KR101716108B1 - Forensic profiling by differential pre-amplification of STR loci - Google Patents

Forensic profiling by differential pre-amplification of STR loci Download PDF

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KR101716108B1
KR101716108B1 KR1020150112362A KR20150112362A KR101716108B1 KR 101716108 B1 KR101716108 B1 KR 101716108B1 KR 1020150112362 A KR1020150112362 A KR 1020150112362A KR 20150112362 A KR20150112362 A KR 20150112362A KR 101716108 B1 KR101716108 B1 KR 101716108B1
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str
dna
amplification
primer
locus
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KR20170018994A (en
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최철용
함선규
우광만
김세용
서보영
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대한민국
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Abstract

Forensic DNA samples are often physically damaged and present at very low concentrations, which is problematic in obtaining STR (Short Tandem Repeats) profiles. The present invention confirms that the pre-amplification method is a powerful means of recovering the STR pattern of long loci of amplification products, and the method of the present invention is very useful for obtaining the STR profile from a damaged DNA sample. In addition, a primer to be used may be selected according to the degree of damage of the DNA or the damaged pattern, and the primer can be selected by selecting the type of primer to be used, and adjusting the ratio of the primer to each primer. The STR pattern can be effectively analyzed even in a sample which is damaged and has a low concentration through the differential amplification of the present invention, and thus it can be widely applied in the related field.

Description

[0002] Forensic profiling by differential pre-amplification of STR loci [

The present invention belongs to the field of genetic detection and forensic typing as an invention relating to a gene detection method by differential amplification of STR (Short Tandem Repeat) genetic loci.

Since the early 1990s, the Amp-FLP (Amplification Fragment Length Polymorphism) typing method using Polymerase Chain Reaction (PCR) has been in full use since the early 1990s. (Forensic typing) or DNA database construction.

In the field of gene detection, short tandem repeats (STRs) are often used. STRs, which are used mainly in the forensic field, exist in the non-coding region of human genome. , Which is a microsatellite in which 2 to 13 nucleotides are repeated as a unit several hundred times. Since STR has a different repeat number of core repeat units for each individual and has a unique value for each individual, STR is widely used for identifying individuals and identifying blood relations.

The STR method is the most frequently used and widely used method of forensic analysis, and its analysis is often used in the court for biological evidence including blood, semen, hair, saliva, etc. that are present in crime scenes. The DNA profile obtained through the STR analysis is particularly useful for identification of individuals in cases where it is difficult to personally identify them by face or fingerprint due to fire, flood, old harm, or slice murder. In addition, this STR method is also applied to classify plant species. It is possible to distinguish not only the marijuana classification found through DNA information of marijuana, but also the location of roots, stem, and seeds.

 In the current forensic science practice, PCR amplification products are separated by electrophoresis, and STR genotypes are analyzed by examining the number of STR repeats according to the difference in length. At this time, multiplex amplification products Amplification PCR (multiplex PCR) is often used. These assays have a resolution that can distinguish microscopic base differences, so that the length of the amplification product can be accurately determined, and the amplified product can be detected easily and quickly from the automated equipment using a fluorescent marker-attached primer. However, this method is not only able to identify the base sequence of the amplification product, but also has limitations on the number of fluorescent markers available and the size of amplification product. The Sanger-based sequencing method, which is a method for analyzing the existing nucleotide sequence, can accurately obtain the nucleotide sequence information, but it is applied to the research field in which a large amount of DNA sequence information such as personal genome analysis is required There were inefficiencies in terms of analysis time, labor, and cost.

In addition, accurate DNA profiling through STR analysis has been hampered by compromised forensic evidence. DNA is subject to physical and chemical damage depending on the duration of exposure to the external environment or the state of the exposed environment. DNA present in human cells and tissues is present in various lesions such as de-purination, nucleotide oxidation, DNA strand cross-liking, or chemical changes in DNA. The state of DNA is also often fragmented due to the physical cleavage of the double helix. Damage or fragmentation due to DNA cleavage enzymes is often seen in forensic evidence derived from biological evidence over a long period of time. STR genotypes with long amplification products are the first and most affected, This is a sensitive area, and when the length of the amplification product is increased by the STR analysis using damaged forensic medical evidence, the height of the fluorescence unit is generally lowered.

In order to overcome this problem, the Mini-STR method has been developed which reduces the length of the amplification product to a certain size or less. This method effectively derives the STR analysis from the damaged DNA, but it is not compatible with the existing STR database Lt; / RTI > Although a method for recovering chemically damaged DNA through DNA repair enzymes has been developed to obtain an effective STR analysis, it is difficult to obtain a complete STR profile even by this method because DNA damage is difficult to repair due to deformation. have. A method of enriching the STR locus through hybridization has also been proposed as a method for increasing the efficiency of damaged DNA, but this method also has a disadvantage in that a very large amount of DNA is required to obtain a clean STR profile.

It is important to develop an experimental method that can effectively restore the STR profile because old biological samples or legal scientific samples are generally damaged and the amount of the sample is very small. The present inventors have developed a new method for obtaining an effective STR profile from damaged DNA by using a biotinylated primer to amplify a target STR locus and then extracting it using streptavidin magnetic beads for STR analysis , And primers, it is possible to recover the STR profile of the long-length amplified product in the damaged DNA and to obtain an effective STR profile in the forensic blood sample that has been damaged by pre-amplification according to the length of the amplified product The present invention has been completed.

It is an object of the present invention to provide a new method of detecting a gene by preliminary amplification of STR (Short Tandem Repeats) genetic loci, thereby providing a more accurate detection result for a gene having a severe damage and a very small sample amount And provide a plan that can be provided.

In order to achieve the above object,

1) determining one or more target STR (Short tandem repeats) genetic loci in a DNA sample to be analyzed;

2) a pre-amplification step of amplifying using the primers corresponding to the STR genetic loci determined in step 1);

3) recovering the target STR locus bound to magnetic beads in the product that has undergone prior amplification of step 2) using a magnetic bead;

4) amplifying the genetic locus through step 3) using the primer; And

5) analyzing the STR from the amplified product in step 4).

The present invention also provides a method for determining a specific organism or identifying a specific person by evaluating the result of analyzing a STR profile using the above method.

The present invention provides a novel method for detecting genes by preliminary amplification of STR (Short Tandem Repeats) genetic loci (loci), thereby providing more accurate detection results for genes that are severely damaged and have a very small sample volume Therefore, it can be useful in various forensic science fields such as identification of individuals, tracking of suspects in a crime scene, and identification.

FIG. 1A is a simplified diagram of a method for pre-amplifying an STR (Short Tandem Repeat) genetic locus.
FIG. 1B is a diagram comparing the STR analysis efficiency when one STR locus is pre-amplified.
FIG. 1C shows multiple pre-amplification results for eight genetic loci of D18S51, Penta E, D16S539, CSF1PO, Penta D, D8S1179, TPOX and FGA.
FIG. 2A is a diagram showing the results of preceding amplification of the primers D18S51 and Penta E at a 1: 1 ratio and a 0.5: 1 ratio, respectively.
FIG. 2B is a diagram showing the results of preliminary amplification of primers D16S539, CSF1PO and Penta D at a ratio of 1: 1: 1 and 0.5: 1: 1.5, respectively.
FIG. 2C shows the result of preliminary amplification of primers D8S1179, TPOX and FGA at a ratio of 1: 1: 1 and 0.5: 1: 1.5, respectively.
FIG. 2d shows the result of preliminary amplification of the primers of eight genetic loci (D18S51 / D16S539 / D8S1179: CSF1PO / TPOX: PentaE / PentaD / FGA) at the ratios of 0.5: 1: 1.5 and 1: 2: (* Indicates pull-up peak due to fluorescence of different colors).
3A shows a result of preliminary amplification in which the amplification products are divided into shorter groups (D18S51, D16S539, D8S1179, TPOX) and longer length groups (Penta E, CSF1PO, Penta D and FGA) Pull-up peak due to color fluorescence).
FIG. 3B is a diagram showing the results of preliminary amplification of more severely damaged DNA.
FIG. 4A shows the results of pre-amplification of multiple genetic loci (Penta E, CSF1PO, Penta D, and FGA) in long stranded amplification products using the damaged blood DNA sample (No. 42) stored in the Supreme Prosecutor's Office .
4B is an analysis of the damaged blood DNA sample (No. 76) stored in the Supreme Prosecutor's Office as shown in FIG. 4A.

Hereinafter, the present invention will be described in detail.

The present invention, in one aspect,

1) determining one or more target STR (Short tandem repeats) genetic loci in a DNA sample to be analyzed;

2) a pre-amplification step of amplifying using the primers corresponding to the STR genetic loci determined in step 1);

3) recovering the target STR locus bound to magnetic beads in the product that has undergone prior amplification of step 2) using a magnetic bead;

4) amplifying the genetic locus through step 3) using the primer; And

5) analyzing the STR from the amplified product in step 4).

Although DNA analysis using STR is very useful for identification of individuals through forensic evidence gathered at the scene of an accident or natural disaster, DNA that has been exposed at the scene of the incident or extracted from damaged biological evidence is damaged, Analysis of the STR is difficult because it is strange. In the present invention, it was confirmed that a method of pre-amplifying the STR genetic locus using a biotinylated primer and then recovering it by magnetic beads and amplifying it again is an effective method for increasing the STR analysis efficiency of damaged DNA (see Experimental Example 1 and FIG. 1 ).

In addition, since it is relatively easy to damage the genetic locus of a long amplification product, it is difficult to accurately analyze the STR. The present inventors adjusted the ratio of each primer according to the length of the amplification product in the previous amplification, It was confirmed that accurate STR analysis was possible even for a long-length genetic locus (see Experimental Example 2 and FIG. 2).

In addition, according to the length of the amplification products, it was possible to effectively increase the STR analysis efficiency in seriously damaged forensic DNA samples by dividing into long and short groups, respectively, by preceding amplification (Experimental Example 3, FIGS. 3 and 4 )

Therefore, even if the DNA sample contains damages and long-length genetic loci, it is clear that the STR profile analysis of the present invention can be effectively performed by pre-amplifying STR genetic loci.

Generally, in the case of the double amplification method, since the amplification is performed once more by using the STR kit, a considerable amount of non-specific peaks are generated. However, in the method of the present invention, only a small amount of STR genetic loci to be identified are selectively amplified and amplified, and STR analysis is performed. Therefore, generation of non-specific peaks is small, There is a big difference from the general amplification method in that it removes the factors that interfere with the PCR process because it is subjected to a washing process when collecting STR loci. In the present invention, it has been shown that the amount of amplified PCR product can be controlled by controlling the relative amount of the primer corresponding to the long-length genetic locus and the short-length genetic locus, rather than merely repeating the amplification twice 2 and Experimental Example 3). These results confirmed that the present invention is a very breakthrough technology for DNA profiling which requires simultaneous analysis of several genetic loci.

The sample is characterized by being a biological sample including DNA.

In addition, the sample may be a blood, a semen, a vaginal cell, a hair, a hair, a nail, a claw, a saliva, a tear, a urine, a feces, a nose, an oral cell, an epithelial cell, a bone tissue, a skin tissue, Amniotic fluid containing fetal cells, and mixtures thereof. However, the present invention is not limited thereto.

The amount of DNA in the sample is preferably 50 pg or more, but is not limited thereto.

In addition, the STR locus includes D18S51, D16S538, D8S1179, TPOX (human thyroid peroxidase gene), CSF1PO (human c-fms proto-oncogene for CSF-1 receptor gene), Penta E, Penta D and FGA ), But the present invention is not limited thereto.

In addition, the primers may be any one or more selected from the group consisting of fluorescent substances labeled, unlabeled, and both.

In addition, the method may further include adjusting the ratio of the primer according to the length of the amplification product in the previous amplification in the step 2), but the present invention is not limited thereto.

The damage may also include physical, chemical, or biological damage.

In addition, the damage is caused by the fact that the fluorescence unit value of the genetic locus having a short product length (100 bp or more and less than 150 bp) of the products amplified by the preceding amplification in the step 2) is longer than 300 bp and 460 bp The value divided by the fluorescence unit value of the genetic locus may be 5 or more and 10 or less, and the larger the value, the more severe the degree of damage may be, but is not limited thereto.

Preliminary amplification of step 2) in the above step may be to amplify the genetic loci longer than 380 bp such as Penata D, Penata E and FGA among the STR genetic loci, but is not limited thereto.

In addition, the preliminary amplification of step 2) distinguishes shorter than 315 bp, such as D18S51, D16S538, D8S1179 and TPOX among STR loci, and 315 bp or longer such as CSF1PO, Penta E, Penta D and FGA, But the present invention is not limited thereto.

According to another aspect of the present invention, there is provided a method of determining a specific organism or identifying a specific person by evaluating a result of analyzing a STR profile using the above method.

Using this method, an effective STR profile can be obtained even if the DNA sample contains an STR having a long damaging or long-length genetic locus. Therefore, the STR analysis and amplification can be performed even in a sample, The identification and genotyping of the identified genes confirmed that identification, identification, and specific biosensing are possible.

The method may further include steps of all of the above-described aspects, but after step 5), the amplified alleles may be evaluated and the respective allelic genotypes may be determined to identify a specific organism or a specific person , But is not limited thereto.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

However, the following examples are illustrative of the present invention, and the content of the present invention is not limited by the following examples and experimental examples.

< Example  1> dielectric ( genomic ) DNA extraction

Genomic DNA was extracted from HeLa cell line.

Specifically, HeLa cells were cultured in a DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10% fetal calf serum for 48 hours at 37 ° C and 5% CO 2 at 37 ° C in a CO 2 incubator. The thus cultured cells were scraped with scrapers, (eppendorf tubes) and washed twice with 1X PBS to remove media components. Genomic DNA was extracted from the prepared cells according to the protocol of QIAamp DNA Mini Kit (Cat. No. 51304, QIAGEN, Hilden, DEU).

In addition, blood DNA samples were extracted from blood stain dropped on a paper towel in 1995 and 20 years old genomic DNA was used. Specifically, 2 to 3 holes were made for a paper towel with blood stains The fragments were transferred to a 1.5 ml tube, and the genomic DNA was extracted from the cells according to the protocol of the QIAamp DNA Mini Kit. The extracted genomic DNA was stored at -20 ° C until used in the next experiment.

< Example  2> DNA Fragmentation Using Ultrasound

DNA was fragmented by ultrasound sonication to create a model of damaged DNA.

Specifically, the sample extracted in Example 1 was subjected to ultrasonic treatment for 15 seconds in ice for 15 seconds by using an ultrasonic processor (Cole Parmer, Vernon Hills, USA) and repeated 60 times to induce fragmentation . The sonicated DNA was electrophoresed on 0.8% agarose gel to confirm its size. That is, DNA fragmentation levels were measured using the Qubit ® dsDNA HS assay kit (Invitrogen, Carlsbad, Calif., USA) and Qubit ® fluorometer (Invitrogen). In the present invention, it was confirmed that fragmented DNA was produced in an average size of about 200 to 300 bp, and the experiment of the present invention was carried out.

< Example  3> primer primer design

Primers were designed to amplify fragmented DNA samples.

Specifically, the primer was designed by referring to the primer sequence information on the PowerPlex ® 16 System kit (Promega, Madison Wis., USA) from STRbase (http://www.cstl.nist.gov/strbase/). All primers were added with biotin at the 5 'position, and the respective primer sequences prepared were as follows.

SEQ ID NO: The STR locus Primer sequence (5'-3 ') One CSF1PO-F CCG GAG GTA AAG GTG TCT TAA AGT 2 CSF1PO-F30 CCG GAG GTA AAG GTG TCT TAA AGT GAG AAA 3 CSF1PO-F35 CCG GAG GTA AAG GTG TCT TAA AGT GAG AAA GAA TA 4 CSF1PO-TG TGC TAA CCA CCC TGT GTC TCA GTT TTC CTA 5 CSF1PO-R ATT TCC TGT GTC AGA CCC TGT T 6 CSF1PO-R30 ATT TCC TGT GTC AGA CCC TGT TCT AAG TAC 7 CSF1PO-R35 ATT TCC TGT GTC AGA CCC TGT TCT AAG TAC TTC CT 8 CSF1PO-R40 ATT TCC TGT GTC AGA CCC TGT TCT AAG TAC TTC CTA TCTA 9 D18S51-F25 TTC TTG AGC CCA GAA GGT TAA GGC T 10 D18S51-F35 TTC TTG AGC CCA GAA GGT TAA GGC TGC AGT GAG CC 11 D18S51-R30 CTA CCA GCA ACA ACA CAA ATA AAC AAA CCG 12 D18S51-R40 CTA CCA GCA ACA ACA CAA ATA AAC AAA CCG TCA GCC TAA G 13 D18S51-Tm60 CCT AAG GTG GAC ATG TTG GCT 14 D18S51-Tm67 ACA GAG AGA AGC CAA CAT GTC CAC C 15 D18S51-GC54 GCC ATG TTC ATG CCA CTG CAC TTC 16 D18S51-TG CGT CAG CCT AAG GTG GAC AT 17 Penta D-F GAA GGT CGA AGC TGA AGT G 18 Penta D-R ATT AGA ATT CTT TAA TCT GGA CAC AAG 19 D21S11 TGT ATT AGT CAA TGT TCT CCA GAG AC 20 D16S539-F GGG GGT CTA AGA GCT TGT AAA AAG 21 D16S539-R GTT TGT GTG TGC ATC TGT AAG CAT GTA TC 22 D16S539-R35 GTT TGT GTG TGC ATC TGT AAG CAT GTA TCT ATC AT 23 Penta E-F ATT ACC AAC ATG AAA GGG TAC CAA TA 24 Penta E-R TGG GTT ATT AAT TGA GAA AAC TCC TTA CAA TTT 25 D8S1179-F ATT GCA ACT TAT ATG TAT TTT TGT ATT TCA TG 26 D8S1179-R ACC AAA TTG TGT TCA TGA GTA TAG TTT C 27 TPOX-F GCA CAG AAC AGG CAC TTA GG 28 TPOX-R CGC TCA AAC GTG AGG TTG 29 FGA-F GGC TGC AGG GCA TAA CAT TA 30 FGA-R ATT CTA TGA CTT TGC GCT TCA GGA

< Example  4> STR (Short Tandem Repeats) Genetic  Pre-amplification

Pre-amplification was performed to verify that the analysis was more effective than general STR analysis through pre-amplification.

Specifically, 2 ng of damaged DNA, 0.6 uM of biotinylated target locus primer at 0.2 uM, 10X PCR Gold ® buffer II (Applied Biosystems, Foster City, CA, USA), 0.05 mM dNTPs mix, 1.5 mM MgCl 2 , 1.25 U Ampli Taq Gold ® DNA polymerase (Applied Biosystems), and distilled water. Pre-amplification was performed using Mycylcer TM The cells were pre-denaturated at 95 ° C for 10 minutes, denatured at 95 ° C for 15 seconds, annealed at 60 ° C for 30 seconds, and incubated for 30 seconds at 72 ° C for 6 to 16 cycles using a thermal cycler (BIO-RAD, USA) , Followed by final extension at 72 ° C for 5 minutes.

After the reaction was completed, the pre-amplified samples were eluted through magnetic beads and then subjected to STR typing. STR typing was done using the PowerPlex ® 16 System kit. PCR products were detected using an ABI 3130XL Genetic Analyzer (Applied Biosystems, CA, USA) containing modified polymer POP-4 ™ (Applied Biosystems, Foster City, Calif., USA) and analyzed using Gene Mapper ID v3.2 Analysis software Applied Biosystems). The PowerPlex 占 Matrix Standards 3100/3130 (Promega, Madison Wis., USA) were used for spectral calibration of the ABI 3130XL Genetic Analyzer. The above process can be briefly expressed as in FIG. 1A.

< Example  5> Magnetic bead  Used Biotin  Tagged DNA fragment  extraction

The pre - amplified products were extracted using streptavidin - coated magnetic beads.

Specifically, the amplification product was gently mixed with 250 μl 1X Pyromark binding buffer (QIAGEN, Hilden, DEU) and 10 μl magnetic beads (Dynabeads ® MyOne ™ Streptavidin T1, Invitrogen) at room temperature for 1 hour to obtain beads and amplified products Respectively. The beads used at this time were previously washed with 1X Pyromark binding buffer. After binding, the bead-target genetic locus complex was washed three times with 800 [mu] l 0.5X Pyromark binding buffer (QIAGEN) at room temperature. After the binding buffer solution was completely removed, the bead complex was again dissolved in 10 μl of distilled water and heated at 90 ° C. for 2 minutes. After heating, the beads were collected with a magnet and the supernatant containing the eluted target DNA was transferred to a clean tube. DNA recovered by bead hybridization was amplified with the PowerPlex ® 16 HS System (Promega) and then detected on an ABI 3130XL Genetic Analyzer.

< Experimental Example  1> of damaged DNA STR Hereditary position  After pre-amplification STR  analysis

1C, the left panel of FIG. 1C shows the results of STR analysis of normal HeLa cell DNA, the middle panel shows the results of STR analysis of DNA damaged by ultrasonication, The seven STR genotypes (D3S1358, TH01, D21S11, D5S818, D13S317, D7S829, vWA (von Willebrand factor A)) with short amplification products except for 8 STR loci were not amplified.

Analysis of FIG. 1C confirmed that the STR profile of the damaged DNA was affected more by longer than the shorter length of the amplification product (FIG. 1C, middle panel).

In order to confirm the sensitivity of the pre-amplification method, we compared the results of general STR analysis and STR analysis after preliminary amplification of one STR locus. Using the 50, 100, and 500 pg of damaged DNA, Analysis efficiency was confirmed. As a result of the preliminary amplification of D8S1179, the STR pattern was observed even though the concentration was lower than 500 pg which is necessary for general STR analysis (FIG. 1B). Similar results were obtained with D18S51. In addition, the sensitivity of Penta D, which is the longest among the 16 STR loci, was also increased (FIG. 1B). We could observe clearly the STR analysis after performing preliminary amplification using 100 pg of the fluorescence unit of the long allele of Penta D, which was difficult to confirm by normal STR analysis using 500 pg of damaged DNA . These results suggest that it is very effective to pre - amplify STR locus specific for STR analysis using damaged DNA.

To extend this approach to multiple STR loci, we selected eight STR loci with amplification products of 200 to 450 bp in length. As a result of STR analysis after pre-amplification, the fluorescence peak height of D18S51, D16S539, D8S1179, and TPOX with relatively short amplification products was significantly increased, (Fig. 1C). It was found that the STR locus of 8 (D18S51, D16S539, D8S1179, TPOX, CSF1PO, Penta D, Penta E and FGA) was enriched specifically. Using these primers, It can be seen that the pre-amplification method is a very specific experiment.

In addition, in this multiplex PCR method, locus having a short amplification product is amplified well, whereas Penta D, Penta E and FGA having a long amplification product are less efficient than a single locus amplification (Fig. 1B).

< Experimental Example  2> Amplified  Long STR Loci  Identify primer ratio conditions to increase efficiency

Unbalanced amplification of STR loci with long amplification products in damaged DNA occurs because the concentration of STR loci in the sample is relatively low. Therefore, in order to solve the other problems of DNA template concentration depending on the length of the amplification product, The ratio of the primers was adjusted by increasing the concentration of loci of long length.

As a result, the fluorescence peak of D18S51 and Penta E at the concentration of 0.4 μM was higher than that of D18S51. However, when the concentration of the primer of D18S51 was reduced to 0.2 uM, the peak height of Penta E was increased (Fig. 2a). Similar results were also observed with the previous amplification of D16S539, CSF1PO, and Penta D (Fig. 2B). In the experiments involving D8S1179, TPOX and FGA, the primer concentration of D8S1179 (0.2 uM), the concentration of FGA primer (0.6 uM), the fluorescence peak height of D8S1179 decreased and the fluorescence peak of FGA increased (Fig. 2C).

Taken together, it can be seen that changing the concentration of the primer during pre-amplification can change the fluorescence unit observed during STR analysis.

These changes were extended to a total of 8 STR loci (D18S51, D16S538, D8S1179, TPOX, CSF1PO, Penta E, Penta D and FGA). In case of D18S51, D16S538 and D8S1179 with relatively short length, the concentration of primer was reduced to half of 0.2 μM and the concentration of primer was increased to 1.5 μM for Penta E, Penta D and FGA having long lengths.

As a result, a change in the fluorescence peak according to the concentration of the primer was observed (FIG. 2d, middle panel). The ratio of the primers added at the previous amplification was changed to 1: 2: 3 (D18S51, D16S538, D8S1179 0.4 uM: TPOX, CSF1PO 0.8 uM: Penta E, Penta D, FGA 1.2 uM) 2 times. As a result, the increase of the fluorescence peak height is not clear and the non-specific peak is also generated, and it is confirmed that the amount of total concentration added as well as the ratio between the primers at the previous amplification affects the prior amplification efficiency of STR loci 2d, right panel).

< Experimental Example  3> By discriminating pre-amplification of damaged DNA STR  Confirmation of improvement of analytical efficiency

The degree of damaged DNA can be represented by the degradation index. In general, the value of the fluorescence unit of the locus in which the length of the amplification product is short is divided by the fluorescence unit of the locus in which the length of the amplification product is long. When the degradation index (DI) Is the most common STR pattern. As shown in FIG. 2d, the STR profile can be obtained by STR analysis when the DNA of 5 <DI <10 is amplified prior to amplification, but in the DNA sample seriously damaged by DI> 10, It was found that the STR pattern was not recovered by the previous amplification.

Referring to FIG. 1B and FIGS. 2A to 2D, it can be seen that STR peak recovery rate is influenced by the number of STR locus amplified when multiple STR locus is amplified simultaneously.

STR loci was divided into short groups (D18S51, D16S539, D8S1179 and TPOX) and long groups (Penta E, CSF1PO, Penta D and FGA) according to the amplification products. The peak of STR loci was effectively improved when only the long group was subjected to fractional pre-amplification (Fig. 3A, right panel).

In order to confirm that this pre-amplification method was also applicable to more severely damaged DNA samples, we proceeded with further ultrasonication to artificially damage at an average of 200 bp. In this more severely damaged DNA, the STR peak pattern of Penta E and Penta D completely disappeared (Fig. 3b, left panel). The STR pattern of Penta E and Penta D was not completely improved (Fig. 3b, middle panel), compared to loci with relatively short amplification products, despite the progress of mutiple pre-amplification. However, it was confirmed that the STR fluorescence peaks of each of the Penta E, CSF1PO, Penta D and FGA primers were completely amplified (FIG. 3B, right panel).

These results show that STR analysis can maximize the efficiency of STR analysis of severely damaged DNA samples by combining pre - amplification and multiple primer amplification according to amplification product length of STR loci.

< Experimental Example  4> Using real DNA samples, STR  Conduct analysis

In order to confirm whether the above-mentioned preliminary amplification method is also applied to the damaged genomic DNA sample, the preliminary amplification method using the forensic blood DNA sample was performed.

Specifically, the forensic DNA samples were subjected to STR analysis at the Supreme Prosecutor 's Office, and the samples suitable for the experiment were selected. A total of 82 blood DNA samples, 42 and 76, were selected for the experiment. 2 ng of 42 samples were used and 8 STR loci, totaling 8 STR loci, were simultaneously amplified in advance, and D18S51, Penta E, D16S539, CSF1PO, Penta D, D8S1179, TPOX and FGA. Previously amplified loci were collected using magnetic beads, and STR patterns of D18S51, D16S539, D8S1179, and TPOX, which were difficult to see through general STR analysis, were confirmed (Fig. 4A, left panel).

Further, only the Penta E, CSF1PO, Penta D and FGA were discriminated and amplified prior to confirming the STR pattern (FIG. 4B, right panel). No accurate fluorescence patterns of Penta E and Penta D were observed when subjected to general STR analysis. However, the other STR loci was precisely matched with the fluorescence pattern after the previous amplification and the normal STR fluorescence pattern. The same experiment was carried out once again using the 76th blood DNA sample and the same result was observed (FIG. 4b). From these results, it was confirmed that the STR analysis by differential amplification is effective for identifying STR patterns of extremely damaged DNA samples that can not confirm the STR pattern by general STR method.

These results suggest that STR loci, which has a long amplification product, can be amplified by preliminary amplification using loci specific primers. Taken together, the pre-amplification method can be applied to forensic DNA samples, and it can be seen that the fluorescence pattern of the STR loci, which is long in the length of the amplification product, can be restored.

<110> THE REPUBLIC OF KOREA (SUPREME PROSECUTORS 'OFFICE) <120> Forensic profiling by differential pre-amplification of STR loci <130> 15p-07-023 <160> 30 <170> Kopatentin 2.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> CSF1PO-F <400> 1 ccggaggtaa aggtgtctta aagt 24 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> CSF1PO-F30 <400> 2 ccggaggtaa aggtgtctta aagtgagaaa 30 <210> 3 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> CSF1PO-F35 <400> 3 ccggaggtaa aggtgtctta aagtgagaaa gaata 35 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> CSF1PO-TG <400> 4 tgctaaccac cctgtgtctc agttttccta 30 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CSF1PO-R <400> 5 atttcctgtg tcagaccctg tt 22 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> CSF1PO-R30 <400> 6 atttcctgtg tcagaccctg ttctaagtac 30 <210> 7 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> CSF1PO-R35 <400> 7 atttcctgtg tcagaccctg ttctaagtac ttcct 35 <210> 8 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> CSF1PO-R40 <400> 8 atttcctgtg tcagaccctg ttctaagtac ttcctatcta 40 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> D18S51-F25 <400> 9 ttcttgagcc cagaaggtta aggct 25 <210> 10 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> D18S51-F35 <400> 10 ttcttgagcc cagaaggtta aggctgcagt gagcc 35 <210> 11 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> D18S51-R30 <400> 11 ctaccagcaa caacacaaat aaacaaaccg 30 <210> 12 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> D18S51-R40 <400> 12 ctaccagcaa caacacaaat aaacaaaccg tcagcctaag 40 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> D18S51-Tm60 <400> 13 cctaaggtgg acatgttggc t 21 <210> 14 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> D18S51-Tm67 <400> 14 acagagagaa gccaacatgt ccacc 25 <210> 15 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> D18S51-GC54 <400> 15 gccatgttca tgccactgca cttc 24 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> D18S51-TG <400> 16 cgtcagccta aggtggacat 20 <210> 17 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Penta D-F <400> 17 gaaggtcgaa gctgaagtg 19 <210> 18 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Penta D-R <400> 18 attagaattc tttaatctgg acacaag 27 <210> 19 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> D21S11 <400> 19 tgtattagtc aatgttctcc agagac 26 <210> 20 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> D16S539-F <400> 20 gggggtctaa gagcttgtaa aaag 24 <210> 21 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> D16S539-R <400> 21 gtttgtgtgt gcatctgtaa gcatgtatc 29 <210> 22 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> D16S539-R35 <400> 22 gtttgtgtgt gcatctgtaa gcatgtatct atcat 35 <210> 23 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Penta E-F <400> 23 attaccaaca tgaaagggta ccaata 26 <210> 24 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Penta E-R <400> 24 tgggttatta attgagaaaa ctccttacaa ttt 33 <210> 25 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> D8S1179-F <400> 25 attgcaactt atatgtattt ttgtatttca tg 32 <210> 26 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> D8S1179-R <400> 26 accaaattgt gttcatgagt atagtttc 28 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TPOX-F <400> 27 gcacagaaca ggcacttagg 20 <210> 28 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> TPOX-R <400> 28 cgctcaaacg tgaggttg 18 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FGA-F <400> 29 ggctgcaggg cataacatta 20 <210> 30 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> FGA-R <400> 30 attctatgac tttgcgcttc agga 24

Claims (12)

1) determining one or more target STR (Short tandem repeats) genetic loci in a DNA sample to be analyzed;
2) pre-amplifying the STR locus determined in step 1) by controlling the ratio of the primer according to the length of the amplification product;
3) recovering the target STR locus bound to magnetic beads in the product that has undergone prior amplification of step 2) using a magnetic bead;
4) amplifying the genetic locus through step 3) using the primer; And
5) analyzing the STR from the amplified product in step 4).
2. The method of claim 1, wherein the sample is all biological samples including DNA.
The method of claim 1, wherein the sample is selected from the group consisting of blood, semen, vaginal cells, hair, hairs, nails, claws, saliva, tears, urine, feces, runny nose, oral cells, epithelial cells, , A placental cell or an amniotic fluid containing a fetal cell, and a mixture thereof.
The method according to claim 1, wherein the amount of DNA in the sample is 50 pg or more and 500 pg or less.
The method according to claim 1, wherein the STR locus is selected from the group consisting of D18S51, D16S538, D8S1179, TPOX (human thyroid peroxidase gene), CSF1PO (Human c-fms proto-oncogene for CSF-1 receptor gene), Penta E, Human fibrinogen alpha chain). &Lt; RTI ID = 0.0 &gt; 21. &lt; / RTI &gt;
The method according to claim 1, wherein the primers are any one or more selected from the group consisting of labeled, unlabeled, and both.
delete The method of claim 1, wherein the damage comprises physical, chemical, or biological damage.
delete 2. The method of claim 1, wherein the pre-amplification of step 2) amplifies a genetic locus longer than 380 bp in the STR locus.
2. The method according to claim 1, wherein the preliminary amplification of step 2) is carried out by differentially amplifying each of the primers separately by discriminating between 315 bp and 315 bp in the determined STR genetic loci.
A method for determining a specific organism or identifying a specific person by evaluating a result of analyzing a STR profile using the method of claim 1.

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KR101008828B1 (en) 2010-03-12 2011-01-19 대한민국 Multiplex pcr system comprising 16 str loci and amelogenin which are highly discriminative in korean population and the method of human identification using them
KR101457983B1 (en) 2014-05-15 2014-11-06 대한민국 Method for Autosomal Analysing Human Subject of Analytes Using Multiplex Gene Amplification

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KR100277289B1 (en) * 1999-04-22 2000-12-15 김태정 Quadruplex PCR systems consisting of STR loci which are high discriminating in Korean population and the methods of human identification using them

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KR101008828B1 (en) 2010-03-12 2011-01-19 대한민국 Multiplex pcr system comprising 16 str loci and amelogenin which are highly discriminative in korean population and the method of human identification using them
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