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CN110499372B - Multiple PCR (polymerase chain reaction) targeted capture typing system and kit based on high-throughput sequencing technology - Google Patents

Multiple PCR (polymerase chain reaction) targeted capture typing system and kit based on high-throughput sequencing technology Download PDF

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CN110499372B
CN110499372B CN201910883090.6A CN201910883090A CN110499372B CN 110499372 B CN110499372 B CN 110499372B CN 201910883090 A CN201910883090 A CN 201910883090A CN 110499372 B CN110499372 B CN 110499372B
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严江伟
范庆炜
张更谦
张君
程凤
王萌春
李万婷
石林玉
张晓梦
张家榕
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Abstract

本发明了公开了一种基于高通量测序技术的多重PCR靶向捕获分型体系及试剂盒,涉及核酸体外检测技术领域。该STR分型系统包括用于扩增58、119或179个连锁常染色体STR基因座的PCR引物,该试剂盒包括PCR引物组合、Index接头序列、IGT‑EM707聚合酶混合物、扩增缓冲增强剂NB、YF缓冲液B和建库试剂等。本发明提供的STR分型系统及其试剂盒能够实现了对58、119或179个连锁常染色体STR基因座的单管扩增,平衡性好、灵敏度高、特异性好、分型结果准确,可用于鉴定同一级不同亲缘关系的复杂亲缘关系。

Figure 201910883090

The invention discloses a multiplex PCR targeted capture typing system and a kit based on high-throughput sequencing technology, and relates to the technical field of nucleic acid in vitro detection. The STR typing system includes PCR primers for amplifying 58, 119 or 179 linked autosomal STR loci, the kit includes a PCR primer set, Index linker sequences, IGT‑EM707 polymerase mix, amplification buffer enhancer NB, YF buffer B and library building reagents, etc. The STR typing system and its kit provided by the invention can realize single-tube amplification of 58, 119 or 179 linked autosomal STR loci, have good balance, high sensitivity, good specificity and accurate typing results, It can be used to identify complex relatives of different relatives at the same level.

Figure 201910883090

Description

Multiple PCR (polymerase chain reaction) targeted capture typing system and kit based on high-throughput sequencing technology
Technical Field
The invention relates to the technical field of forensic detection, in particular to a high-throughput sequencing technology-based composite typing system for multiple PCR (polymerase chain reaction) targeted capture sequencing and a kit thereof.
Background
Short Tandem Repeat (STR) is a special sequence formed by tandem connection of 2-6bp repeat units, the STR locus has large quantity and wide distribution, accounts for about 3 percent of the whole genome, has high polymorphism, and the polymorphism mainly derives from the difference of the repeat times of a core sequence among individuals, and the difference follows Mendelian genetic rules in the genetic process. Therefore, STR amplification detection techniques are widely used in forensic individual identification, genetic identification and population genetics research. The most commonly used STR typing techniques are fluorescence-labeled multiplex amplification combined with Capillary Electrophoresis (CE) and the second generation sequencing technique.
The identification of complex genetic relationship is one of the technical problems which are urgently needed to be solved in the field of judicial identification at present, and the identification is mainly used in multiple fields of lawsuits, inheritance, remains in large-scale disasters, revenues of remains, traffic accidents, immigration cases, relativity identification and the like in judicial practice, and the cases tend to increase in recent years. The success of such case identification is often dependent on the social and bloody relationships of several families, and is a great challenge for forensic geneticists and a significant impact on the personal interests of the general public.
In the complex genetic relationship identification, the identification requirements of different genetic relationships in the same class of the same class between a half-sib and a tertiary nephew, between the half-sib and a grandchild or between the tertiary nephew and a grandchild are met, but the Identity by term (IBD) value and the coordinate coefficient (theta) value derived through Mendel's genetic law in the class of the third class of relationships are consistent, so that the three relationships cannot be distinguished by applying the conventional independent genetic marker and calculation method. It is suggested that although the identification of such relationships can aid in the determination of such problems through age information and other DNA information, such as mitochondrial DNA and sex chromosomal DNA, the linked autosomal genetic markers can better address the above problems in the general case of the same level of different relationships. In theoretical research, Thompson deduces different genetic relationship coefficients among grandfather, half-sibling and tertiary-nephew in 1998 by using recombination rate of linkage genetic law, and with the application of computer technology in the field of forensic physical evidence, Egeland makes a theoretical calculation model by using a certain number of linkage genetic markers according to the different genetic relationship coefficients obtained by Thompson in 2008.
Current state of the art for genetic relationship identification, for example: chinese patent CN104818323B discloses a genotyping detection kit for 20 STR loci of human chromosomes 13, 18 and 21, which can realize single-tube amplification of 20 STR loci; chinese patent CN106906292A discloses a 22 short tandem repeat sequence composite amplification method and a kit thereof, wherein the kit can be used for amplifying 22 STR loci and 1 individual locus. The majority of the chain STR researches are X-STR and Y-STR genetic markers, and the genetic markers can be applied only by special genetic relationship; autosomal STRs that are independently inherited also do not distinguish between complex relationships.
The invention aims at the characteristics of the chain autosomal STR, and designs an STR typing system for multiple PCR targeted capture sequencing, wherein the STR typing system can amplify all STR genetic markers at one time, the adopted primer combination sequence has good balance, all loci in a group can be ensured to be detected, the typing and sequence information of each STR can be obtained through second-generation sequencing and subsequent data analysis, and the STR typing system can be used for identifying the complex genetic relationship of different genetic relationships at the same level.
Disclosure of Invention
The invention aims to provide an STR typing system for identifying complex genetic relationship and a kit thereof. The STR typing system and the kit thereof can realize single-tube amplification of 179 linked autosomal STR loci, have good balance, high sensitivity, good specificity and accurate typing result, and can be used for identifying complex genetic relationships of different genetic relationships at the same level.
On the other hand, in the research process, it is found that the 179 STR genomes can be divided into three groups independent from each other, and the 6 STR locus combinations formed by the three groups of STR loci alone or in pairwise cooperation can achieve the same, similar or equivalent technical effects as the typing system formed by the 179 STR loci.
Based on the above:
the invention provides an STR typing system for identifying complex genetic relationship, which comprises a PCR primer combination 1 for amplifying 60 linked autosomal STR loci;
the corresponding physical positions, chromosome partitions and genetic distances of the 60 linked autosomal STR loci and the reference genome Hg38 are as follows:
Figure BDA0002206469760000021
Figure BDA0002206469760000031
the PCR primer combination 1 comprises a forward primer group and a reverse primer group;
the forward primer group is as follows:
Figure BDA0002206469760000032
Figure BDA0002206469760000041
Figure BDA0002206469760000051
the reverse primer group is as follows:
Figure BDA0002206469760000052
Figure BDA0002206469760000061
secondly, the invention provides an STR typing system for identifying complex genetic relationship, which comprises a PCR primer combination 2 for amplifying 58 linked autosomal STR loci;
the 58 linked autosomal STR loci, the corresponding physical locations on the reference genome Hg38, the chromosomal partitions and the genetic distances are:
Figure BDA0002206469760000071
Figure BDA0002206469760000081
the PCR primer combination 2 comprises a forward primer group and a reverse primer group;
the forward primer group is as follows:
Figure BDA0002206469760000082
Figure BDA0002206469760000091
the reverse primer group is as follows:
Figure BDA0002206469760000101
Figure BDA0002206469760000111
thirdly, the invention provides an STR typing system for identifying complex genetic relationship, which comprises a PCR primer combination 3 for amplifying 61 linked autosomal STR loci;
the 61 linked autosomal STR loci, the corresponding physical locations on the reference genome Hg38, the chromosomal partitions and the genetic distances are:
Figure BDA0002206469760000112
Figure BDA0002206469760000121
Figure BDA0002206469760000131
the PCR primer combination 3 comprises a forward primer group and a reverse primer group;
the forward primer group is as follows:
Figure BDA0002206469760000132
Figure BDA0002206469760000141
the reverse primer group is as follows:
Figure BDA0002206469760000142
Figure BDA0002206469760000151
Figure BDA0002206469760000161
fourthly, the invention provides an STR typing system for identifying complex genetic relationship, which comprises a PCR primer combination 4 for amplifying 118 linked autosomal STR loci; the 118 linked autosomal STR loci are STR loci with STR locus sequence numbers of 1-118 in the description of the invention; the PCR primer combination 4 is a forward primer combination and a reverse primer combination which are used for amplifying STR loci with STR locus sequence numbers of 1-118 in the description of the invention, and specifically consists of primer combinations 1 and 2.
Fifthly, the invention provides an STR typing system for identifying complex genetic relationship, which comprises a PCR primer combination 5 for amplifying 121 linked autosomal STR loci; the 121 linked autosomal STR loci are STR loci with STR locus sequence numbers of 1-60 and 119-179 in the description of the invention; the PCR primer combination 5 is a forward primer combination and a reverse primer combination for amplifying STR loci with STR locus sequence numbers of 1-60 and 119-179 in the description of the invention, and specifically comprises primer combinations 1 and 3.
Sixthly, the invention provides an STR typing system for identifying complex genetic relationship, which comprises a PCR primer combination 6 for amplifying 119 linked autosomal STR loci; the 119 linked autosomal STR loci are STR loci with STR locus sequence numbers of 61-179 in the description of the invention; the PCR primer combination 6 is a forward primer combination and a reverse primer combination for amplifying STR loci with STR locus sequence numbers of 61-179 in the description of the invention, and specifically comprises primer combinations 2 and 3.
Seventhly, the invention provides an STR typing system for identifying complex genetic relationship, wherein the STR typing system comprises a PCR primer combination 7 for amplifying 179 linkage autosomal STR loci; the 179 linked autosomal STR loci are STR loci with STR locus sequence numbers of 1-179 in the description of the invention; the PCR primer combination 7 is a forward primer combination and a reverse primer combination for amplifying STR loci with STR locus sequence numbers of 1-179 in the description of the invention, and specifically comprises primer combinations 1, 2 and 3.
In order to more intuitively embody the technical scheme of the invention, the key points of the first to seventh technical schemes are summarized as follows:
Figure BDA0002206469760000162
Figure BDA0002206469760000171
eighth, the present invention provides a kit for identification of complex relationships, which comprises the PCR primer set 1, 2, 3, 4, 5, 6 or 7 described in the present specification.
In the primer combination, the concentrations of the positive primer and the negative primer are both 0.1 mu M.
Further, the kit also comprises an Index adaptor sequence and DNA polymerase;
further preferably, the Index linker sequences comprise IGT-I5Index and IGT-I7 Index, and the concentration of the working solution is 10. mu.M.
The Index linker sequence is a universal linker sequence for the illiminia sequencing platform and is used for discrimination between samples.
Preferably, the kit further comprises reagents for preparing genomic DNA into a library for sequencing.
The invention also provides a method for identifying complex genetic relationship, which comprises the following steps:
(1) extracting human genome DNA, and quantifying the concentration of the genome DNA to be 1-20 ng/mu L;
(2) multiplex PCR library construction:
A. obtaining a purified multiplex PCR product through a first round of multiplex PCR reaction and a first round of magnetic bead purification;
B. performing a second round of multiplex PCR reaction by taking the purified multiplex PCR product as a template, inserting an Index adaptor sequence, and purifying by a second round of magnetic beads to obtain a multiplex PCR library;
C. carrying out quantitative and quality detection on the obtained multiple PCR library;
(3) and (3) sequencing and analyzing data of the multiple PCR library obtained in the step (2) to obtain STR typing results and forensic parameters corresponding to each individual.
Further preferably, the concentration of genomic DNA is 10-20 ng/. mu.L.
The STR typing system and the kit are suitable for all samples containing DNA, including but not limited to blood, seminal plasma, hair, bones, skin, solid tissues and the like.
Compared with the prior art, the invention has the advantages that:
(1) the STR typing system and the kit thereof for identifying complex genetic relationship provided by the invention can simultaneously detect 60, 58, 61, 118, 121, 119 or 179 linked autosomal STR loci at one time, and the mutual interference between each pair of primers can be increased along with the increase of the number of the detected STR loci in the process of establishing a multiple PCR amplification system.
(2) In the prior art, neither autosomal STR inherited independently nor STR genetic markers linked to sex chromosomes can identify complex relationships such as the same-level different relationships between a half-sib and a tertiary (girl) nephew, a half sib and a grande (milk) grande or a tertiary nephe and a grande. The STR typing system and the kit thereof provided by the invention aim at the characteristics of the chain autosomal STR, can be used for identifying the complex genetic relationship, and have accurate identification result.
(3) The STR typing system and the kit thereof provided by the invention adopt two rounds of PCR reactions to construct the library, have the advantages of short library construction period, high comparison rate, good capture efficiency, good repeatability, simple and convenient operation and the like, have high sensitivity of detection results, good specificity and accurate typing results, and can effectively amplify 60, 58, 61, 118, 121, 119 or 179 linked autosomal STR loci at one time.
It should be clear that, based on the disclosure of the present invention of all 179 STR loci described and their corresponding amplified forward and reverse primers, one skilled in the art can reasonably expect, based on the disclosure of the present invention: the technical scheme of the invention, which is formed by any collocation combination with genetic testing efficiency quantity in 179 STR loci recorded in the invention or the collocation combination of corresponding forward and reverse primers, can obtain the technical effect which is equal to or similar to the technical effect of the invention. The amount of any genetic test potency may specifically be any integer between 3 and 179. Therefore, the technical solutions obtained by the embodiments described in this paragraph are all within the technical extension, the protection scope, and the infringement scope of the present invention.
Drawings
FIG. 1 is a diagram of quality control peaks of a library prepared using the primer combinations disclosed in example 1.
FIG. 2 is a diagram of quality control peaks of a library prepared using the primer combinations disclosed in example 2.
FIG. 3 is a peak inspection diagram of a library prepared using the primer combinations disclosed in example 3.
FIG. 4 is a peak inspection chart of a library prepared by using the primer combination disclosed in example 4.
FIG. 5 is a peak inspection diagram of a library prepared using the primer combinations disclosed in example 5.
FIG. 6 is a peak inspection chart of a library prepared by using the primer combination disclosed in example 6.
FIG. 7 is a peak inspection chart of a library prepared by using the primer combination disclosed in example 7.
Detailed Description
Hereinafter, specific embodiments of the present invention will be described in detail. Well-known structures or functions may not be described in detail in the following embodiments in order to avoid unnecessarily obscuring the details. Unless defined otherwise, technical and scientific terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Example 1: STR typing system for complex genetic relationship identification and kit thereof
1.1 autosomal linked STR locus constitution and characteristics:
(1) the STR typing system in this example includes the following 60 STR loci:
D1S2131, D1S3721, D1S2130, D1S1600, D1S1653, D1S1660, D1S3732, D2S1364, D2S2734, D2S1396, D2S428, D2S435, D2S1387, D2S1792, D2S1399, D2S2959, D3S2431, D3S4547, D4S1643, D4S2408, D4S3326, D4S2368, D5S2845, D5S1473, D5S813, D5S1716, D5S1459, D5S1487, D5S1466, D5S2496, D5S2501, D6S1019, D6S2417, D6S2412, D6S1284, D7S 1280, D7S 376S 821 0, D2S 2328S 1469, D2S 2501, D2S 1019, S14611, S1469, D2S 14611, S1469, D2S 20, S14611, S1469, D2S 1469, D5S 14613, D2S 20, D2S 1469, D2S 20S 1469, D2S 20, D2S 1469, D2S;
it has the following measurable characteristics:
1) the core region repeat unit is tetranucleotide;
2) the heterozygosity of each STR is more than 0.6;
3) each group comprises at least two STRs and the genetic distance of the STRs is less than 3 cM;
4) each STR has the name DXSXX.
(2) Primer set 1 used to simultaneously amplify the 60 STR loci in this example was:
table 1: forward primer set of primer combination 1
Figure BDA0002206469760000191
Figure BDA0002206469760000201
Table 2: reverse primer set of primer set 1
Figure BDA0002206469760000202
Figure BDA0002206469760000211
Figure BDA0002206469760000221
(3) The working concentration of the forward primer in this example was 0.1. mu.M; the working concentration of the reverse primer was 0.1. mu.M.
1.2 a kit for identification of complex relationships:
comprises the PCR primer combination 1, Index adaptor sequence and IGT-EM707 polymerase mixture.
The Index linker sequences include IGT-I5Index and IGT-I7 Index, and the concentration of the working solution is 10. mu.M.
The Index linker sequence is: IGT-I7 index Aijiekang I7 end connector sequence, the working concentration is 10 μ M; one contains 96, linker sequence information as follows
Figure BDA0002206469760000222
Figure BDA0002206469760000231
IGT-I5Index Aijiekang end I5 linker sequence, working concentration is 10 μ M; one contains 4, linker sequence information as follows
Figure BDA0002206469760000232
Note: linker sequences are those commonly used by the illumina sequencing platform for discrimination between samples.
Example 2: STR typing system for complex genetic relationship identification and kit thereof
2.1 autosomal linked STR loci constitution and Properties:
(1) the STR typing system in this example contains the following 58 STR loci:
D1S3736、D1S1665、D1S2127、D1S2138、D1S1642、D1S518、D1S1604、D1S3729、D1S3727、D2S1336、D2S1779、D2S1771、D2S437、D2S2970、D2S2944、D2S1327、D3S2402、D3S1766、D4S2411、D4S3351、D4S243、D4S2426、D4S2373、D5S1490、D5S2856、D5S2495、D5S2855、D5S1722、D5S1725、D6S1048、D6S1275、D7S796、D7S1799、D8S2324、D8S2330、D8S1144、D9S745、D9S301、D9S1124、D10S1246、D10S2485、D11S1983、D11S2363、D11S1368、D12S393、D12S1063、D13S1807、D13S1492、D14S738、D14S301、D14S583、D15S816、D15S1514、D18S872、D18S972、D18S548、D20S1145、D20S477;
it has the following measurable characteristics:
1) the core region repeat unit is tetranucleotide;
2) the heterozygosity of each STR is more than 0.6;
3) each group comprises at least two STRs and the genetic distance of the STRs is less than 3 cM;
4) each STR has the name DXSXX.
(2) Primer set 2 used to simultaneously amplify the 58 STR loci in this example was:
table 3: forward primer set of primer combination 2
Figure BDA0002206469760000233
Figure BDA0002206469760000241
Figure BDA0002206469760000251
Table 4: reverse primer set of primer combination 2
Figure BDA0002206469760000252
Figure BDA0002206469760000261
(3) The working concentration of the forward primer in this example was 0.1. mu.M; the working concentration of the reverse primer was 0.1. mu.M.
2.2 a kit for identification of complex relationships:
the only difference from example 1 is that the PCR primer set used was primer set 2.
Example 3: STR typing system for complex genetic relationship identification and kit thereof
3.1 autosomal linked STR locus constitution and Properties:
(1) the STR typing system in this example comprises the following 61 STR loci:
D1S532、D1S1611、D1S3733、D1S533、D1S1614、D2S2977、D2S1374、D2S1394、D2S2966、D2S2969、D2S1371、D2S434、D2S1338、D3S4016、D3S2388、D4S1626、D4S1653、D5S2858、D5S2796、D5S1463、D5S815、D5S2499、D5S2498、D6S1043、D6S1274、D6S1056、D6S1013、D6S1054、D7S820、D7S2205、D7S3071、D7S2845、D8S2326、D8S1464、D8S2320、D8S1470、D8S588、D8S1471、D9S2026、D9S747、D9S2128、D10S2469、D10S1238、D11S4464、D11S4958、D13S1818、D13S767、D14S615、D14S608、D14S597、D14S302、D14S749、D16S767、D16S3393、D18S537、D18S875、D18S1367、D20S1152、D20S206、D20S607、D20S1146;
it has the following measurable characteristics:
1) the core region repeat unit is tetranucleotide;
2) the heterozygosity of each STR is more than 0.6;
3) each group comprises at least two STRs and the genetic distance of the STRs is less than 3 cM;
4) each STR has the name DXSXX.
(2) Primer set 3 used to simultaneously amplify the 61 STR loci in this example was:
table 5: forward primer set of primer combination 3
Figure BDA0002206469760000271
Figure BDA0002206469760000281
Table 6: reverse primer set of primer combination 3
Figure BDA0002206469760000282
Figure BDA0002206469760000291
Figure BDA0002206469760000301
(3) The working concentration of the forward primer in this example was 0.1. mu.M; the working concentration of the reverse primer was 0.1. mu.M.
3.2A kit for identification of complex relationships:
the only difference from example 1 is that the PCR primer set used was primer set 3.
Example 4: STR typing system for complex genetic relationship identification and kit thereof
4.1 autosomal linked STR loci constitution and Properties:
(1) the STR typing system in this embodiment includes 118 STR loci, specifically including 60 STR loci described in embodiment 1 and 58 STR loci described in embodiment 2.
It has the following measurable characteristics:
1) the core region repeat unit is tetranucleotide;
2) the heterozygosity of each STR is more than 0.6;
3) each group comprises at least two STRs and the genetic distance of the STRs is less than 3 cM;
4) each STR has the name DXSXX.
(2) Primer set 4 for simultaneously amplifying the 118 STR loci in this example is a set of primer sets in examples 1 and 2.
(3) The working concentration of the forward primer in this example was 0.1. mu.M; the working concentration of the reverse primer was 0.1. mu.M.
4.2 a kit for identification of complex relationships:
the only difference from example 1 is that the PCR primer set used was primer set 4.
Example 5: STR typing system for complex genetic relationship identification and kit thereof
5.1 autosomal linked STR loci constitution and Properties:
(1) the STR typing system in this embodiment includes 121 STR loci, specifically including 60 STR loci described in embodiment 1 and 61 STR loci described in embodiment 3.
It has the following measurable characteristics:
1) the core region repeat unit is tetranucleotide;
2) the heterozygosity of each STR is more than 0.6;
3) each group comprises at least two STRs and the genetic distance of the STRs is less than 3 cM;
4) each STR has the name DXSXX.
(2) Primer set 5 for simultaneously amplifying 121 STR loci in this example is a set of primer sets in examples 1 and 3.
(3) The working concentration of the forward primer in this example was 0.1. mu.M; the working concentration of the reverse primer was 0.1. mu.M.
5.2A kit for identification of complex relationships:
the only difference from example 1 is that the PCR primer set used was primer set 5.
Example 6: STR typing system for complex genetic relationship identification and kit thereof
6.1 autosomal linked STR loci constitution and Properties:
(1) the STR typing system in this embodiment includes 119 STR loci, specifically including 58 STR loci described in embodiment 2 and 61 STR loci described in embodiment 3.
It has the following measurable characteristics:
1) the core region repeat unit is tetranucleotide;
2) the heterozygosity of each STR is more than 0.6;
3) each group comprises at least two STRs and the genetic distance of the STRs is less than 3 cM;
4) each STR has the name DXSXX.
(2) Primer combination 6 for simultaneously amplifying the 119 STR loci in this example is a combination of the primer combinations in examples 3 and 2.
(3) The working concentration of the forward primer in this example was 0.1. mu.M; the working concentration of the reverse primer was 0.1. mu.M.
6.2A kit for identification of complex relationships:
the only difference from example 1 is that the PCR primer combination used is primer combination 6.
Example 7: STR typing system for identifying complex genetic relationship
7.1 autosomal linked STR locus constitution and Properties:
(1) the STR typing system in this embodiment includes 179 STR loci, specifically including 60 STR loci described in embodiment 1, 58 STR loci described in embodiment 2, and 61 STR loci described in embodiment 3.
It has the following measurable characteristics:
1) the core region repeat unit is tetranucleotide;
2) the heterozygosity of each STR is more than 0.6;
3) each group comprises at least two STRs and the genetic distance of the STRs is less than 3 cM;
4) each STR has the name DXSXX.
(2) Primer set 7 for simultaneously amplifying the 179 STR loci in this example is a set of primer sets in example 1, example 3, and example 2.
(3) The working concentration of the forward primer in this example was 0.1. mu.M; the working concentration of the reverse primer was 0.1. mu.M.
7.2A kit for identification of complex relationships:
the only difference from example 1 is that the PCR primer set used was primer set 7.
Examples of the experiments
In the following experimental examples, the reagents used were all available from the legal commercial sources as follows:
tissue and blood DNA extraction kit: purchased from Beijing Tiangen Biochemical technology Ltd; the goods number is: DP 304-03;
QIAamp DNA investigetor Kit (50): purchased from qiagen, germany; the goods number is: 5650;
enhancer buffer NB (1N): a PCR reaction enhancer purchased under the name NB of agitta conggins; the goods number is: MT017035
IGT-EM707 polymerase mix: a DNA polymerase cocktail available from Aijiekang under the designation EM 707; the goods number is: MT 017035;
YF Buffer B: magnetic bead rinse buffer purchased from agutazone under the name YF; the goods number is: MT 017035;
the above reagent is Beijing Aikitikang (iGeneTech)TM) The customized composition has the following goods number: MT017035
Primer or Index sequence: hodgkin Eitykang Co., Ltd (iGeneTech)TM) Performing customized synthesis;
1. detecting a sample: 108 unrelated individual samples from the Peking Han nationality, including 48 blood samples and 60 FTA blood cards.
2. Detecting an object: the typing system and the kit described in embodiments 1 to 7 are respectively adopted to detect a detection sample according to a detection flow.
3. And (3) detection flow:
(1) the whole genome DNA was extracted from the blood sample and FTA blood card using tissue, blood DNA extraction Kit and QIAamp DNAinvestigator Kit (50), respectively. The concentration of the genomic DNA was measured by using a nucleic acid quantitative analyzer at 1, 5, 10 and 20 ng/. mu.L.
(2) Multiplex PCR library construction:
library construction was performed using 1, 5, 10, 20 ng/. mu.L of genomic DNA as described above, with library numbers F01, F02, F03 and F04, respectively.
A. First round multiplex PCR reaction: preparing reaction mixed solution according to the table 7, wherein each tube is 25 mu L, and performing multiple PCR reaction according to the reaction conditions of the table 8 to obtain multiple PCR products; wherein the primer combinations referred to in the different detection assays refer to the primer combinations of examples 1-7, respectively;
table 7: first round multiple PCR reaction system
Reagent Volume (μ L)
Double distilled water 4
Enhancer buffer NB(1N) 7
Primer combination 8
Genomic DNA 1
IGT-EM707polymerase mixture 5
Table 8: conditions for the first round of multiplex PCR reactions
Figure BDA0002206469760000321
First round magnetic bead purification:
adding 23 μ L of AMPure XP magnetic beads balanced at room temperature into 25 μ L of multiplex PCR products, sucking and beating the mixture for several times by using a pipettor, after incubating for 5min at room temperature, placing the PCR tube on a DynaMag-96 Side magnetic frame for 3min, completely removing the supernatant, taking the PCR tube off the magnetic frame, adding 40 μ L of YF buffer B (magnetic bead rinsing buffer), sucking and beating the mixture for several times by using a pipettor, incubating for 5min at room temperature, removing the supernatant, adding 180 μ L of 80% ethanol solution, standing for 30s, completely removing the supernatant, standing for 3min at room temperature to completely volatilize the residual ethanol, adding 24 μ L of nucleic-free water or 1 × TE buffer (pH 8.0), sucking and beating and re-suspending the mixture by using a pipettor gently to avoid generating bubbles, standing for 2min at room temperature, placing the PCR tube on the magnetic frame again and standing for 3min, and (4) sucking the supernatant by using a liquid transfer machine, and transferring the supernatant into a new PCR tube, wherein the supernatant in the tube is the purified multiplex PCR product.
B. Second round multiplex PCR reaction: b, taking the purified multiplex PCR product obtained in the step A as a template, performing a second round of multiplex PCR reaction, inserting an Index adaptor sequence, wherein the system of the second round of multiplex PCR reaction is shown in Table 9, and the reaction conditions are shown in Table 10;
table 9: second round multiplex PCR reaction system
Reagent Volume (μ L)
Purified multiplex PCR product 18
IGT-I5 Index(10μM) 1
IGT-I7 Index(10μM) 1
IGT-EM707polymerase mixture 5
Table 10: second round of multiplex PCR reaction conditions
Figure BDA0002206469760000331
And (3) second round of magnetic bead purification:
the specific operation is the same as the first round of magnetic bead purification, and the supernatant transferred to a new PCR tube in the last step is a prepared multiplex PCR library.
C. And (3) carrying out quantitative and quality detection on the obtained multiplex PCR library:
taking 1 mu L of the multiple PCR library obtained in the step B, measuring the library concentration by using a nucleic acid quantifier, and recording the library concentration;
and (4) taking 1 mu L of the multiple PCR library obtained in the step (B), and detecting the length and the purity of the library fragment by using a full-automatic nucleic acid protein analysis system, wherein the distribution interval of the target fragment of the normal library is 300bp-450bp, and the main peak is about 339 bp.
(3) Performing second-generation sequencing on the multiple PCR library obtained in the step (2), performing quality control on a fastq file obtained by sequencing by using FASTQC software, filtering data by using Trimmomatic software, trimming a sequence lower than Q30, removing a sequence with a sequencing fragment lower than 100bp, typing the STR on the fastq file with STRaitRazor3.0 after quality control, and finally obtaining an STR typing file corresponding to each individual.
4. And (3) detection results:
(1) the typing system and the kit disclosed in the embodiments 1 to 7 can accurately realize typing and genetic relationship identification for 108 samples.
(2) The quality control peak patterns of the libraries prepared by using the primer combinations disclosed in examples 1 to 7 are shown in FIGS. 1 to 7: the results show that the 7 STR typing systems and the kits thereof provided by the invention can simultaneously amplify the 60, 58, 61, 118, 121, 119 or 179 STR loci at one time, have good balance, good primer specificity and high sensitivity, and can effectively detect gDNA with the concentration as low as 1 ng/. mu.L.
(3) Effective typing information can be obtained for 108 samples by using the typing system and the kit disclosed in the embodiments 1 to 7, and in order to reasonably simplify the application document, typing information of 3 samples is listed:
analysis software: software STRAIT Razor3.0
Interpretation of the tables: the lower blank represents that the site of the sample is homozygote, and the two types represent heterozygote; the number of reads of the sample after each typing represents the coverage of the typing corresponding to the site obtained by using the configuration file of the STRait Razor 3.0.
The typing results information of STR loci in example 1 in 3 samples are as follows:
Figure BDA0002206469760000341
Figure BDA0002206469760000351
Figure BDA0002206469760000361
the typing results information of STR loci in example 2 in 3 samples are as follows:
Figure BDA0002206469760000362
Figure BDA0002206469760000371
Figure BDA0002206469760000381
Figure BDA0002206469760000391
typing results information of STR loci in example 3 in 3 samples is as follows:
Figure BDA0002206469760000392
Figure BDA0002206469760000401
Figure BDA0002206469760000411
Figure BDA0002206469760000421
example 4 the typing results of the above 3 samples are equivalent to the combination of the results of example 1 and example 2; example 5 the typing results of the above 3 samples are equivalent to the combination of the results of example 1 and example 3; example 6 the typing results of the above 3 samples are equivalent to the combination of the results of example 2 and example 3; example 7 the typing results of the above 3 samples are equivalent to the combination of the results of example 1, example 2 and example 3; to avoid redundancy, the tables are not repeated here.
(4) And typing efficiency:
the forensic parameters of the 60, 58, 61, 118, 121, 119, 179 STR sites contained in the typing systems and kits disclosed in examples 1-7, respectively, are as follows:
an analysis tool: STRAF 1.0.5 (STR Analysis for Forensecs);
analysis data sources: typing result information of the 108 Beijing Han nationality irrelevant individual samples;
interpretation of the tables: locus denotes the name of the STR Locus; n represents the number of alleles, and the total number of 108 Beijing Han nationality unrelated individuals exist, so that each STR genetic marker has 216 alleles; nall represents the total number of genotypes typed at each locus, the range of typing is 5-19; GD (Genetic diversity), ranging from 0.4911 to 0.9013; PIC (polymorphic Information Content) with a range of 0.4368-0.8880; PM (match probability), which ranges from 0.0297-0.5111; PD (power of diagnosis, personal identification probability), its range is 0.4889-0.9703.
Figure BDA0002206469760000431
Figure BDA0002206469760000441
Figure BDA0002206469760000442
Figure BDA0002206469760000451
Figure BDA0002206469760000461
Figure BDA0002206469760000462
Figure BDA0002206469760000471
(5) Library concentration:
Figure BDA0002206469760000472
the RFU (fluorescence signal intensity) values of the gDNA initial amounts were slightly lower at 1ng and 5ng, but both were normal main peaks, and when the gDNA initial amounts were 10ng and 20ng, not only the library was mainly normal main peaks but also the RFU values were better.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
SEQUENCE LISTING
<110> university of Shanxi medical science
<120> multiple PCR targeting capture typing system and kit based on high-throughput sequencing technology
<130>20200220
<160>358
<170>PatentIn version 3.5
<210>1
<211>27
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>1
gctgagattg atggactgaa tccagtg 27
<210>2
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<213> Artificial sequence (artificial sequence)
<400>2
taatcatgtg agcaaaaacc ttataacaa 29
<210>3
<211>22
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<213> Artificial sequence (artificial sequence)
<400>3
gggcagactg tggaactttt ta 22
<210>4
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<213> Artificial sequence (artificial sequence)
<400>4
aaaccccgtc tgtacaaaaa tatt 24
<210>5
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<213> Artificial sequence (artificial sequence)
<400>5
ctaaagaccc caccaacaaa atttt 25
<210>6
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<213> Artificial sequence (artificial sequence)
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acacataaac tagttatata tttgtgggtt atat 34
<210>7
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<213> Artificial sequence (artificial sequence)
<400>7
actcctgctt accactattc tggct 25
<210>8
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<213> Artificial sequence (artificial sequence)
<400>8
gtatgtactc ctgagaatct gattctcag 29
<210>9
<211>36
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>9
aaattttaaa tcttctgaag gaaaagtgtt ctgatc 36
<210>10
<211>32
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>10
taacttgaga ccataagttc acaaatggcc tg 32
<210>11
<211>34
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>11
ctacttatgt tgaatggtca ttaagtcaaa gttt 34
<210>12
<211>32
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>12
cacaagaaca ctgactaata cactatacca ca 32
<210>13
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<213> Artificial sequence (artificial sequence)
<400>13
actgctgtca acacattgta tct 23
<210>14
<211>35
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>14
aatagtcact gctatactaa aatgaataaa ttggt 35
<210>15
<211>26
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>15
ctgctctcaa ggtgaatcta aactac 26
<210>16
<211>27
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>16
agttcttttg ctctggtagt gaaattg 27
<210>17
<211>23
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>17
attctgctcg cactgtagtc caa 23
<210>18
<211>22
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>18
cctgtctcca gagctttgtt tt 22
<210>19
<211>27
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>19
cttcatcatg tgagtcaatt ctgcaga 27
<210>20
<211>26
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>20
actatgattc atttccatag ggtaag 26
<210>21
<211>28
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>21
ttcctccgtg taatgctata tctagcat 28
<210>22
<211>31
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>22
tcatgaaggg attttaggaa ctgatacgat g 31
<210>23
<211>26
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>23
atctcacttc caaaggaaat ctttgt 26
<210>24
<211>30
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>24
agaattaagt ccatttacgt tcaatgttat 30
<210>25
<211>27
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>25
attagtcagg gttctccaaa gagactg 27
<210>26
<211>28
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>26
gtacttgtat cacaacacat aacaacat 28
<210>27
<211>30
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>27
aactaattct gtggttcctg taatatgatt 30
<210>28
<211>30
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>28
gttctttaga aaaacacaac taatcagatg 30
<210>29
<211>25
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>29
aggcacctag gtttgttctg aaggt 25
<210>30
<211>35
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>30
aagtctttta cccattcttt ttatttgaat tgttg 35
<210>31
<211>26
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>31
gaggaaataa agagggcctt tccctt 26
<210>32
<211>36
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>32
gatggattga tttaaaggga attatatata gatata 36
<210>33
<211>27
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>33
catcatgggg ttatgaagct ttggcct 27
<210>34
<211>31
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>34
ctaggtaaat tctacattat ccctttggtt t 31
<210>35
<211>30
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>35
aattttctga gttagccatg aaagattctc 30
<210>36
<211>28
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>36
aggatatggt ttggaagaaa caagattt 28
<210>37
<211>20
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>37
atagagcgag accccctctc 20
<210>38
<211>27
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>38
tcaggaacac tcaactgctc tataagc 27
<210>39
<211>37
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>39
gtagatttta tatatataca cacacgctat atatatt 37
<210>40
<211>35
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>40
gagagattga aattaatata tataaatgaa ttata 35
<210>41
<211>34
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>41
cattgtccat ttttctacag taccattagt cttt 34
<210>42
<211>31
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>42
ttctctttct cattacacct atgtgaaacc a 31
<210>43
<211>28
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>43
ggaggatcat ttgagaattt gagaccag 28
<210>44
<211>37
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>44
attgggtaat tatctcactc atttttatta gtttttg 37
<210>45
<211>29
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>45
cagagaaaca gaacttgtag gatatatag 29
<210>46
<211>30
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>46
gattagaaca ttgtattatt caaggctttc 30
<210>47
<211>28
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>47
gttcaggcct caatatatac ctgtattt 28
<210>48
<211>31
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>48
atatctgaga cttgtagtag aaggccttga g 31
<210>49
<211>31
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>49
tgttatctca agggtactta gaatctttat g 31
<210>50
<211>36
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>50
ccagtcattc atttaaatag ataaagataa atgaaa 36
<210>51
<211>25
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>51
acccatctaa cgcctatctg tattt 25
<210>52
<211>26
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<213> Artificial sequence (artificial sequence)
<400>52
caattcccct actgcctaga cacctt 26
<210>53
<211>24
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>53
tcccagcact cacaatcttg tgag 24
<210>54
<211>23
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>54
ggacttgtaa ggctccacaa ttg 23
<210>55
<211>31
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>55
attggcagac tcacttacaa ataaaacatt c 31
<210>56
<211>27
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>56
agaacacaag atcacatctg ttggcca 27
<210>57
<211>30
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>57
ttccttgtgt acttgttact ggtaattttt 30
<210>58
<211>29
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>58
tcctaacttt tgaggcaatt tgttacaga 29
<210>59
<211>28
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>59
gaaggtagag aaatactgag gaaaaagc 28
<210>60
<211>27
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>60
tagacagaca gatagacaga tcttaac 27
<210>61
<211>31
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>61
cacctataac caatatagaa aatgaggcag g 31
<210>62
<211>20
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>62
ttccgttctc tggagaaccc 20
<210>63
<211>27
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>63
tctatctcca gttggctctg tttctct 27
<210>64
<211>26
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>64
tcctagtagc ctcttcctac aggctt 26
<210>65
<211>36
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>65
tactaatagt gaactacctg caaaagaaat taagaa 36
<210>66
<211>27
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>66
ctgtgttctc ttttctccat ttggtta 27
<210>67
<211>29
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>67
ttacaacatt tctgtatctc tggggtagg 29
<210>68
<211>23
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<213> Artificial sequence (artificial sequence)
<400>68
tgggtaactt gtgtgtcact gag 23
<210>69
<211>26
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<213> Artificial sequence (artificial sequence)
<400>69
tgtctgtctg agtttggaaa tgttta 26
<210>70
<211>26
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>70
attgagggtg cagtaagcta tgatca 26
<210>71
<211>23
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>71
ttccaaggcc aggtacagtg gct 23
<210>72
<211>28
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>72
tttactatga agaagcagct cacacaat 28
<210>73
<211>28
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>73
ttagtcaata aaagcctttc catgaaca 28
<210>74
<211>22
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>74
tcactgcctt cctcatatcc ta 22
<210>75
<211>36
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>75
aacattatat agaatattat acaaaaatga gcaaga 36
<210>76
<211>21
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>76
tccctgagct gccacaatcc t 21
<210>77
<211>32
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>77
aactttaaac aggtcattta agctctctgg gc 32
<210>78
<211>20
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>78
agggcagaaa ccactgttcc 20
<210>79
<211>28
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>79
tctctggaga acatggacta aaacagcc 28
<210>80
<211>35
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>80
gatgaattga agttgaagtt tattgttaat ttagt 35
<210>81
<211>18
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>81
caagggtcag ggatgcca 18
<210>82
<211>28
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>82
ctctgtatag ctattgttct aagtgttg 28
<210>83
<211>28
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>83
aatggctgta ttaatagtgg gaaagaca 28
<210>84
<211>31
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>84
tttcttcttt ctgtgaaggt tattttctcg g 31
<210>85
<211>30
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>85
ggatttatta ggtaaattgg ttcacgtgat 30
<210>86
<211>32
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>86
atgtatgtct gtctgtctat ctatttgtct ca 32
<210>87
<211>35
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>87
gaagtaaatt ctatttatct gctcccttaa atatg 35
<210>88
<211>33
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>88
tatgataagg aattagctca cactgttatg gag 33
<210>89
<211>33
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>89
tattttccag ctttattgag gtattattgg taa 33
<210>90
<211>29
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>90
gtttgttaca taggtttaca acatgccat 29
<210>91
<211>25
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>91
ctaccttttc tatcctcaac cccac 25
<210>92
<211>22
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>92
agatgagaat gaggcactgg ag 22
<210>93
<211>32
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>93
catccactca tctaaaataa aatctcatga aa 32
<210>94
<211>24
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>94
cccttgctac ctttgaaaag actt 24
<210>95
<211>19
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>95
tcccacacca cctgccttt 19
<210>96
<211>31
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>96
atatgcatac tcaccaaagt cattcaattc a 31
<210>97
<211>29
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>97
caccagcaat aataaaactt agcaaccta 29
<210>98
<211>25
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>98
tatccatcca tccacccgtt tttgt 25
<210>99
<211>24
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>99
ggttcaggtt ctgtggaaaa ccct 24
<210>100
<211>24
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>100
aggggaagag tagatacaaa ggaa 24
<210>101
<211>31
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>101
aaatgtgtcc atgtttttca gtattacttt t 31
<210>102
<211>28
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>102
gtttaaattt gcttctggat attggcac 28
<210>103
<211>27
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>103
ttctctgaaa ggtactgtca agaaaat 27
<210>104
<211>30
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>104
cagtgtttac tattagaggt gttttgggtc 30
<210>105
<211>26
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>105
ctcaagcagt tattttggct gacata 26
<210>106
<211>32
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>106
ttcaatacta catcatttac aagctagaga ac 32
<210>107
<211>24
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>107
ataaggaaaa tgacacccca gccc 24
<210>108
<211>27
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>108
actgtcttgt tgaagttggc agtaggg 27
<210>109
<211>31
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>109
ttttgaatgt gtgtaactaa gggaagaatc t 31
<210>110
<211>23
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>110
agagaaagag agacagacag aca 23
<210>111
<211>34
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>111
caagctctct gaatatgttt tgaaaataat gtat 34
<210>112
<211>33
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>112
tgtgaatatc gatgaatgtc aataaaagat tgt 33
<210>113
<211>35
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>113
tgttcatatt atctctttta ttttgagttc tttgc 35
<210>114
<211>33
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>114
ttctctggaa aactctaata aaagtatcaa aga 33
<210>115
<211>28
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>115
aatgttcctt agtcccacct ttctaaga 28
<210>116
<211>26
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>116
atgagtaagt aggcagtgtg catgtg 26
<210>117
<211>22
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>117
gcatccttag ggacagggtt gg 22
<210>118
<211>25
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>118
ttttgttttt ggtaccaggt acatt 25
<210>119
<211>28
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>119
atcatccttt cctctctttc tagtgcaa 28
<210>120
<211>20
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>120
gctgatggaa cctgcttgcc 20
<210>121
<211>35
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>121
cctctctttt tcaatctcta gatagataaa tgtta 35
<210>122
<211>30
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>122
aaattcatca gtgctatgtg gaataaaaaa 30
<210>123
<211>28
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>123
agaaacaagt agtcaaagga gcctttta 28
<210>124
<211>34
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>124
atagtcttta atagtctagt ctgttttgga taac 34
<210>125
<211>29
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>125
tagggataag ttaggacaaa taaaaaatg 29
<210>126
<211>36
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>126
gtcaattcct tgttataaaa ttatatatac atatat 36
<210>127
<211>35
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>127
tactatcaag aaaacaagaa tatttcagaa gaata 35
<210>128
<211>33
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>128
aaaattgagc tactgatctt aactacaaag aac 33
<210>129
<211>25
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>129
ggcgtgtatc tgtagtccta gttac 25
<210>130
<211>30
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>130
aataagataa gagtgtctgg ctcatagaaa 30
<210>131
<211>22
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>131
ctaatgcacc caacattcta ac 22
<210>132
<211>37
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>132
aaccattgat atataggaat catgtgaatt atatctc 37
<210>133
<211>29
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>133
agtggattca tgcagttcaa atccatgct 29
<210>134
<211>22
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>134
cacaggatca gagatgcaaa ta 22
<210>135
<211>26
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>135
ctgcctgaac aaagtaatga aagtgg 26
<210>136
<211>31
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>136
aattctttgc aagttctagg aagagttaaa a 31
<210>137
<211>32
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>137
ataatgagat gaaagaaaga gagaaagaga ga 32
<210>138
<211>25
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>138
accacatgag ccaattctgt ataat 25
<210>139
<211>32
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>139
ggaaaagaaa tcagtatgtc aaagagatat ct 32
<210>140
<211>21
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>140
agccccagga ggattatatt t 21
<210>141
<211>24
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>141
tggcaaaact ctgttagcta ttta 24
<210>142
<211>35
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>142
ttaaaatacc tcaatatgcc acttcataaa cgtat 35
<210>143
<211>24
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>143
aggctgacag tttaccatgt agac 24
<210>144
<211>28
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>144
agccagaata tcatgagcag agaactgg 28
<210>145
<211>21
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>145
catctttgct tggcacactt c 21
<210>146
<211>35
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>146
cattaaaaac attatgaata aaaactgaaa aattc 35
<210>147
<211>37
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>147
ctctttaaaa ttttattgta tctcaggtta tcttttt 37
<210>148
<211>35
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>148
aaagaacaga atcaattgta tatgtatata gatat 35
<210>149
<211>27
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>149
atgtgaaagg cattgtatta gtgttct 27
<210>150
<211>35
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>150
gtctttttaa ataataataa taatacccat gataa 35
<210>151
<211>28
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>151
gtatgttatg agctgagtac attctagg 28
<210>152
<211>23
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>152
tggataaaag gcagctgtag ctg 23
<210>153
<211>25
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>153
atggggcctt ggaaagtata ttagt 25
<210>154
<211>24
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>154
agattcaaca gtggcaaact tttt 24
<210>155
<211>33
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>155
gatatggaat tgacctaagt atccattaat gga 33
<210>156
<211>32
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>156
attgcaatat tcctgaccta aagaaaacat aa 32
<210>157
<211>32
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>157
tgctaaataa cttacacaag ctatcttaag tt 32
<210>158
<211>27
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>158
tctgcaactc tctgaacata tcttcaa 27
<210>159
<211>22
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>159
atctcacacc tccctctctt cc 22
<210>160
<211>32
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>160
accatatagt caggttgtgt tttcataatt tg 32
<210>161
<211>20
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>161
gccagcctgc cttacaggtt 20
<210>162
<211>25
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>162
aagaggggaa tcaaattcaa ccaat 25
<210>163
<211>24
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>163
caagccataa tctcacctca gctt 24
<210>164
<211>24
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>164
ttcagtccat tttccaccag agaa 24
<210>165
<211>28
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>165
ttcagtaatt ctgctgtctc tcccatta 28
<210>166
<211>25
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>166
atttcagagt gaacagtccc acagt 25
<210>167
<211>27
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>167
aagaaaaaca tctcccttct gttttta 27
<210>168
<211>23
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>168
caggcctata tacacaccta tat 23
<210>169
<211>30
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>169
taccatataa gatgctcaat agatagaaat 30
<210>170
<211>31
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>170
aatggtatac ataaattccc atttctgaaa a 31
<210>171
<211>29
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>171
ttttgagtag gcttgcaatc tcttacttt 29
<210>172
<211>18
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>172
cgagtcaggc gagttttt 18
<210>173
<211>35
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>173
gtacaaaatt taggaaaaat taagcaaaac cctac 35
<210>174
<211>27
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>174
atgtgaaatt tggctttacg ctaaatt 27
<210>175
<211>25
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>175
ttttgggtat ctagcctgcc aagag 25
<210>176
<211>35
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>176
acattatgtc tacctatatt tatctctatt tcttt 35
<210>177
<211>30
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>177
tcaccaatta ttaacgtttt gctcaatttg 30
<210>178
<211>26
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>178
actctctgcc cattctgaac tttaca 26
<210>179
<211>25
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>179
agtacatact cacacgcaca cagac 25
<210>180
<211>23
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>180
ttttgtaggt ggcaacaggc cat 23
<210>181
<211>36
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>181
aggataatag tacaaatgat aatgatgatg atgctc 36
<210>182
<211>22
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>182
agtagatagg ggaagcctgt ta 22
<210>183
<211>37
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>183
gtactaaaat tattaaaaat cttattcaat gagctaa 37
<210>184
<211>25
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>184
ctctggagaa ctctaatgca gttgc 25
<210>185
<211>35
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>185
gggaataaaa agcaaaatat tcaagataat gacta 35
<210>186
<211>26
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>186
cctgcttagg aatccaaata agaaac 26
<210>187
<211>30
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>187
atagtcctgc tttcctattt gtactgttca 30
<210>188
<211>31
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>188
ccatgctcat tatttgtaag ttgtaagaaa a 31
<210>189
<211>32
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>189
gattaatcat aaacatttgg gaaggagagt ga 32
<210>190
<211>24
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>190
ggtatgtgtt cttgagcctc ctga 24
<210>191
<211>25
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>191
tgtacatcct ctgcatccgt ttttt 25
<210>192
<211>31
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>192
atcctcctaa gagaccattt atgaaacaat g 31
<210>193
<211>26
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>193
acctaatgat tggatatgga ggtgag 26
<210>194
<211>31
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>194
taacaactct aggaaaagaa ttacaggggg t 31
<210>195
<211>29
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>195
gacttcacca aactgatatt tcgaagtct 29
<210>196
<211>30
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>196
gtttacttac aatattccct tctaggtaac 30
<210>197
<211>25
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>197
ataaagctgg agaacatgtc atgtg 25
<210>198
<211>18
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>198
ctgtctgcgc tggccttt 18
<210>199
<211>26
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>199
gcagggtgat agaaaggtag aaaagg 26
<210>200
<211>28
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>200
aaaaacatac taatcaacca aggcaatt 28
<210>201
<211>25
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>201
ccaactatct gctatctgga aaacc 25
<210>202
<211>25
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>202
atacaaacgc tcctctgact gccac 25
<210>203
<211>23
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>203
ccaacaaaag gactcaccca tca 23
<210>204
<211>23
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>204
ccaacaaaag gactcaccca tca 23
<210>205
<211>24
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>205
gggaattggc tcaatcacag agac 24
<210>206
<211>35
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>206
tataaagaat tggcttacat aattatggat gctga 35
<210>207
<211>25
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>207
gagtgccaat atttgaggac agaag 25
<210>208
<211>26
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>208
tattctgtct gacatcaagc tactac 26
<210>209
<211>26
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>209
gatgatacag tgaggttggg gtatgc 26
<210>210
<211>23
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>210
tctcacccca tgttcttccc acc 23
<210>211
<211>20
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>211
gaagcccagg gaagacaata 20
<210>212
<211>33
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>212
aactatttca atcattggca gatattatga ctt 33
<210>213
<211>25
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>213
tgaaatcatg tcttttgcag caaca 25
<210>214
<211>23
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>214
ggtgtccttg ctcattcctg ggc 23
<210>215
<211>24
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>215
ccatgttatc ttctgggatg caaa 24
<210>216
<211>31
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>216
cattcctctt ttgatgaaca tttaggttct t 31
<210>217
<211>28
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>217
ttaaaaattc ctcaaactgg ctgggtgt 28
<210>218
<211>28
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>218
tcttcaatga gtccgtagtc ttaagaat 28
<210>219
<211>27
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>219
tatagtgtca tctgtttcag cctgaga 27
<210>220
<211>19
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>220
tcacccagct ggagtgcag 19
<210>221
<211>23
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>221
gtttccctaa gttgcccaga ctg 23
<210>222
<211>28
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>222
ttatactaag cttctcgagg gttggagg 28
<210>223
<211>30
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>223
gctattttta tcacggatgt tacatttcat 30
<210>224
<211>34
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>224
cacagtgtta tatttacaca aacctagatt ggtc 34
<210>225
<211>28
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>225
tttagttctg actctgtcac ctaggctg 28
<210>226
<211>25
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>226
gggttgcttt taaacctttg tttaa 25
<210>227
<211>25
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>227
gtcctggtgt catgctttta aagat 25
<210>228
<211>33
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>228
ttgataattt tacaaagatt ctctgcccta cag 33
<210>229
<211>30
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>229
cttatctctc tctacttgtt ttgtagttac 30
<210>230
<211>35
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>230
gtttctattt ttcaggcata aataaattta ctaag 35
<210>231
<211>21
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>231
gcagtgacgc acctaacact c 21
<210>232
<211>31
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>232
cacatctaaa catgcataca cacataaacg a 31
<210>233
<211>31
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>233
ggagaaccct aaaacaaaat aaaaatctat t 31
<210>234
<211>25
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>234
tttctggaga accctgacta ataca 25
<210>235
<211>26
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>235
tgcacacata acctttgatc tgcaga 26
<210>236
<211>30
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>236
atcttggtga ataaaataaa gccaggaaag 30
<210>237
<211>22
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>237
ctggcatcct tagcttgcag aa 22
<210>238
<211>35
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>238
aaggaagtat gttaagtaat ccagaaaaga gatga 35
<210>239
<211>30
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>239
ggtatgtctt tatcagtagt gtgaaaatgg 30
<210>240
<211>35
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>240
tttgtagtgt gatagacaat ttttagtgtc tgcac 35
<210>241
<211>35
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>241
ccaattgata ataatttctg catagtatct gaggt 35
<210>242
<211>21
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>242
tctcctctcc cccaggccag a 21
<210>243
<211>20
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>243
tggagagctg ggagtactac 20
<210>244
<211>28
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>244
aatatagggc tggcattaca aacctgcc 28
<210>245
<211>34
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>245
attggatatc aaattccagg aatatagaat gtat 34
<210>246
<211>33
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>246
cttcctagtt tagacagaag ttttaaaaaa atc 33
<210>247
<211>23
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>247
tagaatacac tgaagggcac cat 23
<210>248
<211>26
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>248
tggaaatcat agccaacttc ctactt 26
<210>249
<211>24
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>249
gtcagtgttc atgcctacat ccct 24
<210>250
<211>31
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>250
gtttacagat aagaggaatc ctttactagg a 31
<210>251
<211>33
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>251
taagggatac tgaaattttt gtctttaaaa tta 33
<210>252
<211>26
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>252
ctgtgaaacc actatagatg ttaaat 26
<210>253
<211>32
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>253
caagtcgttt tccagataga taattagaaa ta 32
<210>254
<211>31
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>254
ttgttgaaca aaacactagt gagtaaaata c 31
<210>255
<211>31
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>255
ttgttgaaca aaacactagt gagtaaaata c 31
<210>256
<211>35
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>256
cattaaaaac attatgaata aaaactgaaa aattc 35
<210>257
<211>37
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>257
ctctttaaaa ttttattgta tctcaggtta tcttttt 37
<210>258
<211>35
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>258
aaagaacaga atcaattgta tatgtatata gatat 35
<210>259
<211>27
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>259
atgtgaaagg cattgtatta gtgttct 27
<210>260
<211>27
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>260
tggcatattg tgaaatttct cagcttc 27
<210>261
<211>35
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>261
ttgtttctaa agtatttttc ttaaaaagga aaaag 35
<210>262
<211>30
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>262
acaattcaag atgagatttg gtgtaggaca 30
<210>263
<211>31
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>263
tttttatggc attcaatgat ttccttactt t 31
<210>264
<211>34
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>264
tatacaacat tacataattg caaatgctat gata 34
<210>265
<211>29
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>265
attgacagaa ttgcaccaaatattggtaa 29
<210>266
<211>25
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>266
gtaaggagga gatttgtgct ggtca 25
<210>267
<211>36
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>267
acaagtggta tatctaacag aataaatgct atatat 36
<210>268
<211>26
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>268
acacttgtac cctaaaaagc tgttga 26
<210>269
<211>29
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>269
acatgctaac tttgaatggt atttcaatg 29
<210>270
<211>29
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>270
cttcaattat acatttctgg gcagatcag 29
<210>271
<211>23
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>271
gaagagggca caaaggattc atc 23
<210>272
<211>25
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>272
cctcaatatg aaaagctgct tataa 25
<210>273
<211>30
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>273
aattctattt tctgtgagct ctcaaaatat 30
<210>274
<211>37
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>274
tgttatcaat ctctttctgt gtctaattta caaatta 37
<210>275
<211>28
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>275
tgctgggatt gcatattcat ttttcgtc 28
<210>276
<211>30
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>276
ctctcaaaat atgaacaaaa tctgaatcca 30
<210>277
<211>25
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>277
gcacaagagg agaggaaaag gaagt 25
<210>278
<211>31
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>278
agaataggca gatacacaga atatgtccag a 31
<210>279
<211>35
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>279
gaaatagtat aaacttactg gtgttttcct aatta 35
<210>280
<211>24
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>280
tcaagtactg ccaggcaatt aaag 24
<210>281
<211>24
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>281
cagcacatca attctgtgtc actc 24
<210>282
<211>30
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>282
gtagaatttg caagcagaaa tgatatcgat 30
<210>283
<211>28
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>283
tttcttatgt aggttgatgc aaaagtaa 28
<210>284
<211>26
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>284
agcctaaaac acctttccca gactaa 26
<210>285
<211>28
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>285
atgaaaagaa agaaaacgtg gtacaggt 28
<210>286
<211>26
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>286
acattttact gcatcctcct gaagaa 26
<210>287
<211>22
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>287
aactggcata gcttgagcac tg 22
<210>288
<211>27
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>288
atagacaagg ggattggatg gatggat 27
<210>289
<211>28
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>289
agcctccata attactagag caaatctc 28
<210>290
<211>19
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>290
cctgggcaat gtggggaga 19
<210>291
<211>29
<212>DNA
<213> Artificial sequence (artificailseq)
<400>291
ggattctcca gacaaataat accaaaagg 29
<210>292
<211>29
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>292
ttttgtacct gtcagaaaag tgaagatat 29
<210>293
<211>36
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>293
taatggtaat ttttcaaact cttgtcataa aatgtt 36
<210>294
<211>31
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>294
caattttaag aagccaaaga caagtttgtt t 31
<210>295
<211>29
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>295
gaagttaata tttcaaggcc acaaattcc 29
<210>296
<211>25
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>296
aattgacaaa tttctgggct ggcgg 25
<210>297
<211>23
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>297
tgatctctgt gtctccctac ccc 23
<210>298
<211>17
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>298
cacaggcttg tccccaa 17
<210>299
<211>31
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>299
aaacaagtag ttgaagaata ctttgctttt t 31
<210>300
<211>29
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>300
acattgtttc tgtgtgtatc tgtcaagtt 29
<210>301
<211>22
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>301
ggggatgtag gaggtgtccc ca 22
<210>302
<211>28
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>302
ggttctattg gatgtaaatt ccattttt 28
<210>303
<211>23
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>303
ccatcagact gttaggacac ata 23
<210>304
<211>27
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>304
atagctcaaa ttacaggagt gagccac 27
<210>305
<211>26
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>305
atgcccacag ttaggagaag aaagta 26
<210>306
<211>36
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>306
taagtgatat tttatatagt catctgtcta cacaga 36
<210>307
<211>27
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>307
cacacctaat ctttaaagcc ctccttc 27
<210>308
<211>36
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>308
ttattctccc ttttctttat ttctttatat cacaca 36
<210>309
<211>23
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>309
acagtacaga tggcagatgt tct 23
<210>310
<211>21
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>310
acagctggat tatgggccag t 21
<210>311
<211>20
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>311
gcagatttcc acggggggca 20
<210>312
<211>30
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>312
gccaaagaca taggttgaaa atattgttat 30
<210>313
<211>24
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>313
ctcgcaaagg ctaccatcat atta 24
<210>314
<211>24
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>314
ctttgaatct caggccattc ggtt 24
<210>315
<211>23
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>315
acagccagac caacaggagg att 23
<210>316
<211>27
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>316
ctaacacacc aagtaaggat aggccag 27
<210>317
<211>31
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>317
tttcacattg ctataaagaa ctacctgaga c 31
<210>318
<211>31
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>318
atattttggg cacatgatca aaaagttttc a 31
<210>319
<211>24
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>319
accgtgacta atacagaagc ctta 24
<210>320
<211>30
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>320
tacttgtagt aaattctacc atgggactgc 30
<210>321
<211>23
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>321
ctggagagct aatacaggca act 23
<210>322
<211>18
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>322
cccttgctct gacccagt 18
<210>323
<211>34
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>323
ccaggaggaa aatataaaga acatagagta atcc 34
<210>324
<211>26
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>324
ttaggaagca tccatttctc cctaaa 26
<210>325
<211>35
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>325
tttaggtatg ttcttattta tgttcctttc tctgt 35
<210>326
<211>29
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>326
atgacaagtg ttctatcata ccctttata 29
<210>327
<211>29
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>327
tgccaataca tgcaggtaaa ataaacaca 29
<210>328
<211>30
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>328
aattatttag ttgggtcaag tatggagcag 30
<210>329
<211>37
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>329
aaatatatct gtaattccta gaaaaatgtc aggttct 37
<210>330
<211>28
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>330
atgactgcca ttgaaaagat agtttatt 28
<210>331
<211>30
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>331
tctaaccaat ttagtcatta gcagtgggct 30
<210>332
<211>31
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>332
tctaaaatta aatatgtgct gccttgacat t 31
<210>333
<211>28
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>333
atggaacaag taggattccc aatagctg 28
<210>334
<211>34
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>334
tccctatctg aaaacaaatt tataagcatg gttt 34
<210>335
<211>29
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>335
tggggctatt gaatcatatc cttatgaac 29
<210>336
<211>23
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>336
ggctatctga aggaacaccc aac 23
<210>337
<211>31
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>337
aaggaaattt aacagaacat gtcttttcca g 31
<210>338
<211>26
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>338
cttttagagc taatccaagc actgat 26
<210>339
<211>34
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>339
tgtagaatta aaatatttct ttaggtggga gccc 34
<210>340
<211>30
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>340
gatcaagagc agcaaagaaa attttaaaga 30
<210>341
<211>30
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>341
taataattgc aacatgactc agtgagggaa 30
<210>342
<211>28
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>342
cattcttgct actggtcttt atcattct 28
<210>343
<211>21
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>343
cctatcccag cagttcaggg t 21
<210>344
<211>22
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>344
taggtcctac tgctcatggc ac 22
<210>345
<211>24
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>345
acccttcctg attacctatt tttg 24
<210>346
<211>36
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>346
ataatggact atataatatg tgacatagca tatata 36
<210>347
<211>30
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>347
atctcacatt atgatttttc tggatggata 30
<210>348
<211>20
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>348
ggtcactgca gcctcgaact 20
<210>349
<211>19
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>349
gaccagggcc agctaggag 19
<210>350
<211>26
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>350
gcagtcccac acatctaaaa atctac 26
<210>351
<211>24
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>351
taactccttc aggcactcca gata 24
<210>352
<211>33
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>352
atagatggat atttactaca aggtattggc tca 33
<210>353
<211>23
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>353
caggctgaga aatcccaaga tct 23
<210>354
<211>30
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>354
gtcttgccat tataatagaa tgctgaagat 30
<210>355
<211>37
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>355
tataacaaat cacataagct tatctatcat ctctctc 37
<210>356
<211>27
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>356
aacaagccct ctatcaaaag atcccct 27
<210>357
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>357
ggctagctag gatggatgtc ctcc 24
<210>358
<211>18
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>358
tgcaggcttg gggcagat 18

Claims (4)

1.一种用于复杂亲缘关系鉴定的试剂盒,其特征在于,所述的试剂盒包括用于58个连锁常染色体的复合分型体系;所述的复合分型体系包括用于扩增58个连锁常染色体STR基因座的PCR引物组合2;1. a test kit for complex kinship identification, is characterized in that, described test kit comprises the composite typing system for 58 linked autosomes; Described composite typing system comprises for amplifying 58 PCR primer set 2 for linked autosomal STR loci; 所述的58个连锁常染色体STR基因座为D1S3736、D1S1665、D1S2127、D1S2138、D1S1642、D1S518、D1S1604、D1S3729、D1S3727、D2S1336、D2S1779、D2S1771、D2S437、D2S2970、D2S2944、D2S1327、D3S2402、D3S1766、D4S2411、D4S3351、D4S243、D4S2426、D4S2373、D5S1490、D5S2856、D5S2495、D5S2855、D5S1722、D5S1725、D6S1048、D6S1275、D7S796、D7S1799、D8S2324、D8S2330、D8S1144、D9S745、D9S301、D9S1124、D10S1246、D10S2485、D11S1983、D11S2363、D11S1368、D12S393、D12S1063、D13S1807、D13S1492、D14S738、D14S301、D14S583、D15S816、D15S1514、D18S872、D18S972、D18S548、D20S1145、D20S477;所述的58个连锁常染色体STR基因座为D1S3736、D1S1665、D1S2127、D1S2138、D1S1642、D1S518、D1S1604、D1S3729、D1S3727、D2S1336、D2S1779、D2S1771、D2S437、D2S2970、D2S2944、D2S1327、D3S2402、D3S1766、D4S2411、 D4S3351、D4S243、D4S2426、D4S2373、D5S1490、D5S2856、D5S2495、D5S2855、D5S1722、D5S1725、D6S1048、D6S1275、D7S796、D7S1799、D8S2324、D8S2330、D8S1144、D9S745、D9S301、D9S1124、D10S1246、D10S2485、D11S1983、D11S2363、D11S1368、 D12S393, D12S1063, D13S1807, D13S1492, D14S738, D14S301, D14S583, D15S816, D15S1514, D18S872, D18S972, D18S548, D20S1145, D20S477; 所述的PCR引物组合2包括正向引物和反向引物;Described PCR primer combination 2 includes forward primer and reverse primer; 所述的正向引物如下:The forward primers are as follows:
Figure FDA0002393663260000011
Figure FDA0002393663260000011
Figure FDA0002393663260000021
Figure FDA0002393663260000021
Figure FDA0002393663260000031
Figure FDA0002393663260000031
所述的反向引物如下:The reverse primers are as follows:
Figure FDA0002393663260000032
Figure FDA0002393663260000032
Figure FDA0002393663260000041
Figure FDA0002393663260000041
Figure FDA0002393663260000051
Figure FDA0002393663260000051
所述的PCR引物组合2中正向引物和反向引物的工作浓度为0.1μM;所述的试剂盒还包括Index接头序列和DNA聚合酶,所述的Index接头序列包括IGT-I5Index和IGT-I7 Index,工作浓度均为10μM。The working concentration of the forward primer and the reverse primer in the PCR primer combination 2 is 0.1 μM; the kit further includes an Index linker sequence and a DNA polymerase, and the Index linker sequence includes IGT-I5Index and IGT-I7 Index, the working concentration is 10 μM.
2.根据权利要求1所述的试剂盒,其特征在于:所述的试剂盒还包括用于扩增61个连锁常染色体STR基因座的PCR引物组合3;2. The test kit according to claim 1, characterized in that: the test kit further comprises a PCR primer combination 3 for amplifying 61 linked autosomal STR loci; 所述的61个连锁常染色体STR基因座为D1S532、D1S1611、D1S3733、D1S533、D1S1614、D2S2977、D2S1374、D2S1394、D2S2966、D2S2969、D2S1371、D2S434、D2S1338、D3S4016、D3S2388、D4S1626、D4S1653、D5S2858、D5S2796、D5S1463、D5S815、D5S2499、D5S2498、D6S1043、D6S1274、D6S1056、D6S1013、D6S1054、D7S820、D7S2205、D7S3071、D7S2845、D8S2326、D8S1464、D8S2320、D8S1470、D8S588、D8S1471、D9S2026、D9S747、D9S2128、D10S2469、D10S1238、D11S4464、D11S4958、D13S1818、D13S767、D14S615、D14S608、D14S597、D14S302、D14S749、D16S767、D16S3393、D18S537、D18S875、D18S1367、D20S1152、D20S206、D20S607、D20S1146;所述的61个连锁常染色体STR基因座为D1S532、D1S1611、D1S3733、D1S533、D1S1614、D2S2977、D2S1374、D2S1394、D2S2966、D2S2969、D2S1371、D2S434、D2S1338、D3S4016、D3S2388、D4S1626、D4S1653、D5S2858、D5S2796、 D5S1463、D5S815、D5S2499、D5S2498、D6S1043、D6S1274、D6S1056、D6S1013、D6S1054、D7S820、D7S2205、D7S3071、D7S2845、D8S2326、D8S1464、D8S2320、D8S1470、D8S588、D8S1471、D9S2026、D9S747、D9S2128、D10S2469、D10S1238、D11S4464、 D11S4958, D13S1818, D13S767, D14S615, D14S608, D14S597, D14S302, D14S749, D16S767, D16S3393, D18S537, D18S875, D18S1367, D20S1152, D20S607, D14S14; 所述的PCR引物组合3包括正向引物和反向引物;Described PCR primer combination 3 includes forward primer and reverse primer; 所述的正向引物如下:The forward primers are as follows:
Figure FDA0002393663260000061
Figure FDA0002393663260000061
Figure FDA0002393663260000071
Figure FDA0002393663260000071
Figure FDA0002393663260000081
Figure FDA0002393663260000081
所述的反向引物如下:The reverse primers are as follows:
Figure FDA0002393663260000082
Figure FDA0002393663260000082
Figure FDA0002393663260000091
Figure FDA0002393663260000091
Figure FDA0002393663260000101
Figure FDA0002393663260000101
所述的PCR引物组合3中正向引物和反向引物的工作浓度为0.1μM。The working concentration of the forward primer and the reverse primer in the PCR primer combination 3 was 0.1 μM.
3.根据权利要求2所述的试剂盒,其特征在于,所述的的试剂盒还包括用于扩增60个连锁常染色体STR基因座的PCR引物组合1;3. test kit according to claim 2, is characterized in that, described test kit also comprises the PCR primer combination 1 for amplifying 60 linked autosomal STR loci; 所述的60个连锁常染色体STR基因座为D1S2131、D1S3721、D1S2130、D1S1600、D1S1653、D1S1660、D1S3732、D2S1364、D2S2734、D2S1396、D2S428、D2S435、D2S1387、D2S1792、D2S1399、D2S2959、D3S2431、D3S4547、D4S1643、D4S2408、D4S3326、D4S2368、D5S2845、D5S1473、D5S813、D5S1716、D5S1459、D5S1487、D5S1466、D5S2496、D5S2501、D6S1019、D6S2417、D6S2412、D6S1284、D7S1820、D7S3050、D7S821、D8S594、D8S1468、D8S2322、D8S569、D8S1475、D9S746、D9S319、D9S2149、D11S2010、D11S1392、D12S376、D12S1052、D13S317、D13S790、D14S1432、D14S1428、D15S644、D15S1507、D16S752、D16S485、D20S481和D20S1151;所述的60个连锁常染色体STR基因座为D1S2131、D1S3721、D1S2130、D1S1600、D1S1653、D1S1660、D1S3732、D2S1364、D2S2734、D2S1396、D2S428、D2S435、D2S1387、D2S1792、D2S1399、D2S2959、D3S2431、D3S4547、D4S1643、 D4S2408、D4S3326、D4S2368、D5S2845、D5S1473、D5S813、D5S1716、D5S1459、D5S1487、D5S1466、D5S2496、D5S2501、D6S1019、D6S2417、D6S2412、D6S1284、D7S1820、D7S3050、D7S821、D8S594、D8S1468、D8S2322、D8S569、D8S1475、D9S746、 D9S319, D9S2149, D11S2010, D11S1392, D12S376, D12S1052, D13S317, D13S790, D14S1432, D14S1428, D15S644, D15S1507, D16S752, D16S485, D205111 and D; 所述的PCR引物组合1包括正向引物和反向引物;Described PCR primer combination 1 includes forward primer and reverse primer; 所述的正向引物如下:The forward primers are as follows:
Figure FDA0002393663260000111
Figure FDA0002393663260000111
Figure FDA0002393663260000121
Figure FDA0002393663260000121
Figure FDA0002393663260000131
Figure FDA0002393663260000131
所述的反向引物如下:The reverse primers are as follows:
Figure FDA0002393663260000132
Figure FDA0002393663260000132
Figure FDA0002393663260000141
Figure FDA0002393663260000141
Figure FDA0002393663260000151
Figure FDA0002393663260000151
所述的PCR引物组合1中正向引物和反向引物的工作浓度为0.1μM。The working concentration of the forward primer and the reverse primer in the PCR primer combination 1 is 0.1 μM.
4.根据权利要求1-3任一项所述的试剂盒,其特征在于,所述的试剂盒还包括将基因组DNA制成可供测序的文库的试剂。4. The kit according to any one of claims 1-3, characterized in that, the kit further comprises a reagent for making genomic DNA into a library available for sequencing.
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