CN112522418A - Genetic marker for detecting human genome DNA - Google Patents
Genetic marker for detecting human genome DNA Download PDFInfo
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- CN112522418A CN112522418A CN201910886259.3A CN201910886259A CN112522418A CN 112522418 A CN112522418 A CN 112522418A CN 201910886259 A CN201910886259 A CN 201910886259A CN 112522418 A CN112522418 A CN 112522418A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6844—Nucleic acid amplification reactions
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Abstract
The invention discloses a genetic marker for detecting human genome DNA. The genetic markers for detecting the human genome DNA disclosed by the invention are a DNA fragment A and a DNA fragment B, wherein the DNA fragment A is a DNA fragment shown as a sequence 3 in a sequence table, and the DNA fragment B is a DNA fragment shown as a sequence weight-retaining sequence 8. The genetic marker for detecting the human genome DNA is used for detecting the human genome DNA, has high sensitivity, can accurately quantify in a human genome DNA concentration range of 10 ng/mu l-0.128 pg/mu l, further can detect the degradation degree of the human genome DNA, and has wide application prospect.
Description
Technical Field
The invention relates to the field of biotechnology, and discloses a genetic marker for detecting human genome DNA.
Background
The existing human genome DNA quantitative method based on fluorescence quantitative PCR can be divided into two types according to the copy number of a detected gene segment in a haploid human genome, one type selects a single copy gene, and the distribution consistency of the copy number in the population is high. However, the detection of a reaction system having a DNA concentration of less than 10 ng/. mu.l is difficult to accurately detect because of the low copy number and low sensitivity of quantitative detection. And the other type selects high repetitive sequence multi-copy genes, mainly Alu, with millions of copies and higher detection sensitivity, which can be used for detecting trace human DNA but is not suitable for detecting human DNA samples with higher concentration.
Disclosure of Invention
The invention aims to provide a genetic marker for detecting human genome DNA, and solves the problems of low sensitivity and narrow applicable sample DNA concentration range of the existing quantitative detection method for detecting the human genome DNA.
The invention firstly provides any one of the following X1) -X13):
x1) the use of a DNA fragment named DNA fragment A for the detection of human genomic DNA; the DNA fragment A is a DNA fragment shown in a sequence 3 in a sequence table or any fragment thereof;
x2) detecting the substance of the DNA fragment A in the detection of human genome DNA;
x3) application of a substance for detecting the DNA fragment A in preparing a product for detecting human genome DNA;
x4) the application of the DNA fragment A in detecting whether human genome DNA is degraded or not;
x5) detecting the substance of the DNA fragment A in detecting whether the human genome DNA is degraded or not;
x6) application of a substance for detecting the DNA fragment A in preparing a product for detecting whether human genome DNA is degraded or not;
x7) the application of the materials used in the method for detecting human genome DNA by taking the DNA fragment A as a human genetic marker in the detection of human genome DNA;
x8) the application of the materials used in the method for detecting human genome DNA by using the DNA fragment A as a human genetic marker in detecting whether the human genome DNA is degraded or not;
x9) the application of the DNA fragment A as a human genetic marker in detecting human genome DNA;
x10) the application of the DNA fragment A as a human genetic marker in detecting whether human genome DNA is degraded or not;
x11) the use of the DNA set for the detection of human female genomic DNA; the complete set of DNA fragments consists of the DNA fragment A and the DNA fragment named as the DNA fragment B, wherein the DNA fragment B is the DNA fragment shown in a sequence 8 in a sequence table or any fragment thereof;
x12) the application of the substance for detecting the DNA fragment set in the preparation of the product for detecting the genome DNA of the human female;
x13) in detecting human female genome DNA.
In the application, the DNA fragment A can be a DNA fragment shown in 66 th to 151 th sites of a sequence 3 in a sequence table or any fragment thereof.
In the above application, the substance for detecting the DNA fragment a may include a primer pair named primer pair a or a primer pair named primer pair B, the primer pair a is composed of two single-stranded DNAs shown in sequences 1 and 2 in the sequence table, and the primer pair B is composed of two single-stranded DNAs shown in sequences 4 and 5 in the sequence table.
The substance for detecting the set of DNA fragments may be composed of the substance for detecting the DNA fragment A and the substance for detecting the DNA fragment B, and the substance for detecting the DNA fragment B includes a primer pair named as primer pair C; the primer pair C consists of two single-stranded DNAs shown as sequences 6 and 7 in a sequence table.
The substance for detecting the DNA fragment A can also comprise other substances required for PCR amplification or real-time fluorescent quantitative PCR.
The substance for detecting the DNA fragment A can specifically consist of the primer pair A or the primer pair B only, or consist of the primer pair A or the primer pair B and other substances required for PCR amplification or real-time fluorescent quantitative PCR.
The substance for detecting the DNA fragment B can also comprise other substances required for PCR amplification or real-time fluorescent quantitative PCR.
The substance for detecting the DNA fragment B can only consist of the primer pair C, and also can consist of the primer pair C and other substances required for PCR amplification or real-time fluorescent quantitative PCR.
The substance may be a kit.
The present invention also provides a method of detecting a human sample, the method comprising: detecting whether the genomic DNA of a sample to be detected contains the DNA fragment A by using the substance for detecting the DNA fragment A, and determining whether the sample to be detected contains a human sample or is the human sample: if the genomic DNA of the sample to be detected contains the DNA fragment A, the sample to be detected contains or is a candidate for containing a human sample or is a candidate for a human sample; if the genomic DNA of the test sample does not contain the DNA fragment A, the test sample does not contain or is not a candidate for a human sample.
In the above method, the detection of whether the genomic DNA of the sample to be detected contains the DNA fragment a may be performed by fluorescent quantitative PCR.
The present invention also provides a method for quantitatively detecting human genomic DNA, the method comprising: detecting the human genome DNA standard products with different concentrations by using the primer pair A or the primer pair B by adopting a fluorescent quantitative PCR method to obtain Ct values of the human genome DNA standard products with different concentrations, and determining the relation between the Ct values and the human genome DNA concentration; and detecting a sample to be detected by using the primer pair A or the primer pair B to obtain a Ct value of the sample to be detected, and determining the content of the human genome DNA in the sample to be detected according to the relation between the Ct value and the human genome DNA concentration and the Ct value of the sample to be detected.
The relationship between the determined Ct value and the human genomic DNA concentration may be a relationship between the Ct value and a male human genomic DNA concentration and/or a relationship between the Ct value and a female human genomic DNA concentration. The relation between the Ct value and the concentration of the male human genome DNA may be a working curve 1 or a working curve 3, wherein the working curve 1 is y ═ 1.396ln (x)) +26.531, the working curve 3 is y ═ 1.45ln (x)) +29.416, x is the concentration of the male genome DNA, and y is the Ct value. The relationship between the Ct value and the concentration of the female human genome DNA may be a working curve 2, where y is-1.432 ln (x) +26.425, x is the concentration of the female genome DNA, and y is the Ct value, in the working curve 2.
The content of the human genome DNA in the sample to be detected is equal to the sum of the contents of the male genome DNA and the female genome DNA.
The invention also provides a method for detecting a female sample, comprising: detecting the content of the human genome DNA in a sample to be detected by adopting the method for quantitatively detecting the human genome DNA; detecting the male human genome DNA standard products with different concentrations by using the primer pair C by adopting a fluorescent quantitative PCR method to obtain Ct values of the male human genome DNA standard products with different concentrations, and determining the relation between the Ct values and the male human genome DNA concentrations; detecting the sample to be detected by using the primer pair C to obtain a Ct value of the sample to be detected, and determining the content of male genome DNA in the sample to be detected according to the relation between the Ct value and the concentration of the male human genome DNA and the Ct value of the sample to be detected;
comparing the content of the human genome DNA in the sample to be detected with the content of the male genome DNA, and determining whether the sample to be detected contains a human female sample: if the content of the human genome DNA in the sample to be detected is equal to the content of the male genome DNA, the sample to be detected does not contain or is candidate to contain the human female sample; and if the content of the human genome DNA in the sample to be detected is larger than that of the male genome DNA, the sample to be detected contains or is candidate to contain the human female sample.
The invention also provides a method for quantitatively detecting the human female genome DNA, which comprises the following steps: detecting the content of the human genome DNA in a sample to be detected by adopting the method for quantitatively detecting the human genome DNA; detecting the male human genome DNA standard products with different concentrations by using the primer pair C by adopting a fluorescent quantitative PCR method to obtain Ct values of the male human genome DNA standard products with different concentrations, and determining the relation between the Ct values and the male human genome DNA concentrations; detecting the sample to be detected by using the primer pair C to obtain a Ct value of the sample to be detected, and determining the content of male genome DNA in the sample to be detected according to the relation between the Ct value and the concentration of the male human genome DNA and the Ct value of the sample to be detected;
comparing the content of the human genome DNA in the sample to be detected with the content of the male genome DNA, and determining the content of the female genome DNA in the sample to be detected according to the following formula: and the content of female genome DNA in the sample to be detected is equal to the content of human genome DNA in the sample to be detected, namely the content of male genome DNA in the sample to be detected.
In the above, the method of fluorescent quantitative PCR can be performed by performing fluorescent quantitative PCR on the genomic DNA of the sample to be tested using each primer pair and 2 × qPCR Master Mix (containing SYBR Green dye). The 2x qPCR Master Mix (containing SYBR Green dye) may be a product of biotechnology limited of nuozokenza, tokyo.
The reaction conditions of the fluorescent quantitative PCR can be as follows: pre-denaturation at 95 ℃ for 5 min; 40 cycles: denaturation at 95 ℃ for 5s and annealing extension at 60 ℃ for 15 s.
The substance for detecting the DNA fragment A also belongs to the protection scope of the invention.
The substance for detecting the DNA fragment set also belongs to the protection scope of the invention.
The complete set of DNA fragments also belongs to the protection scope of the invention.
In the invention, the application of the DNA fragment A in forensic detection, the application of a substance for detecting the DNA fragment A in forensic detection, the application of the set of DNA fragments in forensic detection, or the application of a substance for detecting the set of DNA fragments in forensic detection also belongs to the protection scope of the invention.
Experiments prove that the human genome DNA can be detected by using the genetic marker for detecting the human genome DNA, namely the DNA fragment A, the detection sensitivity is high and can reach 0.128 pg/mu l, accurate quantification can be carried out in a human genome DNA concentration interval of 10 ng/mu l-0.128 pg/mu l, and the degradation degree of the human genome DNA can be further detected; the DNA fragment B can be used for detecting the male human genome DNA, the detection sensitivity is high and can reach 0.128 pg/mu l, and accurate quantification can be carried out in a male human genome DNA concentration range of 10 ng/mu l-0.128 pg/mu l; further, whether the sample to be detected contains a female sample or not can be determined according to the quantitative result of the human total DNA and the quantitative result of the male genome DNA, and the female genome DNA is quantified. The DNA fragment A and the DNA fragment B have wide application prospect.
Drawings
FIG. 1 is a working curve of human genomic DNA detection.
FIG. 2 is the human male genome DNA detection working curve.
FIG. 3 shows the result of the detection of the concentrations of male and female DNAs in the DNA solution to be detected. Ratio of the measured values of female to male DNA concentrations.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.
2x qPCR Master Mix (containing SYBR Green dye) is a product of Nanjing Novozam Biotech Co., Ltd.
Example 1, copy number of DNA fragment shown in SEQ ID No. 3 among different human individuals was stable
The invention finds that the copy number of a DNA fragment (marked as fragment A) shown as a sequence 3 in a sequence table is stable among different human individuals in DNA samples derived from 274 male individuals and 198 female individuals, and a method for detecting the copy number of the DNA fragment by using Alu as a control comprises the following steps:
1. sample to be tested
274 parts of genomic DNA of a blood card sample from a male individual and 198 parts of genomic DNA of a blood card sample from a female individual are extracted, and then the genomic DNAs are diluted by 5 times respectively to obtain DNA solutions of samples to be detected.
2. Fluorescent quantitative PCR
2.1 primer: the primer pair for amplification of the target fragment consists of F1 and R1 (for amplification of the DNA fragment shown at positions 66-151 of sequence 3), and the primer pair for detection of Alu consists of F2 and R2, specifically as follows:
dissolving each primer in 10mM Tris-HCl buffer solution, and mixing pair primers in an equimolar way to obtain primer solutions for respectively amplifying fragments A and Alu, wherein the final concentration of the forward primer and the final concentration of the reverse primer in each solution are both 1.25 mu M.
2.2 reaction solution preparation and qPCR reaction: respectively carrying out fluorescence quantitative PCR on each sample DNA solution to be detected, wherein the reaction system composition is shown in the following table:
| reagent | Volume of |
| 2x qPCR Master Mix (containing SYBR Green dye) | 10μl |
| DNA solution of sample to be tested | 2μl |
| Primer solution | 8μl |
The PCR system amplification reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; 40 cycles: denaturation at 95 ℃ for 5s and annealing extension at 60 ℃ for 15 s.
3. Data analysis
After the fluorescent quantitative PCR in the step 2 is finished, the Ct values of the fluorescent quantitative PCR reaction of the two primers of the male sample (table 1) and the female sample (table 2) are shown in tables 1 and 2, and the difference value of the Ct values in the tables 1 and 2 is the difference value of the Ct value amplified by the primer pair Alu and the Ct value amplified by the primer pair A.
TABLE 1 Ct values of fluorescence quantitative PCR of male samples
TABLE 2 Ct values of fluorescence quantitative PCR of female samples
The results showed that the difference in Ct values obtained was small in the above DNA samples derived from 274 male individuals and 198 female individuals, and there was no significant difference between men and women, and it was useful as a genetic marker for detecting human DNA.
Example 2 sensitivity of detection of human genomic DNA Using the DNA fragment shown in SEQ ID No. 3
On the basis of example 1, human genomic DNA was detected using primer pair A of example 1 by the following method:
1. sample to be tested
Mixing male genome DNA samples: promega Human Genomic DNA: Male (Promega, G1471). The DNA sample was diluted with 10mM Tris-HCl buffer solution in a 5-fold concentration gradient to obtain male DNA dilution solutions having DNA concentrations of 100 ng/. mu.l, 20 ng/. mu.l, 4 ng/. mu.l, 0.8 ng/. mu.l, 0.16 ng/. mu.l, 32 pg/. mu.l, 6.4 pg/. mu.l and 1.28 pg/. mu.l, respectively.
Mixing female genome-wide DNA samples: promega Human Genomic DNA: Female (Promega, G1521). The DNA sample was diluted with 10mM Tris-HCl buffer solution in a 5-fold concentration gradient to obtain female DNA dilution solutions having DNA concentrations of 100 ng/. mu.l, 20 ng/. mu.l, 4 ng/. mu.l, 0.8 ng/. mu.l, 0.16 ng/. mu.l, 32 pg/. mu.l, 6.4 pg/. mu.l and 1.28 pg/. mu.l, respectively.
2. Fluorescent quantitative PCR
Respectively taking each DNA diluted solution in the step 1 as template DNA, and carrying out fluorescent quantitative PCR by using a primer pair A, wherein a reaction system and reaction conditions are as follows:
reaction system (20 μ l): 2x qPCR Master Mix (containing SYBR Green dye) 10. mu.l, DNA dilution 2. mu.l, primer solution 8. mu.l. Wherein, the primer solution is obtained by respectively dissolving two single-stranded DNAs shown as sequences 1 and 2 in a sequence table by using 10mM Tris-HCl buffer solution and then mixing the two single-stranded DNAs in an equimolar manner, and the concentrations of the two single-stranded DNAs shown as sequences 1 and 2 in the solution are both 1.25 mu M.
Reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 5s, annealing extension at 60 ℃ for 15s, and 40 cycles.
3. Data analysis
After the fluorescent quantitative PCR in step 2 is finished, Ct values corresponding to DNA samples of various concentrations for male and female are analyzed, and are shown in Table 3.
TABLE 3 Ct value of DNA of each sample
| Concentration of template DNA in reaction System (ng/. mu.l) | Ct value of male DNA sample | Ct value of |
| 10 | 13.93 | 13.47 |
| 2 | 15.72 | 15.44 |
| 0.4 | 18.05 | 17.77 |
| 0.08 | 20.35 | 20.08 |
| 0.016 | 22.56 | 22.39 |
| 3.2×10-3 | 25.05 | 24.69 |
| 0.64×10-3 | 27.14 | 26.68 |
| 0.128×10-3 | 28.56 | 28.95 |
According to the DNA concentration and Ct value in the reaction system, the working curves for detecting male and female genome DNA are respectively obtained, as shown in FIG. 1. The male genome DNA detection working curve (marked as working curve 1) is y ═ 1.396ln (x) +26.531, R20.9985, x is the male genome DNA concentration, and y is the Ct value; the female genome DNA detection working curve (marked as working curve 2) is y ═ 1.432ln (x) +26.425, R20.9994, x is the female genomic DNA concentration and y is the Ct value. The result shows that the primer pair A can be used for qualitatively and quantitatively detecting the human genome DNA between 10 ng/mu l and 0.128 pg/mu l, has very high sensitivity and is a wide applicable DNA template concentration range.
Example 3 detection of the degree of degradation of human DNA
On the basis of the embodiment 1 and the embodiment 2, another pair of primers is designed, and the primer pair is marked as a primer pair B, and the primer pair consists of two single-stranded DNAs shown as sequences 4 and 5 in a sequence table and can be used for amplifying a DNA fragment shown as a sequence 3. The primer pair can be used for detecting the degradation degree of the human genome, and the detection method comprises the following steps:
1. processing of samples to be tested
A mixed Human DNA sample, Promega Human Genomic DNA, Male (Promega, G1471), was diluted to about 4 ng/. mu.l using 10mM Tris-HCl buffer, and the sample was degraded using DNase I (New England Biolabs) at 37 ℃ for 0 min, 1 min, 2 min, 5min, followed by 20 min heating at 80 ℃ to inactivate DNase I, resulting in a DNA solution with different degradation times.
2. Fluorescent quantitative PCR
Respectively taking the DNA solutions degraded in different time in the step 1 as template DNAs, and performing fluorescent quantitative PCR by using the primer pair A and the primer pair B, wherein the reaction system and the reaction conditions are as follows:
reaction system (20 μ l): 2x qPCR Master Mix (containing SYBR Green dye) 10. mu.l, template DNA 2. mu.l, primer solution 8. mu.l. The primer solution is a primer pair A solution or a primer pair B solution, the primer pair A solution is a solution obtained by respectively dissolving two single-stranded DNAs shown as sequences 1 and 2 in a sequence table by using 10mM Tris-HCl buffer solution and then mixing the two single-stranded DNAs in an equimolar manner, and the concentrations of the two single-stranded DNAs shown as sequences 1 and 2 in the solution are both 1.25 mu M; the primer pair B solution is obtained by respectively dissolving two single-stranded DNAs shown as sequences 4 and 5 in a sequence table by using 10mM Tris-HCl buffer solution and then mixing the two single-stranded DNAs in an equimolar manner, and the concentrations of the two single-stranded DNAs shown as sequences 1 and 2 in the solution are both 1.25 mu M.
Reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 5s, annealing extension at 60 ℃ for 15s, and 40 cycles.
3. Data analysis
The Ct values for different degradation times are as follows:
| time of degradation | Ct value of primer pair A amplification | Ct value of primer pair B amplification | Delta Ct(B-A) |
| 0min | 17.561 | 16.219 | -1.342 |
| 1min | 24.411 | 26.252 | 1.841 |
| 2min | 26.893 | 30.509 | 3.616 |
| 5min | 31.748 | 39.742 | 7.994 |
Note: delta Ct (B-a) ═ Ct value amplified by primer pair B-Ct value amplified by primer pair a.
The length of the amplified fragment of the primer pair A is 86bp, and the length of the amplified fragment of the primer pair B is 226bp, so that the Ct quantified by the primer pair B can be increased more quickly for a degraded sample than the Ct quantified by the primer pair A along with the improvement of the degradation degree of the genomic DNA. The degradation degree of the human gDNA in the sample can be characterized by comparing the Ct values of the primer pair A and the primer pair B when the same sample is amplified, and the greater the Delta Ct (B-A), the greater the degradation degree of the human gDNA.
Example 4, the copy number of the DNA fragment shown in SEQ ID No. 8 was stable between different men
In the invention, the DNA samples from 274 male individuals find that the copy number of the DNA fragment (marked as fragment B) shown as a sequence 8 in a sequence table is stable among different male human individuals, and the method for detecting the copy number of the DNA fragment by using Alu as a control comprises the following steps:
1. sample to be tested
274 parts of genomic DNA of a blood card sample from a male individual and 198 parts of genomic DNA of a blood card sample from a female individual are extracted, and then the genomic DNAs are diluted by 5 times respectively to obtain DNA solutions of samples to be detected.
2. Fluorescent quantitative PCR
2.1 primer: the primer pair for target fragment amplification consists of F3 and R3 (for amplifying the DNA fragment shown in sequence 8), and the primer pair for detecting Alu consists of F2 and R2, which are as follows:
dissolving each primer in 10mM Tris-HCl buffer solution, and mixing pair primers in an equimolar way to obtain primer solutions for respectively amplifying fragments B and Alu, wherein the final concentration of the forward primer and the final concentration of the reverse primer in each solution are both 1.25 mu M.
2.2 reaction solution preparation and qPCR reaction: respectively carrying out fluorescence quantitative PCR on each sample DNA solution to be detected, wherein the reaction system composition is shown in the following table:
| reagent | Volume of |
| 2x qPCR Master Mix (containing SYBR Green dye) | 10μl |
| DNA solution of sample to be tested | 2μl |
| Primer solution | 8μl |
The PCR system amplification reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; 40 cycles: denaturation at 95 ℃ for 5s and annealing extension at 60 ℃ for 15 s.
3. Data analysis
After the fluorescent quantitative PCR in the step 2 is finished, the Ct values of the two primer fluorescent quantitative PCR reactions of the sample are shown in Table 4, and the difference value of the Ct values in the Table 4 is the difference value of the Ct value amplified by the primer pair Alu and the Ct value amplified by the primer pair C.
TABLE 4 Ct values of the sample fluorescent quantitative PCR
The results showed that the difference in Ct values obtained was small in the above DNA samples from 274 male individuals and could be used as a genetic marker for the detection of male DNA.
Example 5 sensitivity of detection of Male human genomic DNA Using the DNA fragment shown in SEQ ID No. 8
On the basis of example 4, the male human genomic DNA was detected using the primer pair C of example 4 by the following method:
1. sample to be tested
Mixing male genome DNA samples: promega Human Genomic DNA: Male (Promega, G1471). The DNA sample was diluted with 10mM Tris-HCl buffer solution in a 5-fold concentration gradient to obtain male DNA dilution solutions having DNA concentrations of 100 ng/. mu.l, 20 ng/. mu.l, 4 ng/. mu.l, 0.8 ng/. mu.l, 0.16 ng/. mu.l, 32 pg/. mu.l, 6.4 pg/. mu.l and 1.28 pg/. mu.l, respectively.
2. Fluorescent quantitative PCR
Respectively taking each DNA diluted solution in the step 1 as a template DNA, and carrying out fluorescence quantitative PCR by using a primer pair C, wherein a reaction system and reaction conditions are as follows:
reaction system (20 μ l): 2x qPCR Master Mix (containing SYBR Green dye) 10. mu.l, DNA dilution 2. mu.l, primer solution 8. mu.l. Wherein, the primer solution is obtained by respectively dissolving two single-stranded DNAs shown as sequences 6 and 7 in a sequence table by using 10mM Tris-HCl buffer solution and then mixing the two single-stranded DNAs in an equimolar manner, and the concentrations of the two single-stranded DNAs shown as sequences 6 and 7 in the solution are both 1.25 mu M.
Reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 5s, annealing extension at 60 ℃ for 15s, and 40 cycles.
3. Data analysis
After the fluorescent quantitative PCR in step 2 is finished, the Ct value corresponding to each concentration of DNA sample is analyzed, and is shown in Table 2.
TABLE 5 Ct value of DNA of each sample
| Concentration of template DNA in reaction System (ng/. mu.l) | Ct value of |
| 10 | 16.41 |
| 2 | 18.27 |
| 0.4 | 20.51 |
| 0.08 | 22.94 |
| 0.016 | 25.36 |
| 0.0032 | 27.69 |
| 0.64×10-3 | 29.85 |
| 0.128×10-3 | 31.84 |
A male genome DNA detection working curve is obtained according to the DNA concentration and the Ct value in the reaction system, as shown in FIG. 2. The male genome DNA detection working curve (marked as working curve 3) is y ═ 1.45ln (x) +29.416, R2X is the male genomic DNA concentration and y is the Ct value, 0.999. The result shows that the primer pair C can be used for qualitatively and quantitatively detecting the male human genome DNA between 10 ng/mu l and 0.128 pg/mu l, has high sensitivity and is a wide applicable DNA template concentration range.
Example 6 detection of specificity of Male human genomic DNA Using the DNA fragment represented by SEQ ID No. 10
1. Sample to be tested
Mixing male genome DNA samples: promega Human Genomic DNA: Male (Promega, G1471). Diluting the DNA sample by using 10mM Tris-HCl buffer solution to obtain a male DNA sample with the DNA concentration of 25 ng/mu l; diluting the male DNA sample by 1000 times by using 10mM Tris-HCl buffer solution to obtain a male DNA sample with the DNA concentration of 25 pg/mu l;
mixing female genome-wide DNA samples: promega Human Genomic DNA: Female (Promega, G1521). Diluting the DNA sample by using 10mM Tris-HCl buffer solution to obtain a female DNA sample with the DNA concentration of 25 ng/mu l; diluting the female DNA sample with 10mM Tris-HCl buffer solution to obtain female DNA samples with DNA concentrations of 12.5 ng/. mu.l, 3.125 ng/. mu.l, 0.625 ng/. mu.l, 0.125 ng/. mu.l and 25 pg/. mu.l respectively;
a male DNA sample with a DNA concentration of 25 pg/. mu.l was mixed in equal volumes with female DNA samples with DNA concentrations of 25 ng/. mu.l, 12.5 ng/. mu.l, 3.125 ng/. mu.l, 0.625 ng/. mu.l, 0.125 ng/. mu.l and 25 pg/. mu.l, giving a ratio of male to female DNA concentrations of 1: 1000,1: 500,1: 125,1: 25,1: 5 and 1: 1, wherein the female DNA content in the six mixed test DNA solutions is 12.5 ng/. mu.l, 6.25 ng/. mu.l, 1.563 ng/. mu.l, 0.313 ng/. mu.l, 0.0625 ng/. mu.l and 12.5 pg/. mu.l, and the male DNA content in the six mixed test DNA solutions is 12.5 pg/. mu.l.
2. Fluorescent quantitative PCR
According to the method of step 2 in example 5, the DNA template "DNA diluted solution" was replaced with the above-mentioned mixed test DNA solution and the male DNA sample and female DNA sample having a DNA concentration of 25pg/μ l, respectively, and fluorescence quantitative PCR was performed without changing the other samples.
The total DNA content was determined by replacing the DNA template "DNA diluted solution" with each of the above-mentioned DNA solutions to be tested and the male DNA sample and female DNA sample having a DNA concentration of 25 pg/. mu.l, respectively, and replacing the primer pair C with the primer pair A in example 2, according to the method of step 2 in example 5.
3. Data analysis
Analyzing Ct values corresponding to each sample to be detected by carrying out fluorescence quantitative PCR on the primer pair C, and calculating the measured value of the male DNA concentration of each sample to be detected according to the working curve 3 of the embodiment 5;
analyzing Ct values corresponding to each sample to be detected by carrying out fluorescence quantitative PCR on the primer pair A, and calculating a detected value y of the total DNA concentration of each sample to be detected by taking each Ct value as x according to a formula y of a male genome DNA detection working curve (working curve 3) which is-1.45 ln (x) + 29.416;
and (3) calculating a female DNA concentration measured value and a female DNA-male DNA concentration ratio according to the male DNA concentration and the total DNA concentration measured value in each mixed DNA solution to be detected, wherein the female DNA concentration measured value in the mixed DNA solution to be detected is the total DNA concentration measured value-male DNA concentration measured value.
The results are shown in Table 6 and FIG. 3.
TABLE 6,
In table 3, 1: 1000,1: 500,1: 125,1: 25,1: 5 and 1: 1 represents that the ratio of the male and female DNA concentrations is 1: 1000,1: 500,1: 125,1: 25,1: 5 and 1: 1, male 2.5pg/μ l and female 2.5pg/μ l represent the DNA concentrations in the reaction system after the male DNA sample and the female DNA sample having a DNA concentration of 25pg/μ l were added to the final reaction solution at a volume ratio of 10%, respectively.
The result shows that the primer pair C can be used for successfully detecting the male human genome DNA and accurately quantifying the male human genome DNA; the primer pair A can be used for successfully detecting and accurately quantifying the human genome DNA, and the primer pair C and the primer pair A are used for quantifying the male genome and the human genome in the sample respectively, so that whether the female genome DNA is contained or not can be further determined and quantified. The specificity and sensitivity of the primer pair C are good, and the primer pair C can be used in the conditions that the ratio of female DNA to male DNA is 1000: 1, accurately detecting the male DNA content in the sample system.
Sequence listing
<110> material evidence identification center of public security department
<120> a genetic marker for detecting human genomic DNA
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tccttccagt tgtcttgaac agcctgactc ctgccagccc tatggaagtt ccttttatgc 180
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Claims (10)
1. Any one of the following X1) -X13):
x1) the use of a DNA fragment named DNA fragment A for the detection of human genomic DNA; the DNA fragment A is a DNA fragment shown in a sequence 3 in a sequence table or any fragment thereof;
x2) detecting the substance of the DNA fragment A in the detection of human genome DNA;
x3) application of a substance for detecting the DNA fragment A in preparing a product for detecting human genome DNA;
x4) the application of the DNA fragment A in detecting whether human genome DNA is degraded or not;
x5) detecting the substance of the DNA fragment A in detecting whether the human genome DNA is degraded or not;
x6) application of a substance for detecting the DNA fragment A in preparing a product for detecting whether human genome DNA is degraded or not;
x7) the application of the materials used in the method for detecting human genome DNA by taking the DNA fragment A as a human genetic marker in the detection of human genome DNA;
x8) the application of the materials used in the method for detecting human genome DNA by using the DNA fragment A as a human genetic marker in detecting whether the human genome DNA is degraded or not;
x9) the application of the DNA fragment A as a human genetic marker in detecting human genome DNA;
x10) the application of the DNA fragment A as a human genetic marker in detecting whether human genome DNA is degraded or not;
x11) the use of the DNA set for the detection of human female genomic DNA; the complete set of DNA fragments consists of the DNA fragment A and the DNA fragment named as the DNA fragment B, wherein the DNA fragment B is the DNA fragment shown in a sequence 8 in a sequence table or any fragment thereof;
x12) the application of the substance for detecting the DNA fragment set in the preparation of the product for detecting the genome DNA of the human female;
x13) in detecting human female genome DNA.
2. Use according to claim 1, characterized in that: the DNA fragment A is a DNA fragment shown in 66 th-151 th site of a sequence 3 in a sequence table or any fragment thereof.
3. Use according to claim 1 or 2, characterized in that: the substance for detecting the DNA fragment A comprises a primer pair named as a primer pair A or a primer pair named as a primer pair B, wherein the primer pair A consists of two single-stranded DNAs shown as sequences 1 and 2 in a sequence table, and the primer pair B consists of two single-stranded DNAs shown as sequences 4 and 5 in the sequence table;
and/or the substance for detecting the set of DNA fragments consists of the substance for detecting the DNA fragment A and the substance for detecting the DNA fragment B, and the substance for detecting the DNA fragment B comprises a primer pair named as a primer pair C; the primer pair C consists of two single-stranded DNAs shown as sequences 6 and 7 in a sequence table.
4. A method of detecting a human sample, comprising: detecting whether the genomic DNA of a test sample contains the DNA fragment A by using the substance for detecting the DNA fragment A as defined in any one of claims 1 to 3, and determining whether the test sample contains a human sample or is a human sample: if the genomic DNA of the sample to be detected contains the DNA fragment A, the sample to be detected contains or is a candidate for containing a human sample or is a candidate for a human sample; if the genomic DNA of the test sample does not contain the DNA fragment A, the test sample does not contain or is not a candidate for a human sample.
5. A method for quantitatively detecting human genomic DNA, comprising: detecting human genome DNA standard products with different concentrations by using the primer pair A or the primer pair B in claim 3 by adopting a fluorescent quantitative PCR method to obtain Ct values of the human genome DNA standard products with different concentrations, and determining the relation between the Ct values and the human genome DNA concentrations; detecting a sample to be detected by using the primer pair A or the primer pair B in the claim 3 to obtain a Ct value of the sample to be detected, and determining the content of the human genome DNA in the sample to be detected according to the relation between the Ct value and the human genome DNA concentration and the Ct value of the sample to be detected.
6. A method of testing a female sample comprising: detecting the content of human genomic DNA in a sample to be tested by using the method of claim 5; detecting male human genome DNA standard products with different concentrations by using the primer pair C in claim 3 by adopting a fluorescent quantitative PCR method to obtain Ct values of the male human genome DNA standard products with different concentrations, and determining the relationship between the Ct values and the male human genome DNA concentrations; detecting the sample to be detected by using the primer pair C in claim 3 to obtain a Ct value of the sample to be detected, and determining the content of the male genome DNA in the sample to be detected according to the relation between the Ct value and the concentration of the male human genome DNA and the Ct value of the sample to be detected;
comparing the content of the human genome DNA in the sample to be detected with the content of the male genome DNA, and determining whether the sample to be detected contains a human female sample: if the content of the human genome DNA in the sample to be detected is equal to the content of the male genome DNA, the sample to be detected does not contain or is candidate to contain the human female sample; and if the content of the human genome DNA in the sample to be detected is larger than that of the male genome DNA, the sample to be detected contains or is candidate to contain the human female sample.
7. A method for quantitatively detecting human female genomic DNA, comprising: detecting the content of human genomic DNA in a sample to be tested by using the method of claim 5; detecting male human genome DNA standard products with different concentrations by using the primer pair C in claim 3 by adopting a fluorescent quantitative PCR method to obtain Ct values of the male human genome DNA standard products with different concentrations, and determining the relationship between the Ct values and the male human genome DNA concentrations; detecting the sample to be detected by using the primer pair C in claim 3 to obtain a Ct value of the sample to be detected, and determining the content of the male genome DNA in the sample to be detected according to the relation between the Ct value and the concentration of the male human genome DNA and the Ct value of the sample to be detected;
comparing the content of the human genome DNA in the sample to be detected with the content of the male genome DNA, and determining the content of the female genome DNA in the sample to be detected according to the following formula: and the content of female genome DNA in the sample to be detected is equal to the content of human genome DNA in the sample to be detected, namely the content of male genome DNA in the sample to be detected.
8. The substance for detecting A as described in any one of claims 1 to 3.
9. The kit of parts for detecting DNA fragments according to any one of claims 1 to 3.
10. A set of DNA fragments according to claim 1 or 2.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001079482A1 (en) * | 2000-04-13 | 2001-10-25 | Hidetoshi Inoko | Gene mapping method |
| CN103103624A (en) * | 2011-11-15 | 2013-05-15 | 深圳华大基因科技有限公司 | Method for establishing high-throughput sequencing library and application thereof |
| US20170002414A1 (en) * | 2014-01-30 | 2017-01-05 | Pécsi Tudományegyetem | Preimplantation assessment of embryos through detection of free embryonic dna |
| CN106868140A (en) * | 2017-03-02 | 2017-06-20 | 北京酷搏科技有限公司 | The method of multiple fluorescence quantitative PCR |
| CN107267643A (en) * | 2017-07-28 | 2017-10-20 | 公安部物证鉴定中心 | Detect reagent set and the application of human sample |
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2019
- 2019-09-19 CN CN201910886259.3A patent/CN112522418A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001079482A1 (en) * | 2000-04-13 | 2001-10-25 | Hidetoshi Inoko | Gene mapping method |
| CN103103624A (en) * | 2011-11-15 | 2013-05-15 | 深圳华大基因科技有限公司 | Method for establishing high-throughput sequencing library and application thereof |
| US20170002414A1 (en) * | 2014-01-30 | 2017-01-05 | Pécsi Tudományegyetem | Preimplantation assessment of embryos through detection of free embryonic dna |
| CN106868140A (en) * | 2017-03-02 | 2017-06-20 | 北京酷搏科技有限公司 | The method of multiple fluorescence quantitative PCR |
| CN107267643A (en) * | 2017-07-28 | 2017-10-20 | 公安部物证鉴定中心 | Detect reagent set and the application of human sample |
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