain a plurality of repetitive sequences, and in consideration of the production cost, the single sequencing can accurately read the given mutation sites, and simultaneously, the requirement of the single-direction sequencing which can be used for Sanger sequencing is met.

The second important point of the invention is the application of a primer group, the design of the primers in the primer group needs to meet a plurality of conditions, for example, the primers need to be capable of carrying out specific amplification, the primers can not contain a plurality of repetitive sequences, the length of the primers is not more than 22bp, and the designed primers can meet the requirement of being used for one-way sequencing in Sanger sequencing; when the product exceeds 800bp, a sequencing primer needs to be designed for segmented sequencing, and the segmented sequencing primer must be capable of being completely overlapped to ensure the reading of the clipping sequence. Traditional commercial company ampliseq design software is used for designing exome sequencing primers, the product size of the exome sequencing primers is within 250bp, a complete sequence is obtained through multiple sequencing and splicing, for identification of a certain point, multiple sequencing is needed, the cost is high, the repetition frequency is higher, and the error probability is higher. The invention protects the primer group, and can read the locus by single sequencing. For example, primers ABCA13-B12 and ABCA13-B13 are essentially the same pair of primers, which covers two exons, and according to the conventional method, two pairs of primers are needed for reading, while in the present invention, one pair of primers is used to control the product size to be within 800bp, and reading can be accomplished by one-way sequencing. For example, primer ABCA13-B17, requires amplification of exon 17, comprising bases more than 5000bp in length. From the perspective of cost, no matter the size of PCR product, the required enzyme is consistent with the cost of BUFFER, more than 20 amplifications are required according to traditional commercial company ampliseq design software, while only one amplification is required by adopting the primer of the invention, so that the method is more economical; the sequence is longer, the whole exon sequence can be read by 8 sequencing primers, and compared with the primers designed by the traditional method, the method has the advantages of less amplification times, less sequencing times and more economy.

Has the advantages that:

1) the detection kit is applied to PCR in-vitro amplification, can be used for pertinently detecting the 2908 th base pathogenic gene of the 17 th exon on the ABCA13 gene by designing a PCR amplification primer and combining Sanger sequencing, and is convenient and quick to detect, accurate in result, less in primer amplification times and sequencing times in the detection process and more economical.

2) The invention also relates to application of a primer group for detecting familial multiple lipoma, wherein the primer group is designed to cover the UTR region, the coding region and the shearing site region, and can be applied to a detection kit or a detection reagent to detect variation of any sites of all UTR regions and coding regions of the ABCA13 gene by considering the influence of a non-coding region on gene transcription and translation, so that the detection range of the familial multiple lipoma is wider, the accuracy is high, the amplification times of the primer in the detection process are less, the sequencing times are less, and the method is more economical.

3) Through intensive research, the invention firstly identifies a new pathogenic gene ABCA13(NM _152701.5) in an autosomal dominant hereditary familial multiple lipoma family, the pathogenic gene is positioned on 7p12.2 of a No. 7 chromosome, and the heterozygous mutation of 2908 th base C of 17 th exon of a wild ABCA13 gene into T is the pathogenic mutation of the family. Based on the discovery of the pathogenic gene, whether a subject carries the mutated ABCA13 gene or not can be screened at an early stage, a bearing guide for good prenatal and postnatal care can be provided, and a molecular diagnosis basis can be provided for the familial multiple lipoma. The novel familial mutant ABCA13 gene encodes ATP binding cassette protein superfamily protein 13, possibly participates in the generation of diseases by participating in cholesterol metabolism, thus providing a new idea for the exploration of subsequent familial multiple lipoma disease mechanisms and the research and development of familial multiple lipoma treatment medicines, and providing a possible medicine target for the subsequent familial multiple lipoma treatment.

Drawings

FIG. 1 is a diagram of FML family.

FIG. 2 is a view of a lipoma in the abdomen of patient II-16.

FIG. 3 is a diagram of a lipoma in the arm of patient III-17.

FIG. 4 is a graph showing the results of linkage analysis (schematic diagram of linkage analysis of chromosomes 1 to 22, in which the LOD value of 0 is used as a threshold value, and the intervals of 0 or more are considered to be linkage, and the intervals of 0 or less are considered to be non-linkage).

FIG. 5 is a graph showing the results of Sanger sequencing in normal humans (II-17, III-1, III-21).

FIG. 6 is a graph showing the results of Sanger sequencing of patients (II-7, II-9, II-14, II-16, III-3, III-17).

Detailed Description

In order to make the technical means, the creation features, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.

It is well known to those skilled in the art that pathogenic genes have many uses. Thus, uses of the mutated ABCA13 gene provided by the present invention include, but are not limited to: as a drug target for the treatment of familial steatoma multiplex; preparing a diagnostic kit of familial multiple lipoma; for generating animal models of disease; provides a new drug action way for treating familial multiple lipoma.

The "kit" described in the present invention refers to a primer provided by the kit for the ABCA13 gene, and the primers provided by the kit are used to react with an enzyme according to the PCR principle, and after Sanger sequencing is assisted, the presence or absence of mutations in the coding region, UTR region and cleavage site of the ABCA13 gene in a biological sample can be detected, so that the presence or absence of the gene mutation site of the present invention in the sample can be detected by using the kit.

The nucleotide sequence of the mutated ABCA13 gene is shown in SEQ ID NO. 139.

The nucleotide sequence of the normal wild-type ABCA13 gene is shown in SEQ ID NO. 140.

Example 1

The mutated ABCA13 gene was identified as follows:

1) collection of study subjects. A four-generation familial multiple lipoma pedigree was collected, 9 of which were collected, 6 patients, 3 normal patients, as shown in FIG. 1, and all exhibited typical multiple lipoma characteristics (as shown in FIGS. 2 and 3, wherein FIG. 2 is two lipomas in the abdomen of patients II-16; FIG. 3 is a lipoma in the arm of proband III-17). After informed consent is obtained, 5mL of venous blood is collected from each person, DNA extraction is carried out by using a DNA extraction kit, and the DNA is diluted to 50 ng/mu L after the concentration of the DNA is measured by a spectrophotometer and is used for subsequent PCR reaction, DNA chip sequencing and exon sequencing.

As shown in fig. 1, the black outer circle and black outer box refer to family members from which the samples were collected; black outer box refers to family members for exome sequencing; +/-refers to heterozygous variant patients carrying the mutation from C2908 to T in the ABCA13 coding region, and +/-refers to normal individuals not carrying the mutation.

2) Exome sequencing. Selecting patients II-16 and III-17 in the pedigree, and normal people II-17 and III-21 in the pedigree to perform exon sequencing. The exon sequencing procedure is briefly described as follows: the genome DNA is randomly ultrasonically crushed into fragments of about 250bp, a sequencing joint is connected, then a DNA sequencing chip is used for capturing and carrying out high-throughput sequencing, and a sequencing instrument is an illumina3000 platform.

3) And (4) screening candidate genes. And (4) performing quality control on the sequencing off-line data, and performing analysis and screening by annotation. The screening criteria were as follows: a. selecting sites with a minimum allele frequency of less than or equal to 0.1%; b. screening out nonsynonymous variation, frame shift, insertion deletion and possible shearing site variation; c. considering that the family is autosomal dominant inheritance, screening heterozygote variation; d. the mutation sites shared by the two patients but not found in the normal were selected. Under this strategy, a total of 179 possible sites were screened, containing over 150 genes.

4) DNA chip sequencing and linkage analysis. All collected samples in the pedigree were selected for DNA chip sequencing. Sequencing is carried out by using an Illumina ASA 750K chip, data are obtained after sequencing, and linkage analysis is carried out by using MERLIN. With LOD value 0 as a threshold, a section above 0 is considered to be linked, and a section below 0 is considered to be unlinked. The final results showed linkage only on

chromosome

1, 3, 6, 7, 10, 17, 19, 20 chromosomal intervals, as shown in fig. 4.

5) And (5) locating pathogenic genes. Combining the data of linkage analysis and the data of exon sequencing candidate genes, the overlapped part of the two has only heterozygous mutation of the 2908 th base C of the ABCA13 gene into T.

6) Designing a primer: variant gene standard sequences of the candidate genes were derived from UCSC GENEME BROSWER (https:// GENOME. UCSC. edu /), numbered NM-024917.6, primers were designed and synthesized using the online Primer design software Primer 3.0(v.0.4.0) (https:// biolnfo. ut. ee/Primer3-0.4.0/), and Primer specificity was predicted online using BLAT (https:// GENOME. UCSC. edu/cgi-bin/hgBlat).

According to the method, the primer group synthesized by the invention and used for the pedigree co-separation comprises 62 primer pairs and 14 sequencing primers; the primers in the primer set can be applied to a detection kit or other detection products in whole or in part.

the sequencing primer is shown as SEQ ID No. M, wherein M is 125-138

The N sets of primer pairs and M sequencing primers are described in Table 1.

TABLE 1

In the practical application process, considering the influence of a non-coding region on gene transcription and translation, a primer is designed to cover a UTR region, a coding region and a shearing site region. Thus, the mutation site existing in any other position of ABCA13 gene can be detected.

Example 2

A detection kit for a familial multiple lipoma pathogenic gene is disclosed, wherein the volume ratio of a primer, DNA amplification enzyme, buffer solution and water is 2:1:2:4, wherein the DNA amplification enzyme is Taq DNA polymerase, and the concentration of the Taq DNA polymerase is 1U/muL; the buffer solution is MgCl2 solution, and the concentration of the MgCl2 solution is 10 mM; the concentration of the dNTP mixture is 10 mM. During detection, the volume ratio of the added DNA template to the buffer solution is 1:1, and the concentration of the DNA template is 50 ng/. mu.L.

The primers comprise a forward amplification primer shown as SEQ ID No.33, a reverse amplification primer shown as SEQ ID No.34 and a sequencing primer shown as SEQ ID No. 126.

The application of the detection kit in detection of familial multiple lipoma pathogenic genes. The following operations were carried out using normal persons (II-17, III-1, III-2) and patients (II-7, II-9, II-14, II-16, III-3, III-17) as subjects, respectively:

1) 5mL of peripheral venous blood of a subject was collected, extracted using a DNA extraction kit, and the DNA was diluted to 50 ng/. mu.L after the concentration was measured with a spectrophotometer.

2) The PCR reaction conditions are as follows: pre-denaturation at 98 ℃ for 2 min; 40 amplification cycles of 98 ℃ for 10 seconds, 59 ℃ for 30 seconds, and 72 ℃ for 1 minute; storing at 4 ℃. The reaction was performed in 10. mu.L total, and 4. mu.L of PCR water, 1. mu.L of forward primer (ABCA13-B17F), 1. mu.L of reverse primer (ABCA13-B17R), 2. mu.L of buffer, 1. mu.L of DNA template, and 1. mu.L of DNA polymerase were added in this order.

And after PCR is finished by using the detection kit, carrying out agarose gel electrophoresis to determine whether a band exists, and sending a corresponding sequencing primer to a sequencing company for Sanger sequencing. Sequencing primer 2 using B17: GGACAAGGATTCAGCTTTAC to obtain AB-I file.

The obtained ABI files were aligned to the standard sequences using the Seqman software to identify the mutation sites, and Sanger sequencing charts are shown in fig. 5 and 6.

FIG. 5 is a graph showing the results of Sanger sequencing in normal humans (II-17, III-1, III-21), and FIG. 6 is a graph showing the results of Sanger sequencing in patients (II-7, II-9, II-14, II-16, III-3, III-17), showing that the patient has heterozygous variation from C to T in the gene coding region 2908 in ABCA13 gene.

Example 3

The invention relates to application of a familial multiple lipoma detection primer group.

The kit is applied to detection of familial multiple lipoma pathogenic genes, wherein the volume ratio of a primer group, DNA amplification enzyme, buffer solution and water is 2:1:2:4, wherein the DNA amplification enzyme is Taq DNA polymerase, and the concentration of the Taq DNA polymerase is 1U/muL; the buffer solution is MgCl2 solution, and the concentration of the MgCl2 solution is 10 mM; the concentration of the dNTP mixture is 10 mM. During detection, the volume ratio of the added DNA template to the buffer solution is 1:1, and the concentration of the DNA template is 50 ng/. mu.L.

The primer set includes 62 sets of primer pairs and 14 sequencing primers shown in table 1.

The application of the detection kit in detection of familial multiple lipoma pathogenic genes. The detection kit can comprise at least one primer pair shown in the primer set, or can comprise all the primer pairs, and the sequencing primer shown in the above is arranged at the corresponding site.

In principle, any site of ABCA13 gene may generate mutation to cause familial multiple lipoma disease-causing gene, so for the subject whose mutation site is unknown, PCR reaction is performed by using different primer pairs. After the PCR is completed, agarose gel electrophoresis is carried out to determine whether a band exists, and corresponding sequencing primers are sent to a sequencing company for Sanger sequencing to obtain an AB-I file.

The obtained AB-I file is compared with a standard sequence by utilizing Seqman software, and all mutation sites on the ABCA13 gene of a subject can be detected.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

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gcattggtcc taggatggtg 20

<210> 61

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 61

gctcctgcaa cttttcctgt 20

<210> 62

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 62

gcacagccct tgtgaatgtt 20

<210> 63

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 63

tggccctcct acctaagtca 20

<210> 64

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 64

gcaaaacaga gaggctcagg 20

<210> 65

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 65

ctgaatttgc aagcaagcag 20

<210> 66

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 66

aaaaccctgc agtttcaacg 20

<210> 67

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 67

tggcttcgct ccttgaatta 20

<210> 68

<211> 23

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 68

gcaaatatcc aaacctcatt ctg 23

<210> 69

<211> 21

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 69

tggccagtga tattgacctt c 21

<210> 70

<211> 21

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 70

ttccctttct ggatcttcct t 21

<210> 71

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 71

agttagggct ccactgcaaa 20

<210> 72

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 72

tcatggcata ctttcccaca 20

<210> 73

<211> 21

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 73

ggaacacatc aacaagcacc t 21

<210> 74

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 74

gcctttcacc cgtgtctcta 20

<210> 75

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 75

aaccaggtca gcagaaggtg 20

<210> 76

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 76

aattcaatgc cttcccacag 20

<210> 77

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 77

tcacgtgtgt gtagcatcca 20

<210> 78

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 78

tgatctggga tgcctcttct 20

<210> 79

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 79

ccctggatct accatcctga 20

<210> 80

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 80

cagaaggcct ccactgagac 20

<210> 81

<211> 22

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 81

cgagatgtca ttgagatgcc ta 22

<210> 82

<211> 21

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 82

cctgtcaggc ctgttaagtt c 21

<210> 83

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 83

tcacaaccgg attaggcaat 20

<210> 84

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 84

gggatgttga ataaggctgg 20

<210> 85

<211> 19

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 85

cttccgcact ttaggtgca 19

<210> 86

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 86

cagcctgaga ggagcaagag 20

<210> 87

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 87

gcagctggtt ctccattctc 20

<210> 88

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 88

tacccataag gcccattcag 20

<210> 89

<211> 21

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 89

tgtaagagct ctgccttctc g 21

<210> 90

<211> 21

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 90

ttcccagaaa cagctaatcc a 21

<210> 91

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 91

tcaggttcgt caaatgcaag 20

<210> 92

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 92

tccatggcat ctatgtggaa 20

<210> 93

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 93

gctgtacctg gtggggttag 20

<210> 94

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 94

tccttctgaa ccctgtgctc 20

<210> 95

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 95

ttgaatcctg cactcaggtg 20

<210> 96

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 96

acggattggt tgccaagtag 20

<210> 97

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 97

gggaatctgc gtagcctgta 20

<210> 98

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 98

agaaatgccc aagtcctgtg 20

<210> 99

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 99

gtctcaaaag gtggctgagg 20

<210> 100

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 100

agatgacccc tttttgtgga 20

<210> 101

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 101

tccacggcta cagaactcct 20

<210> 102

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 102

gtcccttgca gaagcaacat 20

<210> 103

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 103

tgcattttca gggattcacc 20

<210> 104

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 104

gtggccgaaa cagaacattt 20

<210> 105

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 105

gggaagacct ggaacacaga 20

<210> 106

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 106

gggtgactga caccaatcaa 20

<210> 107

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 107

aatcaggatg gcagaactgg 20

<210> 108

<211> 21

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 108

tgaaggctat ttttggctct g 21

<210> 109

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 109

ccattcaagt gccatgatga 20

<210> 110

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 110

tttattccac gggactgagg 20

<210> 111

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 111

agaggcctat tttggggtga 20

<210> 112

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 112

gtgagagagc tgccttggag 20

<210> 113

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 113

gtgggaggta gaagcaggaa 20

<210> 114

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 114

atgctggctt atggcaaaac 20

<210> 115

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 115

tttctttcac ctggggtctg 20

<210> 116

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 116

tccctcatgg cttcagtctc 20

<210> 117

<211> 22

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 117

gcttttgatt tggttaccat ga 22

<210> 118

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 118

ttatcccctc agagccttca 20

<210> 119

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 119

tccctctgtg tgaaaatgga 20

<210> 120

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 120

tagggaggca gggcctatac 20

<210> 121

<211> 23

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 121

catcaataag cattcattgg aga 23

<210> 122

<211> 24

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 122

gaataaagtg gtgaattgaa gcaa 24

<210> 123

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 123

tggtttttag ggcaagttgg 20

<210> 124

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 124

tgggatgtgg tgttttagca 20

<210> 125

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 125

gttcccagtt tccacagcct 20

<210> 126

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 126

ggacaaggat tcagctttac 20

<210> 127

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 127

aaatgtggac tgaaatctgg 20

<210> 128

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 128

tgtaaatacc tcacagagga 20

<210> 129

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 129

aactgaaaac ttgttaaaca 20

<210> 130

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 130

aattgaatta gtatcagata 20

<210> 131

<211> 21

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 131

aaggacagta caattgattt c 21

<210> 132

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 132

gatcaataac ttagctggga 20

<210> 133

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 133

acttgctaat ctaacggatt 20

<210> 134

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 134

ttaacaatga gactcaaaca 20

<210> 135

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 135

aaccaccgga aagaatgtca 20

<210> 136

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 136

atctatttac tagatacatg 20

<210> 137

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 137

cacacatcgc cacacccggc 20

<210> 138

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 138

aattctaaca tagtctagtt 20

<210> 139

<211> 5442

<212> DNA

<213> Homo Sapiens

<400> 139

gagcactgga atttgccttc caggactggg agtgcccaca ccttgctgaa accctacagg 60

catggtttct aatgcactcc actatctgtg ctacctttac atgccaagac attttgaaat 120

ggaattaaat cctaagattc ttctggcata aatagcaaac ttatctttaa taatcataca 180

tattattgtt aaatatgcca ggggaaaata attgagaatg ctttcttttt ggtggtagga 240

aacattttaa gctaaatata tataacttca ttgtgcattt ctaaaacact ttcaaatcct 300

cacattagtt taatttctgt tggaaatcat gttgtctatt aaatgtttca tttgtgtttt 360

actatcaata aatgagtatt aacttggaaa tttgataaat caaatttatt tttttatttt 420

atactaaaat aattctatta atattacagg gctttaaatt tcacaaagca ccttctaatg 480

atggaaaaga agttgcacac ccttgaggat gaacaaatga actttctttt atcatttgtg 540

gaattttttg agaaattatt gttgcctaat ctttttgact cctccattgt tcccagtttc 600

cacagcctcc catctctcac agaggatatt ctgaatataa gttctctgtg gacaaatcat 660

ttaaaaagtt taaagagaga cccatctgcc actgatgctc agaaactctt ggaatttggc 720

aacgaagtga tttggaaaat gcagactctc ggaagtcact ggataaggaa ggaaccaaaa 780

aatcttttga gattcataga attaatactt tttgaaatta atcccaaatt actagaatta 840

tgggcctatg gcatttcaaa aggaaaaaga gctaaattgg aaaacttctt tacactttta 900

aatttttctg ttccagaaaa tgagattctg agtacaagtt ttaacttttc ccagttgttc 960

cattcagatt ggcctaaatc accagctatg aacatagatt ttgtacgttt aagtgaggct 1020

ataataacta gtctccatga atttggattt ttggagcagg aacagatctc agaagctctg 1080

aacacagtct acgctatcag gaatgcatct gatcttttct cagccctttc tgaaccacaa 1140

aaacaagaag ttgataaaat tttgactcac atacacctaa atgtcttcca ggacaaggat 1200

tcagctttac ttctgcaaat ttattcttca ttttactgat atatttatga attattgaat 1260

attcagagta gaggctcttc gttgactttc cttacacaaa tctcaaaaca cattttggat 1320

atcataaaac aatttaattt ccaaaacatc agtaaagcat ttgcattttt atttaagaca 1380

gcagaggttc ttgggggaat ttctaatgta tcttactgtc agcaattgct ttcaattttt 1440

aactttttgg agcttcaggc ccaatccttc atgtctacag agggccaaga actggaagtg 1500

atccacacta ctttgacagg cctcaaacag ctgctcataa ttgatgaaga ttttcgtatt 1560

tctttatttc aatatatgag ccaattcttc aacagttcag tagaagacct attggataat 1620

aaatgcttga tttcggacaa taaacacatt tcttccgtaa attattcaac aagtgaggag 1680

tcttcatttg tttttccatt ggcacaaatt ttttcaaacc tctcagcaaa tgtcagtgtg 1740

ttcaacaagt ttatgtccat tcactgtacc gtttcatggc ttcaaatgtg gactgaaatc 1800

tgggaaacca tatctcaatt atttaagttt gacatgaatg ttttcacatc tcttcatcat 1860

ggtttcactc agcttttgga tgaattggaa gatgatgtga aagtctctaa aagctgccag 1920

ggtatacttc ccacccataa tgttgctaga ctcatattaa atttgtttaa aaatgtaact 1980

caagccaatg acttccataa ttgggaggac ttcctggatc tcagggattt tttggtagct 2040

ttaggtaatg cattagtttc agtaaaaaaa cttaacttgg agcaagtgga gaaatccctt 2100

ttcaccatgg aagctgccct gcatcagttg aagacatttc cattcaacga aagtacaagc 2160

agagagtttt taaattctct gcttgaagtt ttcattgagt ttagcagtac ctcagaatat 2220

atagtcagaa atctagattc aataaatgac tttctttcaa ataatctcac aaattatgga 2280

gaaaaatttg aaaatatcat cactgagcta agagaagcaa tagtatttct tagaaatgta 2340

tcacatgatc gagatttgtt ttcctgtgct gatattttcc aaaatgttac tgagtgtatt 2400

ttagaagatg gctttttata tgtaaatacc tcacagagga tgttacgtat tctagacacg 2460

ttaaattcca cattttcctc tgagaacaca attagcagtc tgaaaggatg cattgtatgg 2520

ttagatgtca taaaccattt gtatttgttg tctaactcca gtttttcaca aggtcatctt 2580

caaaatattt tggggaattt cagagatata gaaaacaaaa tgaactctat attaaaaatt 2640

gtaacttggg tgttaaatat aaaaaaacct ctttgttcat caaatggctc acatataaat 2700

tgtgtcaata tttacttgaa agatgtaact gactttctaa atattgtact tactacagtc 2760

tttgaaaaag agaagaaacc taaatttgag attttattag ctcttttaaa tgattccaca 2820

aagcaagtaa ggatgagtat caacaactta acaacagact ttgattttgc atctcagtcc 2880

aattggagat attttactga attaattcta agaccaatag aaatgtcaga tgaaattcct 2940

aatcagtttc aaaatatttg gcttcattta ataacactgg ggaaggaatt tcagaagctt 3000

gtaaaaggta tttactttaa catcctggaa aataattcct cttctaaaac tgaaaacttg 3060

ttaaacatat ttgccaccag tccaaaagaa aaggatgtaa acagtgtagg caattccatt 3120

tatcacttag ctagttacct tgccttcagc ttatctcatg acctccaaaa ttcaccaaaa 3180

ataataattt cacctgaaat aatgaaagct acaggtcttg gtattcaact gataagggat 3240

gtgttcaact ccttaatgcc tgtagttcat cacactagtc cacaaaatgc aggttatatg 3300

caagctttga agaaggtaac ttctgtcatg cgtaccctta agaaggcaga catagacctt 3360

ttagtggatc agcttgaaca agttagtgta aacctaatgg atttctttaa gaatatcagt 3420

agtgtgggaa ctggcaattt agtggtcaat ttgcttgttg gcttgatgga aaaatttgca 3480

gacagctcac attcttggaa tgttaatcat ctgctgcagc tctcacgcct gtttcctaaa 3540

gatgttgtgg atgctgtgat agatgtgtac tatgtgcttc ctcatgctgt aaggctcctg 3600

cagggagtac ctggtaaaaa catcactgaa ggcctcaagg atgtctacag cttcacactc 3660

cttcatggca taaccatttc aaatatcacc aaggaagact tcgcaattgt gataaaaatt 3720

cttttggata caattgaatt agtatcagat aagccagata ttatttcaga ggctttagct 3780

tgttttcctg tggtttggtg ctggaatcac acaaattctg gatttcggca gaattcaaag 3840

atagacccct gcaatgtcca tgggctcatg tcttcttcct tttatggcaa agtggccagt 3900

atacttgatc atttccacct gtctccccaa ggtgaagatt caccatgttc aaatgaaagc 3960

tcccgaatgg aaataactag gaaagtggtc tgcataattc atgaattagt ggactggaat 4020

tctattcttc tggagctctc tgaagtcttc catgttaaca tttctcttgt gaaaactgtg 4080

cagaaatttt ggcataagat attaccgttt gtcccacctt caataaatca aactagggat 4140

agcatctctg aactctgtcc tagtggttcc ataaagcaag ttgctttgca aatcatagaa 4200

aaacttaaaa atgtcaactt tacaaaagtt acatcaggtg aaaatattct tgacaaacta 4260

agtagtttaa acaagatcct taacattaat gaagacacag agacatctgt tcaaaatatt 4320

atttcctcaa atttggaaag gacagtacaa ttgatttctg aagactggag cctagaaaaa 4380

agtacgcata atctactctc tttattcatg atgctccaga atgcaaatgt cacaggtagc 4440

agtttagaag cattatcaag ttttattgaa aaaagtgaaa caccttacaa ctttgaagaa 4500

ctatggccca agtttcaaca aatcatgaaa gacctaaccc aagattttag aatcagacac 4560

ctgctttctg aaatgaacaa aggaatcaaa agtataaatt caatggctct tcaaaagata 4620

actttgcagt ttgcccattt cctggaaatc ctggattcac cgtcattgaa gacattagaa 4680

attattgaag attttctatt ggtcacaaaa aactggcttc aggaatatgc aaatgaggat 4740

tactccagaa tgatagaaac attattcatt cctgtgacca atgagagttc aactgaagat 4800

atagctttgt tagccaaagc tattgctact ttttggggct ctttaaaaaa tatatctaga 4860

gcaggcaatt ttgatgttgc ctttcttacc catctgctaa atcaagaaca gctgactaat 4920

ttctcagttg ttcagctgct ttttgaaaac atcctaatta atttgatcaa taacttagct 4980

gggaattctc aggaagcagc ttggaactta aatgatactg accttcaaat aatgaatttc 5040

attaacctta tcttgaacca tatgcagtca gaaactagta ggaaaacagt tctctctctg 5100

agaagcatag tagatttcac agaacagttt ttgaaaacat tcttctccct ttttctaaag 5160

gaagattctg agaacaaaat atctcttctg ctgaaatatt tccacaaaga tgttattgca 5220

gagatgaggt gagtatactt ttgctttgtg tcatatatgc agttgcgata tatctcaaac 5280

tactttattt tctggagtat agacatgtca aaaacagtat ttgtatctaa tacctttgaa 5340

agatctttga tgtaaatttg tcttaataaa tattttatca aaatgtcaag gtaaaagaat 5400

tttgattact gtcatctagt aaaatataaa cccagcccag gg 5442

<210> 140

<211> 5442

<212> DNA

<213> Homo Sapiens

<400> 140

gagcactgga atttgccttc caggactggg agtgcccaca ccttgctgaa accctacagg 60

catggtttct aatgcactcc actatctgtg ctacctttac atgccaagac attttgaaat 120

ggaattaaat cctaagattc ttctggcata aatagcaaac ttatctttaa taatcataca 180

tattattgtt aaatatgcca ggggaaaata attgagaatg ctttcttttt ggtggtagga 240

aacattttaa gctaaatata tataacttca ttgtgcattt ctaaaacact ttcaaatcct 300

cacattagtt taatttctgt tggaaatcat gttgtctatt aaatgtttca tttgtgtttt 360

actatcaata aatgagtatt aacttggaaa tttgataaat caaatttatt tttttatttt 420

atactaaaat aattctatta atattacagg gctttaaatt tcacaaagca ccttctaatg 480

atggaaaaga agttgcacac ccttgaggat gaacaaatga actttctttt atcatttgtg 540

gaattttttg agaaattatt gttgcctaat ctttttgact cctccattgt tcccagtttc 600

cacagcctcc catctctcac agaggatatt ctgaatataa gttctctgtg gacaaatcat 660

ttaaaaagtt taaagagaga cccatctgcc actgatgctc agaaactctt ggaatttggc 720

aacgaagtga tttggaaaat gcagactctc ggaagtcact ggataaggaa ggaaccaaaa 780

aatcttttga gattcataga attaatactt tttgaaatta atcccaaatt actagaatta 840

tgggcctatg gcatttcaaa aggaaaaaga gctaaattgg aaaacttctt tacactttta 900

aatttttctg ttccagaaaa tgagattctg agtacaagtt ttaacttttc ccagttgttc 960

cattcagatt ggcctaaatc accagctatg aacatagatt ttgtacgttt aagtgaggct 1020

ataataacta gtctccatga atttggattt ttggagcagg aacagatctc agaagctctg 1080

aacacagtct acgctatcag gaatgcatct gatcttttct cagccctttc tgaaccacaa 1140

aaacaagaag ttgataaaat tttgactcac atacacctaa atgtcttcca ggacaaggat 1200

tcagctttac ttctgcaaat ttattcttca ttttaccgat atatttatga attattgaat 1260

attcagagta gaggctcttc gttgactttc cttacacaaa tctcaaaaca cattttggat 1320

atcataaaac aatttaattt ccaaaacatc agtaaagcat ttgcattttt atttaagaca 1380

gcagaggttc ttgggggaat ttctaatgta tcttactgtc agcaattgct ttcaattttt 1440

aactttttgg agcttcaggc ccaatccttc atgtctacag agggccaaga actggaagtg 1500

atccacacta ctttgacagg cctcaaacag ctgctcataa ttgatgaaga ttttcgtatt 1560

tctttatttc aatatatgag ccaattcttc aacagttcag tagaagacct attggataat 1620

aaatgcttga tttcggacaa taaacacatt tcttccgtaa attattcaac aagtgaggag 1680

tcttcatttg tttttccatt ggcacaaatt ttttcaaacc tctcagcaaa tgtcagtgtg 1740

ttcaacaagt ttatgtccat tcactgtacc gtttcatggc ttcaaatgtg gactgaaatc 1800

tgggaaacca tatctcaatt atttaagttt gacatgaatg ttttcacatc tcttcatcat 1860

ggtttcactc agcttttgga tgaattggaa gatgatgtga aagtctctaa aagctgccag 1920

ggtatacttc ccacccataa tgttgctaga ctcatattaa atttgtttaa aaatgtaact 1980

caagccaatg acttccataa ttgggaggac ttcctggatc tcagggattt tttggtagct 2040

ttaggtaatg cattagtttc agtaaaaaaa cttaacttgg agcaagtgga gaaatccctt 2100

ttcaccatgg aagctgccct gcatcagttg aagacatttc cattcaacga aagtacaagc 2160

agagagtttt taaattctct gcttgaagtt ttcattgagt ttagcagtac ctcagaatat 2220

atagtcagaa atctagattc aataaatgac tttctttcaa ataatctcac aaattatgga 2280

gaaaaatttg aaaatatcat cactgagcta agagaagcaa tagtatttct tagaaatgta 2340

tcacatgatc gagatttgtt ttcctgtgct gatattttcc aaaatgttac tgagtgtatt 2400

ttagaagatg gctttttata tgtaaatacc tcacagagga tgttacgtat tctagacacg 2460

ttaaattcca cattttcctc tgagaacaca attagcagtc tgaaaggatg cattgtatgg 2520

ttagatgtca taaaccattt gtatttgttg tctaactcca gtttttcaca aggtcatctt 2580

caaaatattt tggggaattt cagagatata gaaaacaaaa tgaactctat attaaaaatt 2640

gtaacttggg tgttaaatat aaaaaaacct ctttgttcat caaatggctc acatataaat 2700

tgtgtcaata tttacttgaa agatgtaact gactttctaa atattgtact tactacagtc 2760

tttgaaaaag agaagaaacc taaatttgag attttattag ctcttttaaa tgattccaca 2820

aagcaagtaa ggatgagtat caacaactta acaacagact ttgattttgc atctcagtcc 2880

aattggagat attttactga attaattcta agaccaatag aaatgtcaga tgaaattcct 2940

aatcagtttc aaaatatttg gcttcattta ataacactgg ggaaggaatt tcagaagctt 3000

gtaaaaggta tttactttaa catcctggaa aataattcct cttctaaaac tgaaaacttg 3060

ttaaacatat ttgccaccag tccaaaagaa aaggatgtaa acagtgtagg caattccatt 3120

tatcacttag ctagttacct tgccttcagc ttatctcatg acctccaaaa ttcaccaaaa 3180

ataataattt cacctgaaat aatgaaagct acaggtcttg gtattcaact gataagggat 3240

gtgttcaact ccttaatgcc tgtagttcat cacactagtc cacaaaatgc aggttatatg 3300

caagctttga agaaggtaac ttctgtcatg cgtaccctta agaaggcaga catagacctt 3360

ttagtggatc agcttgaaca agttagtgta aacctaatgg atttctttaa gaatatcagt 3420

agtgtgggaa ctggcaattt agtggtcaat ttgcttgttg gcttgatgga aaaatttgca 3480

gacagctcac attcttggaa tgttaatcat ctgctgcagc tctcacgcct gtttcctaaa 3540

gatgttgtgg atgctgtgat agatgtgtac tatgtgcttc ctcatgctgt aaggctcctg 3600

cagggagtac ctggtaaaaa catcactgaa ggcctcaagg atgtctacag cttcacactc 3660

cttcatggca taaccatttc aaatatcacc aaggaagact tcgcaattgt gataaaaatt 3720

cttttggata caattgaatt agtatcagat aagccagata ttatttcaga ggctttagct 3780

tgttttcctg tggtttggtg ctggaatcac acaaattctg gatttcggca gaattcaaag 3840

atagacccct gcaatgtcca tgggctcatg tcttcttcct tttatggcaa agtggccagt 3900

atacttgatc atttccacct gtctccccaa ggtgaagatt caccatgttc aaatgaaagc 3960

tcccgaatgg aaataactag gaaagtggtc tgcataattc atgaattagt ggactggaat 4020

tctattcttc tggagctctc tgaagtcttc catgttaaca tttctcttgt gaaaactgtg 4080

cagaaatttt ggcataagat attaccgttt gtcccacctt caataaatca aactagggat 4140

agcatctctg aactctgtcc tagtggttcc ataaagcaag ttgctttgca aatcatagaa 4200

aaacttaaaa atgtcaactt tacaaaagtt acatcaggtg aaaatattct tgacaaacta 4260

agtagtttaa acaagatcct taacattaat gaagacacag agacatctgt tcaaaatatt 4320

atttcctcaa atttggaaag gacagtacaa ttgatttctg aagactggag cctagaaaaa 4380

agtacgcata atctactctc tttattcatg atgctccaga atgcaaatgt cacaggtagc 4440

agtttagaag cattatcaag ttttattgaa aaaagtgaaa caccttacaa ctttgaagaa 4500

ctatggccca agtttcaaca aatcatgaaa gacctaaccc aagattttag aatcagacac 4560

ctgctttctg aaatgaacaa aggaatcaaa agtataaatt caatggctct tcaaaagata 4620

actttgcagt ttgcccattt cctggaaatc ctggattcac cgtcattgaa gacattagaa 4680

attattgaag attttctatt ggtcacaaaa aactggcttc aggaatatgc aaatgaggat 4740

tactccagaa tgatagaaac attattcatt cctgtgacca atgagagttc aactgaagat 4800

atagctttgt tagccaaagc tattgctact ttttggggct ctttaaaaaa tatatctaga 4860

gcaggcaatt ttgatgttgc ctttcttacc catctgctaa atcaagaaca gctgactaat 4920

ttctcagttg ttcagctgct ttttgaaaac atcctaatta atttgatcaa taacttagct 4980

gggaattctc aggaagcagc ttggaactta aatgatactg accttcaaat aatgaatttc 5040

attaacctta tcttgaacca tatgcagtca gaaactagta ggaaaacagt tctctctctg 5100

agaagcatag tagatttcac agaacagttt ttgaaaacat tcttctccct ttttctaaag 5160

gaagattctg agaacaaaat atctcttctg ctgaaatatt tccacaaaga tgttattgca 5220

gagatgaggt gagtatactt ttgctttgtg tcatatatgc agttgcgata tatctcaaac 5280

tactttattt tctggagtat agacatgtca aaaacagtat ttgtatctaa tacctttgaa 5340

agatctttga tgtaaatttg tcttaataaa tattttatca aaatgtcaag gtaaaagaat 5400

tttgattact gtcatctagt aaaatataaa cccagcccag gg 5442

Claims

1. A familial multiple lipoma detection kit is characterized by comprising a forward amplification primer shown as SEQ ID No.33, a reverse amplification primer shown as SEQ ID No.34 and a sequencing primer shown as SEQ ID No. 126.

2. The familial multiple lipoma detection kit according to claim 1, wherein the detection kit is used for detecting a pathogenic gene of the familial multiple lipoma, the pathogenic gene is a mutated ABCA13 gene, the difference between the mutated ABCA13 gene and a wild ABCA13 gene comprises 1 mutation, the mutation is that the 2908 th base C of the 17 th exon in the coding region of the wild ABCA13 gene is mutated into T, and the nucleotide sequence of the mutated ABCA13 gene is shown as SEQ ID No. 139.

3. The familial multiple lipoma detection kit according to claim 1, wherein the detection kit further comprises a DNA amplification enzyme for PCR, a buffer solution, and a dNTP mixture, wherein the DNA amplification enzyme is Taq DNA polymerase, and the concentration of the Taq DNA polymerase is 1U/μ L; the buffer solution is MgCl2 solution, and the concentration of the MgCl2 solution is 10 mM; the concentration of the dNTP mixture is 10 mM.

4. The familial multiple lipoma detection kit according to claim 3, wherein the volume ratio of the primer group, the DNA amplification enzyme, the buffer solution and the water in the detection kit is 2:1:2: 4.

5. The kit for detecting familial multiple lipomas according to claim 4, wherein the volume ratio of the added DNA template to the buffer is 1:1, and the concentration of the DNA template is 50ng/μ L.

6. An application of a primer group in a familial multiple lipoma pathogenic gene detection product is characterized in that,

the primer group comprises 62 primer pairs and 14 sequencing primers;

the sequencing primer is shown as SEQ ID No. M, wherein M is 125-138.

7. The primer group of claim 6, wherein the primer group is used for detecting the pathogenic gene of the familial multiple lipoma, the pathogenic gene is a base mutation of a wild-type ABCA13 gene at any position, and the nucleotide sequence of the wild-type ABCA13 gene is shown as SEQ ID No. 140.