AU2020104011A4 - Snp-str multiplex system for unbalanced dna mixtures analysis - Google Patents

Abstract

The invention provides a multiplex system, a method and an application of the multiplex system for detecting unbalanced DNA mixtures. The multiplex system includes primers for amplifying 18 SNP-STR loci, each locus includes a universal reverse primer and two specific forward primers with mutation introduced, and the specific sequences are shown in SEQ ID NO.1-54. The SNP-STR multiplex amplification system established by this invention simplifies the operation process, can be widely used for forensic genetics, such as individual identification, detecting biological evidence including samples of two persons mixed, samples with same STR profiles, and can also be applied to non-invasive prenatal diagnosis and non-invasive prenatal paternity testing. The present invention provides a new technical method for forensic genetics, can serve as a valuable tool to trace clues and evidence for solving forensic cases.

Description

SNP-STR MULTIPLEX SYSTEM FOR UNBALANCED DNA MIXTURES ANALYSIS TECHNICAL FIELD

The present invention relates to the technical field of forensic detection, and in

particular to a multiplex system, a method and an application of the multiplex system

for detecting unbalanced DNA mixtures.

BACKGROUND

DNA mixtures are often obtained in forensic casework. The complexity of DNA

mixtures increases with the number of contributors, and the difficulty of detection

varies with the proportion of contributors' DNA.

The key of DNA mixtures analysis is to effectively separate the information of

different components. Currently, there are three main methods to analyze DNA

mixtures in practice cases.

(1) Physical separation of each component during DNA extraction, including

differential extraction, laser capture microdissection (LCM), fluorescence-assisted

cell sorting (FACS). The differential extraction procedure aims to separate sperm and

vaginal epithelial cells from vaginal swabs in sexual offence cases, thereby avoiding

the mixture of male-female DNA. However, the effectiveness of this method is

limited to the analysis of mixtures consisting of male (i.e., sperm) and female cells.

Both LCM and FACS offer precise separation of distinct cell types in forensic

mixtures, but their workflows are technically demanding, time-consuming, and labor

intensive; neither can be used routinely in forensic laboratories.

(2) Separation at the DNA amplification stage. The separation is achieved mainly

by using special genetic markers, such as using male-specific Y chromosome-short

tandem repeat (Y-STR) genetic markers to analyze the male Y chromosome

information in male-female DNA mixtures. However, these markers cannot be

directly used for individual identification, nor can they be used for male-male and female-female DNA mixtures because such markers are paternal inheritance and haplotype. In addition to Y-STR, there is mitochondrial DNA (mtDNA), but the mtDNA is maternal inheritance and haplotype, it cannot be directly used for individual identification.

DIP-STR is a highly sensitive genetic marker composed of an insertion-deletion

polymorphism (DIP) located in close proximity to an STR, enabling the specific

detection of minor DNA in the presence of up to a 1000-fold excess of the major

contributor's DNA. Both DIP and STR genotypes can be obtained simultaneously

regardless of the gender combinations of the mixture, and the polymorphism is higher

than that of STR. However, DIP-STR is rarely distributed in genome, and there are no

DIPs distributed in the flanking region of STR loci commonly used in forensic DNA

analysis. Therefore, their association with the existing forensic DNA database is

insufficient for further application in forensic casework.

(3) Separation and analysis of components after profiling results of DNA

mixtures are obtained. This method mainly depends on forensic statistics and

commercial probabilistic genotyping software, and is easily affected by the

unbalanced proportion of DNA mixtures and random effects resulted in the

amplification process. If the proportion of major and minor components in a mixture

is too large, causing insufficient minor DNA amplification in the presence of

excessive major component amplification, little to no resultant genotype information

of the minor component will be represented in the mixture's profile. At that point,

statistical methods and software analysis cannot be applied effectively. Currently,

there are no effective solutions and means for analyzing unbalanced DNA mixtures,

and the separation and analysis of the minor component in unbalanced DNA mixtures

are still under study.

SUMMARY

In view of the above deficiencies in the prior art, the present invention provides a

multiplex system and a method and an application for forensic genetics, which can be used to detect mixed samples in forensic field cases. In order to achieve the above objective, the technical solution adopted by the present invention to solve the technical problem is as follows. A multiplex system, including primers for amplifying 18 SNP-STR loci, each locus includes a universal reverse primer and two specific forward primers with mutation introduced. SNP-STR loci include rs11642858-D16S539, rs58390469-D2S441, rs2325399-D6S1043, rs2070018-FGA, rs25768-D5S818, rs9531308-D13S317, rs17651965-CSF1PO,rs4847015-D1S1656,rs7962284-D12S391,rs7275705-PentaD, rs7786079-D7S820,rs57346531-D8S1179,rs8031604-PentaE,rs2246512-D10S1248, rs17077990-D3S1358, rs6736691-D2S1338, rs13413321-TPOX and rs9362476-SE33; The specific sequences of primers are shown as follows: rs11642858-D16S539-R:GGCAGATCCCAAGCTCTTCCTC; (SEQ ID NO.1) rs11642858-D16S539-F-A:GCATGTATCTATCATCCATCTCTaT; (SEQ ID NO.2) rs11642858-D16S539-F-C:GCATGTATCTATCATCCATCTCTtG; (SEQ ID NO.3) rs58390469-D2S441-R:GCTAAGTGGCTGTGGTGTTA; (SEQ ID NO.4) rs58390469-D2S441-F-A:TGAAAGGAGTGCAAGAGAAGcTA; (SEQ ID NO.5) rs58390469-D2S441-F-C:TGAAAGGAGTGCAAGAGAAGcTC; (SEQ ID NO.6) rs2325399-D6S1043-R:GAGCCACTTCCCATAATAAATCCT; (SEQ ID NO.7) rs2325399-D6S1043-F-C:AAGTACCCTAACAAGTAACTCATCcT; (SEQ ID NO.8) rs2325399-D6S1043-F-G:AAGTACCCTAACAAGTAACTCAgGcTC; (SEQ ID NO.9) rs2070018-FGA-R:CCAAAATAAAATTAGGCATATTTACAAGCTAG; (SEQ ID NO.10) rs2070018-FGA-F-C:GCCTTCCTTTTCCCTCTACTCcG; (SEQ ID NO.11) rs2070018-FGA-F-T:GCCTTCCTTTTCCCTCTACTCcA; (SEQ ID NO.12) rs25768-D5S818-R:AGCCACAGTTTACAACATTTGTATCT; (SEQ ID NO.13) rs25768-D5S818-F-A:GGGTGATTTTCCTCTTTGGTATCCTTcT; (SEQ ID

NO.14)

rs25768-D5S818-F-G:GGGTGATTTTCCTCTTTGGTATCCTTcC; (SEQ ID

NO.15)

rs9531308-D13S317-R:CTCTGGACTCTGACCCATCTAACG; (SEQ ID

NO.16)

rs9531308-D13S317-F-C:GTGGGGAAATTTGTACATTCATTAATATACAgG;

(SEQ ID NO.17)

rs9531308-D13S317-F-A:GTGGGGAAATTTGTACATTCATTAATATACAgT;

(SEQ ID NO.18)

rs8031604-PentaE-R:TTTGGGTTATTAATTGAGAAAACTCCTTAC; (SEQ ID

NO.19)

rs8031604-PentaE-F-T:GGGTACCAATAACAAGAAAATTGTGtA; (SEQ ID

NO.20)

rs8031604-PentaE-F-G:GGGTACCAATAACAAGAAAATTGTtGC; (SEQ ID

NO.21)

rs4847015-DIS1656-R:GAGAAATAGAATCACTAGGGAACC; (SEQ ID

NO.22)

rs4847015-DIS1656-F-C:TGTGTTGCTCAAGGGTCAACTcTG; (SEQ ID

NO.23)

rs4847015-DIS1656-F-T:TGTGTTGCTCAAGGGTCAACTGcA; (SEQ ID

NO.24)

rs7962284-D12S391-R:TCCATATCACTTGAGCTAATTCCTCT; (SEQ ID

NO.25)

rs7962284-D12S391-F-T:CACCACTGCACTCCAGTtT; (SEQ ID NO.26)

rs7962284-D12S391-F-C:CACCACTGCACTCCtGCG; (SEQ ID NO.27)

rs7275705-PentaD-R:GAGCAAGACACCATCTCAAGAAA; (SEQ ID NO.28) rs7275705-PentaD-F-G:GGTTAAATATCTCTTCAAATCTTTTGCaC; (SEQ ID

NO.29)

rs7275705-PentaD-F-C:GGTTAAATATCTCTTCAAATCTTTTGtCG; (SEQ ID

NO.30) rs7786079-D7S820-R:AAGGGTATGATAGAACACTTGTCATAG; (SEQ ID

NO.31)

rs7786079-D7S820-F-C:CCTCATTGACAGAATTGCACCt; (SEQ ID NO.32)

rs7786079-D7S820-F-A:CCTCATTGACAGAATTGCACCtA; (SEQ ID NO.33)

rs57346531-D8S1179-R:TACCTATCCTGTAGATTATTTTCACTGTG; (SEQ

ID NO.34)

rs57346531-D8S1179-F-A:GAGCATAACAGAGGCACTGAaA; (SEQ ID

NO.35)

rs57346531-D8S1179-F-G:GAGCATAACAGAGGCACTGAaG; (SEQ ID

NO.36) rs2246512-D1OS1248-R:CATATTAATGAATTGAACAAATGAGTGAGT;

(SEQ ID NO.37)

rs2246512-D1OS1248-F-A:CCCACCCCTGGATATTATAATTAAaAT; (SEQ ID

NO.38) rs2246512-D1OS1248-F-G:CCCACCCCTGGATATTATAATTAACg; (SEQ ID

NO.39) rs17077990-D3S1358-R:CAGAGCAAGACCCTGTCTCAT; (SEQ ID NO.40)

rs17077990-D3S1358-F-C:CTCAGCTTCAGCCCATACaC; (SEQ ID NO.41)

rs17077990-D3S1358-F-G:CTCAGCTTCAGCCCATACaG; (SEQ ID NO.42)

rs17651965-CSF1PO-R:TTGCTAACCACCCTGTGTCTCAG; (SEQ ID NO.43)

rs17651965-CSF1PO-F-G:GCTCMCACTCCGATGAGgTG; (SEQ ID NO.44)

rs17651965-CSF1PO-F-C:GCTCMCACTCCGATGAGgT; (SEQ ID NO.45)

rs6736691-D2S1338-R:GGAGGGAGCCAGTGGATTT; (SEQ ID NO.46) rs6736691-D2S1338-F-C:CTGCAGGTGGCCCATAAaC; (SEQ ID NO.47)

rs6736691-D2S1338-F-A:CTGCAGGTGGCCCATAtTA; (SEQ ID NO.48)

rs13413321-TPOX-R:GGCACAGAACAGGCACTTAGG; (SEQ ID NO.49) rs13413321-TPOX-F-G:GGGGAGGAACTGGGAACtC; (SEQ ID NO.50) rs13413321-TPOX-F-T:GGGGAGGAACTGGGAACgA; (SEQ ID NO.51) rs9362476-SE33-R:GTCATGCCATTGCACTCCAAT; (SEQ ID NO.52) rs9362476-SE33-F-C:gGCTGGAGCAGTTGTCtACtA; (SEQ ID NO.53) rs9362476-SE33-F-T:gGCTGGAGCAGTTGTCGgTtA; (SEQ ID NO.54)

In the above sequences, lowercase letters indicate that an original base at this site

is changed to an introduced mismatched base, and underlined letters represent the

bases of allele-specific primers matched to SNP.

A method for detecting unbalanced DNA mixtures using the multiplex system,

including the following steps:

(1) Extracting DNA of the mixture samples waiting to be tested, and the

reference samples of the victim and suspect, respectively;

(2) Performing a polymerase chain reaction (PCR) amplification on the reference

DNA of the victim and suspect using the multiplex system of claim 1, respectively;

detecting amplified products using capillary electrophoresis (CE) to obtain SNP-STR

genotypes of the victim and the suspect respectively; on this basis, screening out

specific SNP alleles of the suspect compared with the victim, and the linked

SNP-STR alleles are effective loci;

(3) Performing a PCR amplification on the DNA of the mixtures waiting to be

tested using the primers corresponding to the selected effective loci in claim 1,

respectively. To detect whether the specific SNP alleles of the suspect can be

separated from the DNA mixtures, the specific processes are as follows.

For example, as shown in FIG. 1, when the SNP genotype of the major DNA

component is homozygous-AA and the minor DNA component in the unbalanced

DNA mixtures is BB or AB, the SNP B-specific primer, "B-SNP," could be used to

specifically target the B genotype of the minor DNA component in the mixtures,

while failing to amplify the major DNA component, and the haplotype is B3/B5 or

B5.

Further, an amplification system in step (2) is as follows: 5 L of 2xQIAGEN

Multiplex PCR Master Mix, 0.5 L of primer mix, 1 L of DNA (1 ng/L), and finally supplement to 10 L with RNase-free water.

Further, amplification conditions in step (2) are as follows: an initial denaturation

at 95°C for 15 min, followed by 31 cycles of a denaturation at 94°C for 30 s, an

annealing at 58°C for 90 s, and an extension at 72°C for 60 s, and a final extension at

°C for 35 min.

Further, an amplification system in step (3) is as follows: 5 L of 2xQIAGEN

Multiplex PCR Master Mix, 0.5 L of a primer working solution PA/Pb (3 M), 2 L

of DNA, and finally supplement to 10 L with RNase-free water.

Further, amplification conditions in step (3) are as follows: an initial denaturation

at 95°C for 15 min, followed by 31 cycles of a denaturation at 94°C for 30 s, an

annealing at 58°C for 90 s, and an extension at 72°C for 60 s, and a final extension at

°C for 35 min.

An application of the above-mentioned method in individual identification,

non-invasive prenatal diagnosis, non-invasive paternity test or unbalanced DNA

mixtures identification.

A kit for detecting mixed stains, including the multiplex system.

More specifically, the kit includes the following components:

a) reaction mixed solution: including PCR buffer, MgCl 2 solution, dNTP, DNA

polymerase, etc.; b) primer mix: the specific sequences are shown in SEQ ID

No.1-54.

The reaction mixed solution and the purification reagent for amplification

product can be prepared according to the formula commonly used in the field or

according to the molecular biology manual, or commercial products can be directly

used. Genomic DNA can be extracted by using various commercial kit or other

existing conventional methods.

The advantages of the present invention are as follows.

1. The present invention screens 18 SNP-STRs, which is a novel compound

genetic marker. Based on capillary electrophoresis (CE), both SNP and STR

genotypes can be obtained in a single PCR. On the basis of the high polymorphism of

STRs, combined with the advantage of bi-allelic genotyping of SNPs, the forensic performance is better than that of the STR contained in the genetic marker.

2. In conjunction with a PCR technique based on amplification refractory

mutation system (ARMS) technique, SNP-STRs have obvious advantages in forensic

unbalanced DNA mixtures analysis. The SNP allele-specific primers of 18 SNP-STRs

are not affected by the major components when detecting the minor components in

unbalanced DNA mixtures.

3. The SNP-STR typing results detected by the present method can be directly

compared with the existing data in the current STR database, providing more effective

clues and evidence for case investigation.

4. The SNP-STRs are not affected by the gender of contributors in the mixture

analysis because these loci in this study are screened based on autosomal STR.

5. The present invention establishes a SNP-STR multiplex amplification system,

simplifies the operation process. Based on the routine equipment, this invention

provides a new technical method, can be widely used for forensic evidence detecting,

such as detecting samples of two persons mixed, samples with same STR profiles,

provides clues and evidence for solving cases.

6. The present method depends on the identification of the cell-free fetal DNA

(cffDNA) in maternal peripheral blood, can be applied to non-invasive prenatal

diagnosis, non-invasive prenatal paternity testing and other fields. Invasive techniques,

such as chorionic or direct chorionic villus sampling, are associated with a certain

degree of harm to the mother and the fetus. These problems can be avoided by using

the present invention. And this method also could be the solution for early pregnancy

tests.

7. The present method can also be used for analysis of the peripheral blood DNA

microchimerism resulting from clinical hematopoietic stem cell transplantation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of SNP-STR allele-specific primers for analyzing

the minor DNA of two-person mixtures;

FIG. 2 is a schematic diagram of calculation of probability of informative

genotypes (I value);

FIG. 3 is a schematic diagram of allele-specific primer design for SNP-STR

markers using the ARMS technique;

FIG. 4 is a diagram showing distribution and fluorescent-dye colors of primers

for 18 SNP-STRs in multiplex panels, where, FIG. 4a is Panel-A, FIG. 4b is Panel-B,

and FIG. 4c is Panel-C.

FIG. 5 is a diagram showing detection results of the effective loci in mixtures;

where, FIG. 5a is rs58390469-D2S441-A12, FIG. 5b is rs2325399-D6S1043-G19, FIG.

c is rs17077990-D3S1358-G16, and FIG. 5d is rs13413321-TPOX-T11.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The specific embodiments of the present invention are described below to

facilitate those skilled in the art to understand the present invention. However, it

should be clear that the present invention is not limited to the scope of specific

embodiments. For ordinary technical personnel in the art, these modifications are

obvious as long as they are within the spirit and scope of the present invention to

which the claims are limited and determined, and all inventions conceived by the

present invention are protected.

Embodiment 1: Loci screening

1. DNA extraction: Genomic DNA was isolated using a whole blood extraction

kit (BioTeke, Beijing, China) according to the manufacturer's instructions and

quantified spectrophotometrically using a NanoDrop TM 1000 Spectrophotometer

(Thermo Fisher Scientific, Waltham, MA, USA). According to the resulting

concentration, the obtained DNA solution is diluted with TE buffer into a working

solution with a concentration of 1 ng/L, and stored at 4°C.

2. SNP-STR loci screening

(1) screening criteria: SNP-STR genetic markers can be used to analyze

unbalanced DNA mixtures on the premise that the genotype combinations of major and minor components in the mixtures are informative genotypes. The informative genotypes refer to the genotype of the minor component DNA with alleles that do not exist in the major component, and their occurrence probability is related to the frequency of SNP alleles. According to FIG. 2, four informative genotype combinations are obtained: 1) the minor DNA is AA, and the major DNA is BB; 2) the minor DNA is AB, and the major DNA is AA; 3) the minor DNA is AB, and the major DNA is BB; and 4) the minor DNA is BB, and the major DNA is AA. The other combinations do not form informative genotypes.

The frequency of each genotype is calculated according to the allele frequency,

that is, AA frequency is A 2, BB frequency is B 2, AB frequency is 2AB, so the 2 2 3 3 2 2 probabilities of different combination are A2B2, 2A B, 2AB and B2A2. In conclusion,

the formula for calculating probability of informative genotypes (I value) is: I= A 2B 2 3 3 2 2 +2AB+2AB +BA.

Due to the limited number of the commonly used STR loci, the minimum value

of I value is not excessively limited during SNP-STR loci screening. Meanwhile, it is

necessary to consider the length range of STR loci core repeat sequence and the

length detection limit of target amplicons in CE analysis to determine the maximum

physical distance between SNP and STR core repeat region. In conclusion, based on

the common STR loci, the SNP-STR screening criteria were as follows: 1) SNP minor

allele frequency in CHB (Han Chinese in Beijing, China), CHB and JPT (Japanese in

Tokyo, Japan), or EAS (East Asia) populations, higher than 0.02, namely I> 0.04; 2)

SNP located within 400 bp of STR region; and 3) targeted amplicons shorter than 550

bp.

(2) Screening basic STR Loci: the basic STR loci for screening SNP-STR of the

present invention are autosomal STR loci commonly used in forensic DNA analysis,

mainly including: 1) the expanded Combined DNA Index System (CODIS) core loci

STR set; 2) Extended European Standard Set (ESS); and 3) other forensic commonly

used STR kits.

(3) Screening of single SNP-STR locus: according to the screening criteria in

step (1), the STR loci selected in step (2) are used to further select SNP. Finally, 18

SNP-STR loci were screened, including: rs11642858-D16S539, rs58390469-D2S441,

rs2325399-D6S1043, rs2070018-FGA, rs25768-D5S818, rs9531308-D13S317, rs17651965-CSF1PO, rs4847015-D1S1656, rs7962284-D12S391, rs7275705-Penta D,

rs7786079-D7S820, rs57346531-D8S1179, rs8031604-Penta E, rs2246512-D10S1248, rs17077990-D3S1358, rs6736691-D2S1338,

rs13413321-TPOX and rs9362476-SE33. Detailed information is shown in Table 1.

Table 1 Information of the 18 SNP-STR loci

SNP MAF Chr. Distance SNP SNP-STR SNP MAF (HCB/CHB+JPT Ivalue Location (bp) Allele /EAS)

rs17651965-CSFlPO 5q33.1 271 C/G C=0.3508 C=0.4583 0.352

rs2070018-FGA 4q28 260 C/T G=0.1100 C=0.0568 0.177 rs13413321-TPOX 2p25.3 147 G/T G=0.4249 G=0.4302 0.369

rs17077990-D3S1358 3p2l.31 271 C/G G=0.2440 G=0.2222 0.301 rs25768-D5S818 5q23.2 12 A/G A=0.1571 G=0.0889 0.23 rs7786079-D7S820 7q21.11 64 A/C C=0.0797 C=0.0333 0.136

rs57346531-D8S1l79 8q24.13 270 A/G G=0.2873 G=0.3083 0.326 rs9531308-D13S317 13q31.1 114 A/C C=0.3590 A=0.4167 0.354 rsl1642858-D16S539 16q24.1 15 A/C C=0.2592 C=0.4390 0.31

rs4847015-DlS1656 1q42 5 C/T T=0.1989 T=0.1333 0.268 rs58390469-D2S441 2p14 107 A/C C=0.4599 A=0.4417 0.373 rs6736691-D2S1338 2q35 34 A/C A=0.2041 A=0.1125 0.272

rs2246512-D10S1248 10q26.3 69 A/G G=0.1524 G=0.3690 0.225 rs7962284-D12S391 12p13.2 192 C/T C=0.4044 C=0.3095 0.366 rs2325399-D6S1043 6q15 145 C/G G=0.3818 G=0.3837 0.361

rs7275705-Penta D 21q22.3 302 C/G G=0.2810 G=0.1833 0.322 rs8031604-Penta E 15q26.2 279 G/T T=0.0711 T=0.0341 0.123 rs9362476-SE33 6q14 169 C/T T=0.4898 T=0.2907 0.375 Chr. Location: the location of the SNP-STR on the chromosome; distance: number of bases

between SNP and STR core area; SNP Allele: allele of SNP in SNP-STR locus; SNP MAF: minor

allele frequency of SNP in dbSNP; CHB: Han Chinese in Beijing, China; JPT: Japanese in Tokyo,

Japan; EAS: East Asia; I value: probabilities of informative genotypes for SNP-STR calculated

using SNP frequencies from MAF.

Embodiment 2: Primer design and synthesis

Amplification refractory mutation system (ARMS) primers are designed for

SNP-STRs to obtain both SNP and STR genotypes in a single PCR. Based on the

principle of ARMS (see FIG 3), the design of SNP-STR loci primers is divided into

two steps: (1) the first step, general forward primer with the 3' end starting with the

SNP and a reverse primer downstream of the STR were designed for each SNP-STR

locus using Primer3-web version 4.0.0 (http://primer3.ut.ee), and the amplicons

shorter than 550 bp; (2) SNP allele-specific primers are designed on the basis of the

general forward primers. Based on the forward primers obtained in (1), allele-specific

primers were designed by introducing deliberate mismatch at the antepenultimate or

penultimate base at the 3' end of the forward primers.. Two allele-specific forward

primers and one standard reverse primer were obtained for each SNP-STR locus. Two

specific forward primers are labeled with different fluorescein and the reverse primers

are not labeled.

After repeated screening and optimization, the 54 primer sequences and

fluorophores of the 18 SNP-STR loci obtained in embodiment 1 are shown in Table 2.

Each locus contains a universal reverse primer (R) and two specific forward primers

with mutation introduced. The lowercase letters are bases mismatched to original

sequence, and the underlined letters represent the bases of allele-specific primers

matched to SNP.

Table 2 Primer sequences and fluorophores of 18 SNP-STRS

SNP-STR Primer Sequence 5'Dye

rs11642858-D16S539 R GGCAGATCCCAAGCTCTTCCTC

F-A GCATGTATCTATCATCCATCTCTaT FAM

F-C GCATGTATCTATCATCCATCTCTtG JOE rs58390469-D2S441 R GCTAAGTGGCTGTGGTGTTA

F-A TGAAAGGAGTGCAAGAGAAGcTA FAM

F-C TGAAAGGAGTGCAAGAGAAGcTC JOE rs2325399-D6S1043 R GAGCCACTTCCCATAATAAATCCT

F-C AAGTACCCTAACAAGTAACTCATCcTC FAM

F-G AAGTACCCTAACAAGTAACTCAgGcTC JOE rs2070018-FGA R CCAAAATAAAATTAGGCATATTTACAAGCTAG F-C GCCTTCCTTTTCCCTCTACTCeG FAM

F-T GCCTTCCTTTTCCCTCTACTCcA JOE rs25768-D5S818 R AGCCACAGTTTACAACATTTGTATCT F-A GGGTGATTTTCCTCTTTGGTATCCTTcT FAM

F-G GGGTGATTTTCCTCTTTGGTATCCTTcC JOE rs9531308-D13S317 R CTCTGGACTCTGACCCATCTAACG F-C GTGGGGAAATTTGTACATTCATTAATATACAgG FAM

F-A GTGGGGAAATTTGTACATTCATTAATATACAgT JOE rs8031604-Penta E R TTTGGGTTATTAATTGAGAAAACTCCTTAC F-T GGGTACCAATAACAAGAAAATTGTGtA JOE

F-G GGGTACCAATAACAAGAAAATTGTtGC FAM

rs4847015-DIS1656 R GAGAAATAGAATCACTAGGGAACC F-C TGTGTTGCTCAAGGGTCAACTcTG TAM

F-T TGTGTTGCTCAAGGGTCAACTGcA ROX rs7962284-D12S391 R TCCATATCACTTGAGCTAATTCCTCT F-T CACCACTGCACTCCAGTtT TAM

F-C CACCACTGCACTCCtGCG ROX rs7275705-Penta D R GAGCAAGACACCATCTCAAGAAAG F-G GGTTAAATATCTCTTCAAATCTTTTGCaC TAM

F-C GGTTAAATATCTCTTCAAATCTTTTGtCG ROX rs7786079-D7S820 R AAGGGTATGATAGAACACTTGTCATAG F-C CCTCATTGACAGAATTGCACCtC TAM

F-A CCTCATTGACAGAATTGCACCtA ROX rs57346531-D8S179 R TACCTATCCTGTAGATTATTTTCACTGTG F-A GAGCATAACAGAGGCACTGAaA TAM

F-G GAGCATAACAGAGGCACTGAaG ROX rs2246512-D1OS1248 R CATATTAATGAATTGAACAAATGAGTGAGT F-A CCCACCCCTGGATATTATAATTAAaAT FAM

F-G CCCACCCCTGGATATTATAATTAACgC JOE rs17077990-D3S1358 R CAGAGCAAGACCCTGTCTCAT F-C CTCAGCTTCAGCCCATACaC FAM

F-G CTCAGCTTCAGCCCATACaG JOE rs17651965-CSFlPO R TTGCTAACCACCCTGTGTCTCAG F-G GCTCMCACTCCGATGAGgTG FAM

F-C GCTCMCACTCCGATGAGgTC JOE

rs6736691-D2Si338 R GGAGGGAGCCAGTGGATTT F-C CTGCAGGTGGCCCATAAaC ROX

F-A CTGCAGGTGGCCCATAtTA TAM

rs13413321-TPOX R GGCACAGAACAGGCACTTAGG F-G GGGGAGGAACTGGGAACtC TAM

F-T GGGGAGGAACTGGGAACgA ROX

rs9362476-SE33 R GTCATGCCATTGCACTCCAAT F-C gGCTGGAGCAGTTGTCtACtA TAM

F-T gGCTGGAGCAGTTGTCGgTtA ROX

After repeated experiments, optimization and adjustment, the PCR reaction

system of these 18 SNP-STR loci includes: 5 pL of 2xQIAGEN Multiplex PCR

Master Mix, 0.5 L of a primer working solution PA/Pb (3 M), 2 L of DNA, and

finally supplement to 10 L with RNase-free water. The amplification procedure is as

follows: an initial denaturation at 95°C for 15 min, followed by 31 cycles of a

denaturation at 94°C for 30 s, an annealing at 58°C for 90 s, and an extension at 72°C

for 60 s, and a final extension at 60°C for 35 min.

Embodiment 3: Establishment and optimization of a multiplex system

After the primer design of 18 SNP-STR loci is finished, according to the

amplicon length and fluorophore label for each locus, three multiplex panels for 18

SNP-STRs were established: Panel-A, Panel-B, and Panel-C. Distribution and

fluorescent-dye colors of SNP allele-specific primers for 18 SNP-STRs in multiplex

panels are shown in FIG. 4. Each block represents one pair of allele-specific forward

and reverse primers of a SNP-STR locus, the width of each block indicates the length

range of amplicons, and the color indicates fluorophore label of each allele-specific

primer, and the bottom vertical band is the fragment distribution of the size standard

HiDi GeneScan T M 600 LIZ®Size Standard (Thermo Fisher Scientific, USA) (the

shortest fragment is 20 bp, the longest fragment is 600 bp). The concentrations of

each primer in the three panels of SNP-STR multiplex amplification system are shown in Table 3. The PCR reaction system includes: 5 pL of 2xQIAGEN Multiplex

PCR Master Mix, 1 L of DNA (1 ng/L), and supplement to 10 L with RNase-free

water. The amplification procedure is as follows: an initial denaturation at 95°C for 15

min, followed by 31 cycles of a denaturation at 94°C for 30 s, an annealing at 58°C for

s, and an extension at 72°C for 60 s, and a final extension at 60°C for 35 min.

Table 3 Concentration of each primer in the SNP-STR multiplex Primer Mix

Conc Conce Conce Prim entrat Pri ntratio Prime ntratio Panel-A Panel-B Panel-C er ion( mer n( r n(

pM) pM) piM) rs11642858 F-A 2 rs11642858C- F-C 1.6 rs224651 FA 3.6 A-D16S539 R 2 D16S539 R 1.6 2-D10S12 FG 1.2 rs58390469 F-A 5 rs58390469C- F-C 4.2 48 R 3.6 A-D2S441 R 5 D2S441 R 4.2 rs170779 FC 3.6 rs2325399 F-C 1.2 rs2325399G- F-G 0.5 90-D3S13 FG 3 C-D6S1043 R 1.2 D6S1043 R 0.5 58 R 3.6 rs2070018 F-C 2.6 rs2070018T-F F-T 2.6 rs176519 FG 1.6 C-FGA R 2.6 GA R 2.6 65-CSF1 FC 1.5 rs25768G- F-G 1.2 rs25768A-D5 F-A 2 PO R 1.6 D5S818 R 1.2 S818 R 2 rs673669 FA 3.4 rs9531308 F-A 1.4 rs9531308C- F-C 3.2 1-D2S133 FC 4 A-D13S317 R 1.4 D13S317 R 3.2 8 R 4 rs8031604T F-T 2 rs8031604G-P F-G 1.6 FG 4 rs134133 -Penta E R 2 enta E R 1.6 FT 3 21-TPOX rs4847015 F-C 1.4 rs4847015T-D F-T 3 R 4 C-DIS1656 R 1.4 IS1656 R 3 FC 8 rs936247 rs7962284T F-T 3 rs7962284C- F-C 5.4 FT 8 6-SE33 -D12S391 R 3 D12S391 R 5.4 R 4 rs7275705 F-G 1 rs7275705C-P F-C 3 G-Penta D R 1 enta D R 3 rs7786079 F-A 3 rs7786079C- F-C 1.8 A-D7S820 R 3 D7S820 R 1.8 rs57346531 F-G 4 rs57346531A- F-A 4 G-D8Sl179 R 4 D8S1l79 R 4

Embodiment 4: Detection of unbalanced DNA mixtures

The present invention is used to detect the unbalanced mixed stains obtained in forensic casework (the principle is shown in FIG. 1). DNA of mixed stains are obtained from crime scene, as well as the reference DNA of the victim and suspect.

According to the preliminary investigation of the case, the major component of the

mixed stains is the victim's DNA, while the minor component is an unknown

contributor.

1. The SNP-STR multiplex system prepared by embodiment 3 is used for

amplifying the reference DNA of the victim and suspect. The PCR reaction system

and reaction conditions are shown in embodiment 3.

2. The amplified products are analyzed by CE. The PCR products were prepared

for separation and detection by adding 1 L of product to 9 L of a 40:1 mixture of

HiDi GeneScan T M 600 LIZ® Size Standard (Thermo Fisher Scientific, USA).

Electrophoretic conditions were 9 seconds at 3 kV for injection and 1500 seconds at

15 kV for the run. The initial data were analyzed using GeneMapper ID-X vi.2

software (Thermo Fisher Scientific), with the peak height threshold for each

fluorescent dye set at 50 RFU. The SNP-STR genotypes of the victim and the suspect

are obtained as shown in Table 4.

Table 4 SNP-STR profiling results of the victim and suspect

SNP-STR locus Major component (victim) enor component I No.(sset All All 1 rsll642858-D16S539 All A12 C1O A12 2 rs58390469-D2S441 C11.3 Cl2 C13 Cl2 3 rs2325399-D6S1O43 C14 G19 122 116 4 rs2070018-FGA T22 T123

G9 G12 rs25768-D5S818 G10 G12 A8 C11 6 rs9531308-Dl3S317 C1 Cl Cll Cl12 G16 G1O 7 rs8031604-Penta E G20 G12 C11 C15 8 rs4847015-DlS1656 C13 C17 C 13 C 17 120 117 9 rs7962284-D12S391 T20 T18 120 118

G14 C9 rs7275705-Penta D C9 Cl2 A9 A10 11 rs7786079-D7S820 Al All All All

12 rs57346531-D8Sll79 Al3 A15

A15 A15 13 rs2246512-D1OS1248 A16 A15 A16 A15 C14 C16 14 rsl7077990-D3S1358 C15 G16 C15 G16 G12 G9 rsl7651965-CSFlPO C13 C1 Cl13 Cil C19 C23 16 rs6736691-D2S1338 C23 C23 C23 C23 G8 G8 17 rsl3413321-TPOX G8 Tl G8 Ill C23.2 C24.2 18 rs9362476-SE33 C30 C30 C30 C30

3. The specific-effective loci of the suspect are screened out, as shown in bold type in Table 5. Table 5 effective loci

Effective loci Major component Minor component (victim) (suspect) 1 rs58390469-D2S441 3 A12

C13 Cl2 G19 2 rs2325399-D6S1043 Cl4

Cl4 C16 3 rsl7077990-D3S1358 C15 G16

4 rsl3413321-TPOX G8 G8

4. The specific primers for SNP-STR corresponding to the selected effective loci are used to target the minor component from the DNA mixtures. The PCR reaction system and reaction conditions are shown in embodiment 2, and the results are shown in FIG. 5, in which FIG. 5a is rs58390469-D2S441-A12, FIG. 5b is rs2325399-D6S1043-G19, FIG. 5c is rs17077990-D3S1358-G16, and FIG. 5d is rs13413321-TPOX-T11. The profiling results of the four loci in the mixed stains are consistent with those of the suspect. The specific SNP-STR haplotype of the suspect is successfully detected in the mixed stains. Therefore, the method of the present invention can provide more effective evidence for case investigation, and will be conducive to the investigation and analysis of the forensic case.

Claims (8)

CLAIMS WHAT IS CLAIMED IS:

1. A multiplex system, comprising primers for amplifying 18 SNP-STR loci,

each locus comprises a universal reverse primer and two specific forward primers

with mutation introduced; wherein the SNP-STR loci comprise rs11642858-D16S539,

rs58390469-D2S441, rs2325399-D6S1043, rs2070018-FGA, rs25768-D5S818,

rs9531308-D13S317, rs17651965-CSF1PO, rs4847015-D1S1656,

rs7962284-D12S391, rs7275705-PentaD, rs7786079-D7S820, rs57346531-D8S1179,

rs8031604-PentaE, rs2246512-D10S1248, rs17077990-D3S1358,

rs6736691-D2S1338, rs13413321-TPOX and rs9362476-SE33;

the specific sequences of the primers are shown in SEQ ID NO.1-54.

2. A method for detecting unbalanced DNA mixtures using the multiplex system

according to claim 1, comprising the following steps:

(1) extracting DNA of the mixture samples waiting to be tested, and the

reference samples of the victim and suspect, respectively;

(2) performing a polymerase chain reaction (PCR) amplification on the reference

DNA of the victim and suspect using the multiplex system of claim 1 respectively, to

obtain SNP-STR genotypes of the victim and suspect; on this basis, screening out

SNP-STR effective loci;

(3) performing a PCR amplification on the DNA of the mixtures waiting to be

tested using the primers corresponding to the selected effective loci in claim 1,

respectively.

3. The method according to claim 2, wherein, an amplification system in step (2)

is as follows: 5 L of 2xQIAGEN Multiplex PCR Master Mix, 0.5 L of a primer mix,

1 L of DNA (1 ng/L), andfinally supplement to 10 L with RNase-free water.

4. The method according to claim 2, wherein, amplification conditions in step (2)

are as follows: an initial denaturation at 95°C for 15 min, followed by 31 cycles of a

denaturation at 94°C for 30 s, an annealing at 58°C for 90 s, and an extension at 72°C

for 60 s, and a final extension at 60°C for 35 min.

5. The method according to claim 2, wherein, an amplification system in step (3)

is as follows: 5 L of 2xQIAGEN Multiplex PCR Master Mix, 0.5 L of a primer

working solution PA/Pb (3 M), 2 L of DNA, and finally supplement to 10 L with

RNase-free water.

6. The method according to claim 2, wherein, amplification conditions in step (3)

are as follows: an initial denaturation at 95°C for 15 min, followed by 31 cycles of a

denaturation at 94°C for 30 s, an annealing at 58°C for 90 s, and an extension at 72°C

for 60 s, and a final extension at 60°C for 35 min.

7. An application of the method according to any one of claims 2-6 in individual

identification, mixtures analysis, non-invasive prenatal diagnosis or non-invasive

prenatal paternity test.

8. A kit for detecting mixed stains, comprising the multiplex system according to

claim 1.