CN114410798B - System for detecting composite amplification of chain STR loci on human chromosome one and chromosome two and application thereof - Google Patents

System for detecting composite amplification of chain STR loci on human chromosome one and chromosome two and application thereof Download PDF

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CN114410798B
CN114410798B CN202111565920.4A CN202111565920A CN114410798B CN 114410798 B CN114410798 B CN 114410798B CN 202111565920 A CN202111565920 A CN 202111565920A CN 114410798 B CN114410798 B CN 114410798B
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刘秋玲
吕德坚
赵虎
杨建广
黄斌
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Sun Yat Sen University
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Abstract

The invention relates to the technical field of nucleic acid detection, in particular to a composite amplification detection system for a chain STR (short tandem repeat) locus on a first chromosome and a second chromosome of a human and application thereof. The invention screens closely linked STR loci with high polymorphism from chromosome 1 and chromosome 2 respectively, and establishes 2 groups of complex amplification systems. The closely linked STR composite amplification detection system developed by the invention is not only suitable for genetic identification, but also can distinguish single parent diploid with multiple mutation sites from other genetic relationship, is used for prenatal diagnosis of chromosome abnormality, and has wide market application prospect.

Description

System for detecting composite amplification of chain STR loci on human chromosome one and chromosome two and application thereof
Technical Field
The invention relates to the technical field of nucleic acid detection, in particular to a composite amplification detection system for a chain STR (short tandem repeat) locus on a first chromosome and a second chromosome of a human and application thereof.
Background
Paternity testing is a difficulty in forensic testing. Identification of the identity of the nameless, identification of the misshapen, identification of the casualty or death of the traffic accident, and identification of the identity of even the victim of the catastrophic event may involve genetic identification. For genetic identification without parental participation, in addition to the fact that the Y chromosome and mitochondrial DNA genetic markers can exclude father or mother genetic relationships, when the genetic markers which are not linked on autosomes are applied, genetic conditions of alleles cannot be directly observed among identified individuals, so that the genetic relationships cannot be directly excluded, and the genetic relationships can only be evaluated by comparing the probability that the genetic and unrelated individuals share the alleles. Because of the genetic background of STRs, it appears that many STRs have allele frequencies greater than 0.125 or greater, which is greater than a value where the tertiary relatives have 12.5% or the secondary relatives have 25% of the same alleles, and thus cannot distinguish tertiary relatives or unrelated individuals, which have suffered from bottlenecks in the identification of the secondary or tertiary relatives. Obviously, the autosomal unlinked STR has a good effect on the identification of the first-level relative (isotactic cells) only, but cannot distinguish the second-level relative, the third-level relative or the unrelated individuals, so that the genetic relationship identification cannot be performed.
In the human genome, there is a class of closely linked STR loci, linked STR constitutive haplotypes (haplotyes) inherited from the parent to the offspring, closely linked STR haplotypes having a high degree of polymorphism. The probability of having the same haplotype among unrelated individuals is very small, the interference of low polymorphism and genetic background of the conventional non-linkage STR can be overcome, and the difficult problem that the two-level or more genetic relationships can not be identified and different genetic relationships can not be distinguished at present is overcome. If the polymorphism of a closely linked STR group haplotype is infinitely high, the probability of having the same haplotype between unrelated individuals is very small, and only individuals with genetic relationship will have the same haplotype, so that individuals with genetic relationship and individuals without genetic relationship can be distinguished.
However, except for the X chromosome STR gene locus, the existing STR kit applied to forensic identification is not closely linked STR, and a closely linked STR composite amplification system which can be applied to actual paternity identification is not reported at home and abroad.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a composite amplification detection system for linkage STR loci on a first chromosome and a second chromosome of a human and application thereof.
The invention screens out closely linked STR loci with high polymorphism from chromosome one and chromosome two, and establishes a composite amplification system. The closely linked STR composite amplification system developed by the invention is not only suitable for genetic identification, but also can distinguish the single parent diploid with multiple mutation sites from the exclusion of genetic relationship. And the kit is used for prenatal diagnosis of chromosome abnormality, and has wide market application prospect.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: a28-locus multiplex amplification detection system closely linked on a first chromosome and a second chromosome of a human is provided, wherein the system I is used for amplifying 15 loci on the first chromosome and the system II is used for amplifying 13 loci on the second chromosome.
The 15 loci on chromosome 1 are respectively: D1S3721, D1S2130, D1S3467, D1S3724, D1S1631, GATA133a08, D1S1642, D1S518, D1S3731, D1S3468, D1S1670, D1S1656, D1S3462, D1S1634 and D1S1609;
the 13 loci on chromosome 2 are respectively: D2S2696, D2S410, D2S437, D2S2970, D2S1339, D2S1399, D2S2987, D2S1391, D2S1361, D2S1371, D2S434, D2S1338, D2S427.
As a preferred embodiment of the detection system of the present invention, the primer has a sequence as shown in SEQ ID NO: 1-56.
As a preferred embodiment of the detection system of the present invention, at least one of the primers corresponding to each locus is fluorescently labeled at the 5' end.
As a preferred embodiment of the detection system of the present invention, the primer is labeled with FAM, HEX, TAM or ROX fluorescein, respectively.
As a preferred embodiment of the detection system of the present invention, the primers used in System 1 for amplifying the 15 loci located on chromosome 1 are labeled with the following luciferin, respectively: FAM fluorescein markers GATA133a08, D1S1634, D1S3731 and D1S3468; HEX fluorescein markers D1S1656, D1S1609, D1S3462, and D1S1670; TAMRA fluorescein markers D1S3724, D1S3721, D1S1642 and D1S3467; ROX fluorescein markers D1S1631, D1S518 and D1S2130.
As a preferred embodiment of the detection system of the present invention, the primers used for amplifying 13 loci located on chromosome 2 of System II are labeled with fluorescein as follows: FAM fluorescein label: D2S2978, D2S410, D2S437, and D2S1339; HEX fluorescein markers D2S1399, D2S2970 and D2S2696; TAMRA fluorescein markers D2S1391, D2S1361 and D2S427; ROX fluorescein labels D2S1371, D2S1338, and D2S434.
As a preferred embodiment of the detection system of the present invention, the 28 loci are 9 linkage groups in total;
wherein 15 loci on chromosome 1 can constitute 5 linkage groups: i: D1S3721-D1S2130; II: D1S3467-D1S3724; III: D1S1631-GATA133a08; IV: D1S1642-D1S518-D1S3731-D1S3468; v: D1S1670-D1S1656-D1S3462-D1S1634-D1S1609;
the 13 loci on chromosome 2 can constitute 4 linkage groups: i: D2S2696-D2S410-D2S437-D2S2970; II: D2S1339-D2S1399; III: D2S2978-D2S1391-D2S1361; IV: D2S1371-D2S434-D2S1338-D2S427.
As a preferred embodiment of the detection system of the present invention, the amplification system is: 5. Mu.l of PCR reaction buffer, 3. Mu.l or 4. Mu.l of primer mixture (system 1: 4. Mu.l; system II: 3. Mu.l), 1. Mu.l of DNA polymerase, 2. Mu.l of template DNA, and sterile water were added to 15. Mu.l.
As a preferred embodiment of the detection system of the present invention, the amplification procedure is: 95 ℃ for 11min; 15sec at 95 ℃, 15sec at 60 ℃ for 30 cycles; and at 60℃for 5min.
The invention also provides application of the detection system in paternity test or paternity test.
The invention has the beneficial effects that:
(1) The invention provides an AS-STR locus fluorescence labeling composite amplification system which is suitable for Chinese crowds, can rapidly and conveniently distinguish secondary relatives, tertiary relatives or irrelevant individuals aiming at non-closely linked autosomes STR, Y-STR and X-STR, and can not identify the alternate relatives and the application.
(2) Tightly interlocked: the 28 loci are positioned on the first chromosome and the second chromosome and are tightly linked, the 28 loci can form 9 linkage groups, and haplotype data of the 9 linkage groups can be analyzed simultaneously.
(3) The individual recognition rate is high: the haplotype individual recognition rate of the 9 linkage groups of 28 linkage loci in the fluorescent marker compound amplification system reaches more than 0.99999999;
(4) The multiplex amplification system of the present invention has been used in the inventor's laboratory to detect samples of multiple generations of families and multiple child families. The result shows that the linked STR gene locus forms a monomer type and is stably inherited from a parent to a offspring, and the result shows that the invention can break through the difficult problem that three-level and more-level genetic relationships can not be identified at present and different genetic relationships can not be distinguished, and provides scientific basis for accurately evaluating the different genetic relationships. The locus selected by the invention has good stability, low mutation rate and accurate parting result, and can meet the requirement of actual examination;
(5) The invention firstly develops a group of 15 and 13 loci linked on chromosome 1 and chromosome 2 respectively in China, and simultaneously a five-color fluorescent-labeled multiplex amplification detection system for multiplex amplification in a single reaction tube.
(6) The fluorescent-labeled primer composite amplification technology is quick and convenient, PCR products can be electrophoresed by using genetic analyzers such as ABI 3500, 3130, 3100, 310 and 377, and the results are automatically analyzed, so that the method can be standardized and internationalized, and the accuracy of data comparison in different laboratories is ensured.
(7) The invention can prepare two kits, can be commercialized, can fill the blank of the fluorescent composite amplification kit without the interlocked loci in China, can also supplement the deficiency of the international composite amplification kit, can be used AS the interlocked AS-STR locus composite amplification kit with Chinese characteristics, can be used for paternity test, individual identification, is particularly suitable for deducing complex relationships with more than two levels, improves the identification capability of three-level or higher-level relationships, and provides important scientific evidence for the identification of the special cases.
(8) The linked AS-STR locus fluorescent marker composite amplification system can provide a prenatal diagnosis method for chromosome-linked genetic diseases, also provides a new identification method for the paternity test which is difficult to distinguish secondary relatives, tertiary relatives and the like from unrelated individuals only by non-linked autosomes STR, Y-STR and X-STR, and provides a prenatal diagnosis of abnormal inheritance of the first chromosome and the second chromosome and a method for distinguishing whether multi-site mutation is derived from a single parent diploid or is removed from the paternity test, and has wide application prospect.
Drawings
FIG. 1 (A) is a 9947A electropherogram of 15 STR loci multiplex amplification positive samples on chromosome 1; FIG. 1 (B) is a 9947A electropherogram of a multiplex amplification positive sample of 13 STR loci on chromosome 2.
FIG. 2 (A) is a composite amplification electropherogram of 15 linked STR loci on chromosome 1 of a master sample; FIG. 2 (B) is a composite amplification electropherogram of 15 linked STR loci on chromosome 1 of a maternal sample; FIG. 2 (C) is a composite amplification electropherogram of 15 linked STR loci on chromosome 1 of a suspected grandchild sample.
FIG. 3 (A) is a composite amplification electropherogram of 13 linked STR loci on chromosome 2 of a master sample; FIG. 3 (B) is a composite amplification electropherogram of 13 linked STR loci on chromosome 2 of a maternal sample; FIG. 3 (C) is a composite amplification electropherogram of 13 linked STR loci on chromosome 2 of suspected grandchild sample;
FIG. 4 (A) is a family analysis chart of typing results of 15 STR loci on chromosome 1 for a ancestor, mother, and grandson sample; FIG. 4 (B) is a family analysis of typing results of 13 STR loci on chromosome 2 for ancestor, mother, and grandson samples.
FIG. 5 is a family analysis chart of the typing results of example 2:
from FIG. 5, it was found that the linked STRs on chromosome 2 are stably inherited in the fourth generation family as haplotypes (e.g., haplotypes 29/15.2/5/16/12/13/13/12/22 for 25 individuals/were transmitted from a once-outside ancestor by a generation, haplotype 13/12/23/13 was transmitted from a once-outside ancestor by a generation.)
Note that: alleles of 15 loci in the chromosome 1 multiplex amplification map are not completely sequenced, and are temporarily represented by bp values. In fig. 5, D2S410 genotypes 152, 140, 120, 130, 142 are values of 10 for the actual allele genotypes, since the analysis software typing values cannot have decimal points. 152. 140, 120, 130, 142 are 15.2, 14, 12, 13, 14.2.
Detailed Description
In order to more clearly describe the technical solution of the present invention, the following description is further given by way of specific examples, but not by way of limitation, only some examples of the present invention.
Example 1 selection of loci in a Complex amplification System
(1) Tightly interlocked
And screening closely linked STR loci positioned on the same chromosome from the genome, wherein a linked pair or a linked group consisting of the closely linked STRs is stably inherited according to monomers, and the genetic distance of the STR loci in each linked group is less than or equal to 3cM. According to the standards of the literature (InturriS, et al, forensic Sci intGenet.2011; 5:152-4), a genetic distance of 3cM or less can be considered to be tightly linked. Different STR linkage groups are positioned on different chromosomes, or a plurality of linkage groups are positioned on the same chromosome but far apart, so that the STR linkage groups can be freely recombined, and a composite amplification system is constructed by using STR loci of the linkage groups. The use of these systems to detect samples for genetic inference allows for the combined use of linked loci (a linked group can be considered as a locus) to enhance the polymorphism of genetic markers; and considering the free recombination between the chain STR groups, the forensic genetics statistics can be carried out between the groups according to the product rule.
Of the 28 loci of the invention, 15 on chromosome 1, 6 on the short arm (GATA 133a08, D1S1631, D1S3467, D1S3724, D1S3721 and D1S 2130) and 9 on the long arm (D1S 518, D1S1642, D1S3731, D1S3468, D1S1656, D1S3462, D1S1670, D1S1634 and D1S 1609) can constitute 5 linkage groups:
Ⅰ：D1S3721-D1S2130；
Ⅱ：D1S3467-D1S3724；
Ⅲ：D1S1631-GATA133A08；
Ⅳ：D1S1642-D1S518-D1S3731-D1S3468；
Ⅴ：D1S1670-D1S1656-D1S3462-D1S1634-D1S1609。
on chromosome 2 there are 13 loci D2S2696, D2S410, D2S437, D2S2970, D2S1339, D2S1399, D2S2987, D2S1391, D2S1361, D2S1371, D2S434, D2S1338 and D2S427, all located on the long arm, which can constitute 4 linkage groups:
Ⅰ：D2S2696-D2S410-D2S437-D2S2970；
Ⅱ：D2S1339-D2S1399；
Ⅲ：D2S2978-D2S1391-D2S1361；
Ⅳ：D2S1371-D2S434-D2S1338-D2S427。
closely linked loci constitute haplotypes and are useful in genetic analysis of the genetic relationship between a parent and offspring.
(2) Easy to amplify
In addition to the 28 loci described above, the inventors have selected additional loci linked to loci D1S1660, D1S547, D1S2131, D1S2539, D1S1595, etc. located on chromosome 1 at the beginning of the experiment. However, single locus amplification results have found that there are problems with amplification of these loci, some of which have a small amount of amplified product, which is too shallow to give amplified product, such as D1S2539, in some cases; some are prone to non-specific bands (e.g., shadow bands), such as D1S547, D1S1595; the 15 loci are easy to amplify, have good stability, clear results and few nonspecific bands.
Thus, finally, D1S3721, D1S2130, D1S3724, D1S3467, GATA133A08, D1S1631, D1S1642, D1S518, D1S3731, D1S3468, D1S1670, D1S1656, D1S3462, D1S1634 and D1S1609 were selected, and these 15 loci on chromosome 1 were readily amplified, with better polymorphism and better typing established a composite amplification system I. And 13 loci of chromosome 2 on long arms, D2S2696, D2S410, D2S437, D2S2970, D2S1339, D2S1399, D2S2987, D2S1391, D2S1361, D2S1371, D2S434, D2S1338 and D2S427, are easy to amplify, have better polymorphism and better typing, and establish a composite amplification system II.
(3) The fragment lengths of the amplified products are not overlapped
The difference of the sizes of the amplified gene locus fragments is larger than 20bp, and the alleles of the gene loci are not overlapped and interfere with typing.
According to the length of amplified product fragments of each locus, 28 loci in the multiplex amplification system of the invention are marked with loci with different amplified product fragment lengths (the difference is more than 20 bp) by the same fluorescence, so that the alleles of the loci are amplified in a multiplex manner, and the alleles of the loci are not overlapped and the typing is not interfered. Four different colored fluorescent markers are used for marking the primers of a plurality of loci, so that the same reaction system can amplify a plurality of loci at the same time without affecting the typing of each locus. The 28 locus primers of the invention were respectively fluorescently labeled with FAM, HEX, TAMRA, ROX four different colors.
The fluorescence combination mode of 15 loci on chromosome 1 in the system I:
FAM fluorescein markers GATA133a08, D1S1634, D1S3731 and D1S3468;
HEX fluorescein markers D1S1656, D1S1609, D1S3462, and D1S1670;
TAMRA fluorescein markers D1S3724, D1S3721, D1S1642 and D1S3467;
ROX fluorescein markers D1S1631, D1S518 and D1S2130.
15 loci can constitute 5 linkage groups:
Ⅰ：D1S3721-D1S2130；
Ⅱ：D1S3467-D1S3724；
Ⅲ：D1S1631-GATA133A08；
Ⅳ：D1S1642-D1S518-D1S3731-D1S3468；
Ⅴ：D1S1670-D1S1656-D1S3462-D1S1634-D1S1609。
the fluorescence combination mode of 13 loci on chromosome 2 in system II:
FAM fluorescein label: D2S2978, D2S410, D2S437, and D2S1339;
HEX fluorescein markers D2S1399, D2S2970 and D2S2696;
TAMRA fluorescein markers D2S1391, D2S1361 and D2S427;
ROX fluorescein labels D2S1371, D2S1338, and D2S434.
13 loci can constitute 4 linkage groups:
Ⅰ：D2S2696-D2S410-D2S437-D2S2970；
Ⅱ：D2S1339-D2S1399；
Ⅲ：D2S2978-D2S1361-D2S1391；
Ⅳ：D2S1371-D2S434-D2S1338-D2S427。
example 2 amplification of multiplex amplified loci
This example provides a 2-set fluorescent-labeled multiplex amplification system for analyzing the linked STR loci on chromosome 1 and chromosome two, respectively, comprising a system I for multiplex amplification of primers for analyzing 15 loci on chromosome 1, with all of the locus primers mixed in a single tube. System II was used to amplify primers for 13 loci on chromosome 2, with all locus primers mixed in one tube. The locus in the multiplex amplification system is amplified by a pair of primers flanking the locus, wherein the 5' ends of the forward primer strands in each pair of primers are labeled with the corresponding luciferin. The sequences of the amplification primers are shown in the following table:
the 28 locus fluorescent labeling multiplex amplification system of the invention adopts multiplex PCR technology to simultaneously amplify 15 loci linked on chromosome 1 (system I) in one reaction tube and simultaneously amplify 13 loci linked on chromosome 2 (system II) in another reaction tube.
(1) PCR reaction system
The total reaction volume was 15. Mu.l: 5. Mu.l of PCR reaction buffer, 3. Mu.l or 4. Mu.l of primer mixture, 1. Mu.l of DNA polymerase, 2. Mu.l of template DNA, and sterile water was added to 15. Mu.l.
The DNA polymerase can be selected from DNA polymerases commonly used by those skilled in the art, such as gold-plate DNA polymerase (Ampli Taq)，Goldeneye TM And (3) in a PCR reaction buffer solution and matched DNA polymerase.
(2) PCR amplification parameters:
(3) ABIPRISM 3500 genetic analyzer electrophoresis typing amplification product
(1) Sample preparation:
Goldeneye TM molecular weight internal standard ORG 500: 0.4. Mu.l. Times.sample number;
deionized formamide: 9.6. Mu.l. Times.sample number;
after mixing, the mixture was centrifuged transiently, 10.0. Mu.l of the mixture was added to a 96-well sample plate, and 1.0. Mu.l of the PCR product was added thereto, followed by centrifugation transiently. Denaturation at 95℃for 3min, cooling in an ice box for 3min, and loading into an ABIPRISM 3500 genetic analyzer for electrophoresis.
(2) Analysis of results:ID-X software.
Example 3 data statistics
820 samples were tested using a two-set system, of which 168 multiple child samples (97 for 2 children, 50 for 3 children, 14 for 4 children, 5 for 5 children, and 2 for 6 children) were used, and the allele frequencies and haplotype frequencies of 386 unrelated individuals were counted and calculated using Arlequin v3.5.1.3software (Excofier, L., lischer, H.E., mol ecl resource.2010, 10, 564-567.). Allele frequencies at 13 loci in the second set of multiplex amplification systems (2 AS 13) are shown in Table 1 and haplotype frequencies for 4 linkage groups are shown in tables 2-5. The individual recognition rate of the haplotypes of the 4 linkage groups is 0.999999962816973. The results show that the loci have higher polymorphism, are suitable for detection of Chinese population, and are applied to actual detection cases to obtain ideal results. The 15 locus alleles were not all sequenced in the first set of multiplex amplification systems and were not statistically data.
Table 1: alleles and frequencies of 13 loci in 2AS13 system
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Table 2: frequency of haplotype D2S2696/D2S410/D2S437/D2S2970
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Table 3: frequency of haplotype D2S1339/D2S1399
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Table 4: frequency of haplotype D2S2987/D2S1361/D2S1391
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Table 5: frequency of haplotype D2S1371/D2S434/D2S1338/D2S427
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Example 4 alternate paternity testing using a two-set fluorescent-tagged multiplex amplification System containing 28 linked AS-STR loci
1. Principle of
In the human genome, there is a class of closely linked STR loci, linked STR constitutive haplotypes (haplotyes) inherited from the parent to the offspring, closely linked STR haplotypes having a high degree of polymorphism. The probability of having the same haplotype among unrelated individuals is very small, the interference of low polymorphism and genetic background of the conventional non-linkage STR can be overcome, and the difficult problem that the two-level or more genetic relationships can not be identified and different genetic relationships can not be distinguished at present is overcome. If the polymorphism of a closely linked STR group haplotype is infinitely high, the probability of having the same haplotype between unrelated individuals is very small, and only individuals with genetic relationship will have the same haplotype, so that individuals with genetic relationship and individuals without genetic relationship can be distinguished.
2. Operating procedure
(1) DNA extraction
Chelex-100 method: cutting 4X 4mm blood streak or saliva spot, adding 200 μl 10% Chelex-100, oven overnight at 56 deg.C, placing in PCR instrument at 100deg.C for 10min, oscillating for 30sec, centrifuging at 10000r/min for 2min, and storing at 4deg.C.
(2) PCR amplification
The total PCR amplification reaction was 15. Mu.l: 5. Mu.l of PCR reaction buffer, 3. Mu.l or 4. Mu.l of primer mixture, 1. Mu.l of DNA polymerase, 2. Mu.l of template DNA, and sterile water was added to 15. Mu.l.
In the primer mixture, the primers of each locus are shown as SEQ ID NO. 1-56, and FAM fluorescein marks GATA133A08, D1S1634, D1S3731 and D1S3468; D2S2978, D2S410, D2S437, and D2S1339.HEX fluorescein markers D1S1656, D1S1609, D1S3462, and D1S1670; D2S1399, D2S2970, and D2S2696.TAMRA fluorescein markers D1S3724, D1S3721, D1S1642 and D1S3467; D2S1391, D2S1361, and D2S427.ROX fluorescein markers D1S1631, D1S518 and D1S2130; D2S1371, D2S1338, and D2S434.
(3) PCR amplification parameters: 95 ℃ for 11min; 15sec at 95 ℃, 15sec at 60 ℃ for 30 cycles; and at 60℃for 5min.
(4) ABIPRISM 3500 genetic analyzer electrophoresis
1) Starting: starting a stabilized voltage power supply, starting 3500 a host power supply (the instrument enters a self-checking state), starting a computer, and starting 3500DATA Collection DATA acquisition software.
2) The device preparation: and (5) cleaning and installing regularly.
Removing capillary tube and Buffer tank, cleaning with pure water, air drying, and pouring adhesive; the Buffer tank contains 1 Xbuffer solution. Double-click Run 3500Data CollectionVersion2.0 software- & gt click Wizards in menu bar- & gt click Fill Capillary Wizards, glue injection to capillary- & gt capillary positioning and sample editing.
3) Sample preparation:
Goldeneye TM molecular weight internal standard ORG 500: 0.4. Mu.l. Times.sample number;
deionized formamide: 9.6. Mu.l. Times.sample number;
after mixing, the mixture was centrifuged instantaneously, 10.0. Mu.l of the mixture was added to each tube, and 1.0. Mu.l of the PCR product was added thereto, followed by instantaneous centrifugation. Denaturation at 95℃for 3min, cooling in an ice box for 3min, transferring 10.0. Mu.l of denatured sample to a 96-well sample plate, and loading into 3500 genetic analyzer for electrophoresis.
4) Analysis of results: genotyping was performed using GeneMapper ID-X software.
3. Results
The 28 STR loci composite amplification electropherograms of ancestor, mother and suspected grandson are as follows: the electropherograms of 15 loci on chromosome 1 are shown in fig. 2 (a) to 2 (C); the electropherograms of 13 loci on chromosome 2 are shown in FIGS. 3 (A) to 3 (C). Statistical analysis of the typing results is shown in FIG. 4 (A) and FIG. 4 (B). From FIG. 2, it can be seen that 14 loci out of the 15 loci on chromosome 1 of the suspected grandchild sample, except D1S1631, are typed from the grandmother. From FIG. 4 (A), it is clear that the grandchild is linked to group I on chromosome 1: D1S3721-D1S2130; II: D1S3467-D1S3724; IV: D1S1642-D1S518-D1S3731-D1S3468; v: haplotypes (211/251; 306/157;252/206/226/296; 367/142/257/161/193) of D1S1670-D1S1656-D1S3462-D1S1634-D1S1609 were all derived from grandparent, linkage group III: the D1S1631-GATA133A08 haplotype (145/110) was derived from grandparent, 10 of the 13 loci on chromosome 2 were typed from grandparent, but haplotypes on chromosome 2 were all derived from grandparent when the pedigree was analyzed in linkage.
For this case, it is a case of postmortem source approval by public security commission: 1 female cadaver is found in a park, and identity identification is needed for the cadaver. Because the unique son is not in the world, the unique relatives grandson and the son are found. Only the generation-related relationship identification can be performed. As the milk and grandchildren are identified as not being applicable to Y-STR or X-STR, the method can only rely on autosomal STR when using Goldeney TM 25A, the cumulative grandchild relationship index (CGI) is calculated to be 0.04971, the detection Microreader 23sp is increased, the CGI is 1.8999, the AGCU 21+1 is additionally detected, the CGI is 20.0154, and the Investigator HDplex is finally additionally detected, the total number of 62 unlinked STR loci of 4 sets of autosomal STR kits are detected, the CGI is calculated to be 342.5, and the conclusion of supporting the grandchild relationship cannot be reached. For unlinked STRs, it appears that many STRs have allele frequencies greater than 0.125 or greater 0.25 due to the genetic background of the STRs, which is greater than 12.5% for tertiary relatives or 25% for secondary relatives% of the same allele, and thus cannot distinguish secondary relatives, tertiary relatives or unrelated individuals, and thus cannot identify relatives. When the 28 closely linked STR multiplex amplification systems developed by the invention are used for detection, 24 loci can be found from grandma for the result typing of grandchildren, and the linked group I of the grandchildren on chromosome 1: D1S3721-D1S2130; II: D1S3467-D1S3724; IV: D1S1642-D1S518-D1S3731-D1S3468; v: the monomers of D1S1670-D1S1656-D1S3462-D1S1634-D1S1609 are derived from ancestor; so that the relationship between ancestor and grandson is supported.
The result shows that the closely linked STR developed by the invention can make up for the defect of unlinked STR, has extremely high polymorphism of closely linked STR group haplotype, can overcome the interference of low polymorphism and genetic background of the conventional unlinked STR, and breaks through the difficult problem that the two-level or more genetic relationship can not be identified and different genetic relationships can not be distinguished at present.
Example 5
The four-generation family is detected by using the linkage STR composite amplification system of the invention, and the linkage group of the linkage STR is proved to be according to the monomer genetic condition. The procedure of example 4 was used for the specific authentication procedure: DNA extraction, PCR amplification product electrophoresis and result analysis.
As shown in FIG. 5, it was revealed from FIG. 5 that the 13 linkage STR loci on chromosome 2 form 4 linkage groups (I: D2S2696-D2S410-D2S437-D2S2970; II: D2S1339-D2S1399; III: D2S2978-D2S1361-D2S1391; IV: D2S1371-D2S434-D2S1338-D2S 427) which were stably inherited in the third or fourth generation.
Wherein individual 25 in figure 5 is in linkage group i: D2S2696-D2S410-D2S437-D2S2970; II: D2S1339-D2S1399; III: the monomers of D2S2978-D2S1361-D2S1391 are from the great grandfather; IV: the D2S1371-D2S434-D2S1338-D2S427 monomers are from Zengzu.
The 28 loci of the invention are applied to the population study in the inventor laboratory and the typing result of Chinese population is obtained, and the results prove that the loci have higher polymorphism, are suitable for the detection of Chinese population, and are applied to the actual examination table to obtain the ideal result. The 28-linkage locus fluorescent marker composite amplification system can be prepared into a kit, a closely-linked STR locus haplotype database is established by detecting multiple generations of families, and based on the linked STR haplotype, complex genetic relationships of more than two levels and prenatal diagnosis of abnormal inheritance of chromosome I and chromosome II are deduced, and whether multi-site mutation in paternity identification is derived from a single parent diploid or is excluded from the paternity is distinguished.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.
SEQUENCE LISTING
<110> university of Zhongshan
<120> human chromosome I and chromosome II linkage STR gene locus composite amplification detection system and application thereof
<130> 2021.12.13
<160> 56
<170> PatentIn version 3.3
<210> 1
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agagaacgga accaacagaa 20
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tgcccgtatc tgaagtacct 20
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gattgccaat tatctcgcac 20
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tgcagatctt gggacttctc 20
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aaaaagagtg tgggcaactg 20
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atgtagaaga gtcagaattt caacc 25
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tatcatgtga gcccattctg 20
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aaattttgag gcaggtgaca 20
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cattggtcca ggtaaactgc 20
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ctcactgtct ggatttcttg g 21
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taaggacaag ttaaaaaagc tgg 23
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taagaagccg ttcttggatg 20
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atgtagactg ctaacagtgt ctgc 24
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ttggtcaaca tgtcaattta gg 22
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gttgggggaa gcattaaaat 20
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ccagtggatt tggaaacaga 20
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taaatcacta gcctttgccg 20
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gccatctgta ctgttcccag 20

Claims (9)

1. A28-locus composite amplification detection system closely linked on a first chromosome and a second chromosome of a human is characterized by comprising a system I for amplifying 15 loci on a first chromosome and a system II for amplifying 13 loci on a second chromosome;
the 15 loci on chromosome 1 are respectively: D1S3721, D1S2130, D1S3467, D1S3724, D1S1631, GATA133a08, D1S1642, D1S518, D1S3731, D1S3468, D1S1670, D1S1656, D1S3462, D1S1634 and D1S1609;
the 13 loci on chromosome 2 are respectively: D2S2696, D2S410, D2S437, D2S2970, D2S1339, D2S1399, D2S2987, D2S1391, D2S1361, D2S1371, D2S434, D2S1338, D2S427;
the sequences of the amplification primers of the 28 loci are shown in SEQ ID NO: 1-56.
2. The detection system of claim 1, wherein at least one of the primers corresponding to each locus is fluorescently labeled at the 5' end of the primer.
3. The detection system of claim 2, wherein the primers are labeled with FAM, HEX, TAM or ROX fluorescein, respectively.
4. The detection system of claim 3, wherein the primers used in system I to amplify the 15 loci on chromosome 1 are labeled with fluorescein as follows:
FAM fluorescein labels primers for amplification of GATA133a08, D1S1634, D1S3731 and D1S3468;
HEX fluorescein labels primers for amplification of D1S1656, D1S1609, D1S3462, and D1S1670;
TAMRA fluorescein labels primers for amplification of D1S3724, D1S3721, D1S1642 and D1S3467;
ROX fluorescein labels primers for amplification of D1S1631, D1S518 and D1S2130.
5. The detection system of claim 3, wherein the primers used in system ii to amplify the 13 loci on chromosome 2 are labeled with fluorescein as follows:
FAM fluorescein labels primers for amplification of D2S2978, D2S410, D2S437 and D2S1339;
HEX fluorescein labels primers for amplification of D2S1399, D2S2970 and D2S2696;
TAMRA fluorescein labels primers for amplification of D2S1391, D2S1361 and D2S427;
ROX fluorescein labels primers for amplification of D2S1371, D2S1338 and D2S434.
6. The detection system of claim 1, wherein the 28 loci are 9 linked groups;
wherein 15 loci on chromosome 1 in the system i constitute 5 linkage groups:
Ⅰ：D1S3721-D1S2130；
Ⅱ：D1S3467-D1S3724；
Ⅲ：D1S1631-GATA133A08；
Ⅳ：D1S1642-D1S518-D1S3731-D1S3468；
Ⅴ：D1S1670-D1S1656-D1S3462-D1S1634-D1S1609；
the 13 loci on chromosome 2 in the system ii constitute 4 linkage groups:
Ⅰ：D2S2696-D2S410-D2S437-D2S2970；
Ⅱ：D2S1339-D2S1399；
Ⅲ：D2S2978-D2S1391-D2S1361；
Ⅳ：D2S1371-D2S434-D2S1338-D2S427。
7. the detection system of claim 1, wherein the amplified system is: system i: 5 mu L of PCR reaction buffer solution, 4 mu L of primer mixture, 1 mu L of DNA polymerase, 2 mu L of template DNA and 15 mu L of sterilizing water; system II: 5. Mu.L of PCR reaction buffer, 3.0. Mu.L of primer mixture, 1. Mu.L of DNA polymerase, 2. Mu.L of template DNA, and 15. Mu.L of sterile water.
8. The detection system of claim 1, wherein the amplification procedure is: 95 ℃ for 11min; 15sec at 95 ℃, 15sec at 60 ℃ for 30 cycles; and at 60℃for 5min.
9. Use of the detection system according to any one of claims 1 to 8 in paternity testing or paternity testing.
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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101956004A (en) * 2010-08-13 2011-01-26 司法部司法鉴定科学技术研究所 Fluorescent-marked 16 X-STR loci multiplex amplification system and application thereof
CN104911257A (en) * 2015-04-21 2015-09-16 中山大学 AS-STR locus fluorescent labeling composite amplification system and application thereof
CN104946632A (en) * 2015-05-28 2015-09-30 宁波海尔施基因科技有限公司 Autosome STR gene locus fluorescent labeling composite amplification kit having enhanced identification ability, and applications thereof
WO2016188331A1 (en) * 2015-05-27 2016-12-01 宁波海尔施基因科技有限公司 Multiplex amplification kit for thirty-four loci of human genome dna
CN107904317A (en) * 2017-12-27 2018-04-13 无锡中德美联生物技术有限公司 Mankind's euchromosome STR polymorphic site composite amplification reagent kit and its application
CN109880911A (en) * 2019-03-07 2019-06-14 基点认知技术(北京)有限公司 The composite amplification reagent kit of 25 human chromosomal locus and its application
CN110499373A (en) * 2019-09-18 2019-11-26 山西医科大学 Identify the high-throughput STR classification system and kit of complicated affiliation
CN110734982A (en) * 2019-09-18 2020-01-31 山西医科大学 High-throughput sequencing technology-based linkage autosomal STR typing system and kit

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008002672A2 (en) * 2006-06-28 2008-01-03 The Cleveland Clinic Foundation Targets for use in diagnosis, prognosis and therapy of cancer

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101956004A (en) * 2010-08-13 2011-01-26 司法部司法鉴定科学技术研究所 Fluorescent-marked 16 X-STR loci multiplex amplification system and application thereof
CN104911257A (en) * 2015-04-21 2015-09-16 中山大学 AS-STR locus fluorescent labeling composite amplification system and application thereof
WO2016188331A1 (en) * 2015-05-27 2016-12-01 宁波海尔施基因科技有限公司 Multiplex amplification kit for thirty-four loci of human genome dna
CN104946632A (en) * 2015-05-28 2015-09-30 宁波海尔施基因科技有限公司 Autosome STR gene locus fluorescent labeling composite amplification kit having enhanced identification ability, and applications thereof
CN107904317A (en) * 2017-12-27 2018-04-13 无锡中德美联生物技术有限公司 Mankind's euchromosome STR polymorphic site composite amplification reagent kit and its application
CN109880911A (en) * 2019-03-07 2019-06-14 基点认知技术(北京)有限公司 The composite amplification reagent kit of 25 human chromosomal locus and its application
CN110499373A (en) * 2019-09-18 2019-11-26 山西医科大学 Identify the high-throughput STR classification system and kit of complicated affiliation
CN110734982A (en) * 2019-09-18 2020-01-31 山西医科大学 High-throughput sequencing technology-based linkage autosomal STR typing system and kit

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
John M. Butler.Short tandem repeat typing technologies used in human identity testing.《BioTechniques》.2007,第43卷(第4期),第i-v页. *
刘晓军等.瘢痕疙瘩候选基因与染色体2q23微卫星扫描及连锁分析的多家系研究.《中国组织工程研究与临床康复》.2008,第12卷(第33期),第6484-6487页. *
吴微微 等.中国28个省/区汉族人群41个STR基因座多态性数据分析.《中国法医学杂志》.2016,第31卷(第1期),第27-32页. *

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