CN104357561B - A kind of method and system obtaining the Chinese population female individual age - Google Patents
A kind of method and system obtaining the Chinese population female individual age Download PDFInfo
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Abstract
The present invention provides a kind of method and system obtaining the Chinese population female individual age, and described method includes the DNA extracting described female individual；Obtain the methyl rate in 11 CpG sites of described DNA；By the methyl rate in described each CpG site, use SPSS17 software, 11 CpG sites and age are carried out regression analysis, build regression model, to obtain the described Chinese population female individual age.The scheme that the present invention provides can obtain the age of Chinese population female individual exactly, biological material blood or blood stain sample that in public security under battle conditions, scene of a crime is left over can be utilized, carry out the deduction of Individual Age, it is beneficial to quick lock in suspect's scope, give a clue for cracking of cases, improve cracking of cases speed.
Description
Technical field
The present invention relates to a kind of method and system obtaining the Chinese population age, particularly relate to a kind of acquisition Chinese population female
The method and system of property Individual Age.
Background technology
Individual Age infers always one of medicolegal important subject, and the assessment of Individual Age presently mainly depends on
Become with the timing that the age increases according to the physics of all kinds of materials and chemical characteristic etc. in the tissue such as skeleton, tooth and tissue
Change.Change due to the physiological of organism each histoorgan growth promoter and influence factor is extremely complex, the different deduction ages
Method all have its using value, but the most all have some limitations.Such as, 1) the six of Ministry of Public Security's material evidence center research application
The stone age is inferred in big joint, and the age is applicable to 12～20 years old, mainly concludes 14,16,18 3 age points；2) Chinese's metacarpal bone is inferred
Age method i.e. CHN, is mainly developed by the State Physical Culture and Sports Commission, and for athlete's Age Estimation, but surveyor need to be through system training
With repeat；3), ischial tuberosity, ilium ridge infer age 16～23 years old；4), tooth infers June at age～12 years old.And in many
Criminal-scene residual sample only have blood or blood stain, make the most then to cannot be carried out the deduction of Individual Age.
How to provide a kind of method may be used for biological material blood or blood stain sample that in public security under battle conditions, scene of a crime is left over
This, it is achieved the deduction of Individual Age that blood or blood stain are originated, it is beneficial to quick lock in suspect's scope, for cracking of cases
Give a clue, improve cracking of cases speed, become the focus of research.
Existing researcher proposes to utilize blood to extract DNA, measures telomeric dna length and judges Individual Age, and telomeric dna is long
Degree is confirmed by numerous experiments with the dependency at age, but the difference that telomeric dna length is in the different cells of same individuality
The biggest, there is tissue specificity；And same tissue-derived cell shortens with the age in the telomeric dna length of all age group
Degree the most unbalanced.Therefore, utilize the span at this index prediction age often more than 5-10 year.Also researcher is had to utilize
MtDNA oxidative damage judges the age of individuality, and progressive mitochondrial DNA oxidative damage also has been found to be had with aging closely
Relation, but mtDNA fragment deletion or other sudden change use detection method and the hybridization technique of PCR-based technology more, deposit equally
In problems such as technology limitation and judgement age range are big.
It is known that DNA methylation is a kind of chemical modification important in epigenetic, refer to that dnmt rna is urged
Change the 5th carbon atom of cytosine ring and form 5-methylcytosine (5mC).Mankind's haploid genome there are about 5 × 107Individual CpG,
It is widely distributed in transposable element, other DNA repeated and the coding region of most of functional gene, the almost all of born of the same parents that methylate
Pyrimidine all occurs at CpG dinucleotide.
Existing numerous studies find the change age-related property disease of the DNA methylation level in a lot of sites, such as metabolism
Generation of property disease, autoimmune disease and tumor etc. is closely related.Along with being widely used of DNA methylation chip, from base
Get more and more because organizing the report of the dependency that horizontal detection methylated with the age.Hannum etc. are by detecting from Caucasia and west
Methylation signature in the blood sample of 656 independent individuals of class's tooth ethnic group, narrows down to prediction age error about 4 years old,
Also by the DNA methylation level of detection different tissues sample, demonstrate age and methylated dependency.
But during growth in humans and aging, the DNA methylation in different normal structures is as well as the age
Increase and difference occurs, and there is obvious histological difference.DNA methylation not only has tissue specificity simultaneously, also has
There is colony's specificity.Fraser Hunterd etc. find the DNA methylation pattern between different crowd in full-length genome level
There is larger difference.At present, most research is all with American-European crowd as object of study, for Chinese population, and especially Chinese
In Qun the research of female individual almost without.
Therefore, one how is provided can be obtained by detection Chinese population female individual genomic DNA methylation level level
The method and system at Chinese population female individual age becomes problem to be solved.
Summary of the invention
The invention provides a kind of method obtaining the Chinese population female individual age, by obtaining described female individual
The methyl rate in 11 CpG sites of DNA, in conjunction with using SPSS17 software, carries out returning to 11 CpG sites and age and divides
Analysis, builds regression model, to obtain the age of described Chinese population female individual, it is achieved that utilize blood or blood stain to individual year
The deduction in age.
Present invention also offers a kind of system obtaining the Chinese population female individual age, utilize the compound expansion in this system
Increasing detection system can quickly obtain the methyl rate in 11 CpG sites of the DNA of described female individual, and then utilizes recurrence mould
Type, it is thus achieved that described female individual age.
Present invention also offers a kind of composite amplification detection system, can quickly obtain 11 of DNA of described female individual
The methyl rate in CpG site, provides data support for obtaining the age of described female individual.
The method obtaining the Chinese population female individual age that the present invention provides, including:
1) DNA of described female individual is extracted；
2) obtaining the methyl rate in described 11 CpG sites of below DNA, described 11 CpG sites are: X21, X25, X27,
X28, X68, X70, X76, X77, X92, X93 and X95；
3) utilize the methyl rate in described 11 CpG sites, use SPSS17 software, to 11 CpG sites and age
Carry out regression analysis, build regression model, to obtain the described Chinese population female individual age.
In the solution of the present invention, described regression model can be:
Y (regression models in 11 CpG sites)=73.518-45.008X21 '-27.493X25 '+59.265X27 '-
16.307X28′-19.231X68′-6.554X70′-13.057X76′-26.705X77′+2.790X92′+60.252X93′+
39.703X95 ', wherein X21 ', X25 ', X27 ', X28 ', X68 ', X70 ', X76 ', X77 ', X92 ', X93 ', X95 ' are respectively institute
State the methyl rate in 11 CpG sites；Or
Y (regression models in 8 CpG sites)=68.40-46.562X21 '-27.991X25 '+59.545X27 '-
15.985X28 '-26.465X68 '-35.773X77 '+65.322X93 '+42.313X95 ', wherein X21 ', X25 ', X27 ',
X28 ', X68 ', X77 ', X93 ' and X95 ' are respectively the first of 8 CpG sites X21, X25, X27, X28, X68, X77, X93 and X95
Base rate.
In the scheme of the application, described SPSS17 software is the software building regression model that this area is conventional, ability
Field technique personnel can as required (such as according to the individual difference of Age Estimation to be carried out) from above-mentioned 11 CpG sites
A part is selected to utilize other model of this software building, as long as its requirement to Age Estimation accuracy can be met.
In the application detailed description of the invention, the regression model in above-mentioned 11 CpG sites is used to carry out the deduction at age, its
R2=0.741, P < 0.0001, use the regression model in 8 CpG sites to carry out the deduction at age, its R2=0.7983, P=
1.158×10-11.In above-mentioned regression model, R2For correlation coefficient or the polynary coefficient of determination, represent degree of fitting, the biggest expression side
Journey degree of fitting is the best, and the accuracy of its also representative model, the such as accuracy rate of the regression model in 11 CpG sites is 74%, 8
The regression model accuracy rate in CpG site is 80%；P is significance, the most meaningful, less than 0.05 for judging the equation built
Just it may be said that the equation built is statistically significant.
According to the detailed description of the invention of the application, use above-mentioned two model can realize Chinese population female individual,
The especially deduction of Han women Individual Age, and can be to 20 to 80 years old, the individuality of even more than 80 years old carries out Age Estimation, pushes away
The mean error at disconnected age was at about 6.4 years old.
Further, described step 2) include using and described 11 CpG sites 11 pairs of amplimers pair one to one
Its step carrying out expanding to obtain amplified production.
Further, the nucleotides sequence of SEQ ID No.1 to SEQ ID No.22 during described amplimer is sequence table
Row.
Further, wherein step 2) be additionally included in acquisition amplified production after, use Manganic pyrophosphate complex initiation method or mass spectrum inspection
Survey method obtains the step of the methyl rate in described 11 CpG sites.In the solution of the present invention, Manganic pyrophosphate complex initiation method (such as profit
Methylate the method for order-checking detection of platform with Sequenom) or mass spectroscopic assays be conventional the methylating water for detection in this area
Flat method, its using method is known in the art, and implementing the method is to realize to those skilled in the art
's.
A kind of system obtaining the Chinese population female individual age that the present invention provides, including DNA extraction system, compound expansion
Increase detection system, data acquisition system；
Described DNA extraction system is for extracting the DNA of described female individual；
Described composite amplification detection system is for expanding 11 CpG sites of described DNA, and utilizes amplified production
Obtaining the methyl rate in CpG site, described 11 CpG sites are: X21, X25, X27, X28, X68, X70, X76, X77, X92,
X93 and X95；
Described data acquisition system, for by the methyl rate in described each CpG site, uses SPSS17 software, to 11
CpG site and age carry out regression analysis, build regression model, to obtain the described Chinese population female individual age.
Further, in the system, described regression model can be:
Y=73.518-45.008X21 '-27.493X25 '+59.265X27 '-16.307X28 '-19.231X68 '-
6.554X70 '-13.057X76 '-26.705X77 '+2.790X92 '+60.252X93 '+39.703X95 ', wherein X21 ',
X25 ', X27 ', X28 ', X68 ', X70 ', X76 ', X77 ', X92 ', X93 ', X95 ' are respectively the methyl in described 11 CpG sites
Rate；Or
Y=68.40-46.562X21 '-27.991X25 '+59.545X27 '-15.985X28 '-26.465X68 '-
35.773X77 '+65.322X93 '+42.313X95 ', wherein X21 ', X25 ', X27 ', X28 ', X68 ', X77 ', X93 ' and X95 '
It is respectively the methyl rate of 8 CpG sites X21, X25, X27, X28, X68, X77, X93 and X95.
In a scheme of the present invention, described composite amplification detection system is for using and described 11 CpG sites one
11 pairs of amplimers of one correspondence expand to obtain amplified production to it, and utilize described amplified production to be surveyed by pyrophosphoric acid
Sequence method or mass spectroscopic assays obtain the methyl rate in described 11 CpG sites.
Further, the nucleotide sequence of SEQ ID No.1 to SEQ ID No.22 during described amplimer is sequence table.
A kind of composite amplification detection system that the present invention provides, including described female individual DNA, 11 CpG sites, and
Amplimer,
Described composite amplification detection system is for utilizing 11 CpG positions of the DNA of the amplimer described female individual of amplification
Point is to obtain amplified production, and utilizes described amplified production to obtain described 11 by Manganic pyrophosphate complex initiation method or mass spectroscopic assays
The methyl rate in CpG site；
Described 11 CpG sites are: X21, X25, X27, X28, X68, X70, X76, X77, X92, X93 and X95；
Described amplimer is formed by with described 11 CpG sites 11 pairs of amplimers one to one, and described amplification is drawn
Thing is the nucleotide sequence of SEQ ID No.1 to SEQ ID No.22 in sequence table.
Present invention also offers a kind of detection kit, it is characterised in that include described composite amplification detection system.
In the solution of the present invention, the present invention utilizes described composite amplification detection system to carry out the methyl in 11 CpG sites
The method of rate, including: 1) using extract described female individual DNA as template；2) use described amplimer to as mould
The described female individual DNA of plate carries out pcr amplification reaction to obtain amplified production；3) described amplified production is utilized to pass through pyrophosphoric acid
Sequencing or mass spectroscopic assays obtain the methyl rate in described 11 CpG sites.
The combination in above-mentioned 11 CpG sites be applicant by consulting a large amount of list of references, by Chinese population, especially
It is that the living environment of Chinese population female individual, ethnic origin etc. are comprehensively analyzed, investigates the phenotype of each department nationality population
Feature difference, including resemblance, physical signs etc., carries out document and network data base investigation for these differences, existing
The combination in the specific CpG site that can carry out Age Estimation obtained on the basis of research.
Further, in the solution of the present invention, described female individual is the female individual from Chinese han population, institute
The DNA sample stating female individual is the DNA sample being derived from blood sample.
The information in described 11 CpG sites of the present invention is as shown in table 1:
Table 1
The preferred amplimer sequence that the present invention provides is as follows.11 couples of amplimer PRIMER PREMIER 5.0
(PREMIER Biosoft, Palo Alto, CA, USA) designs, and described 11 pairs of amplimers and corresponding CpG site thereof are such as
Shown in table 2 below, PCRU represents forward primer, and PCRL represents downstream primer；
The solution of the present invention has the advantage that
1, the inventive method and system can be in concerning foreign affairs, anti-terrorism cases, particularly with extract in above-mentioned case
Women blood or blood stain sample carry out Age Estimation, infer suspect or victim the range of age, for the detection of case
Give a clue, reduce seeking scope, improve efficiency of solving a case.
2, the present processes and system, uses the combination in 11 CpG sites, and Chinese population female individual can be entered by composition
The detection system of the effective composite amplification of row, and it is used in combination Manganic pyrophosphate complex initiation method or Mass Spectrometry detection method, can quickly obtain institute
State the methyl rate in 11 CpG sites of the DNA of female individual, provide data support for obtaining the age of described female individual.
3, by embodiment data it can be seen that the present processes and system are applied to 49 all ages and classes in Chinese population
The Age Estimation of unrelated female individual, inferred results with 49 individual known ages closely, illustrates by the present invention's
Method and system can relatively accurately obtain age of Chinese population female individual, it is achieved that utilize blood or blood stain to individual
The deduction at body age.
4, according to the detailed description of the invention of the application, the application can realize China for the scheme of two regression models
Crowd's female individual, the especially deduction of Han women Individual Age, and can between 20 to 80 years old, even more than 80 years old
Body carries out Age Estimation, and the mean error at the age of deduction, at about 6.4 years old, has good deduction accuracy.
Detailed description of the invention
In Chinese population, the blood sample of the unrelated female individual of 49 all ages and classes comes from China Han healthy women
Individual.
The method and system that embodiment 1 uses the present invention to provide obtains the Chinese population female individual age
The implementation process of the present invention program is described below as a example by a female individual.
1, the DNA of individuality to be detected is extracted as template
DNA blood midi test kit (QIAGEN, Hilden, Germany) is used to extract said one women respectively
The DNA solution of body.
2, the DNA profiling extracted is carried out multiplexed PCR amplification reaction
2.1, primer pond configuration
The configuration in amplimer pond, is that described 11 CpG sites are 11 right one to one in wherein said amplimer group
Amplimer, every pair of amplimer can expand and include its corresponding CpG site on DNA to be detected；In the present embodiment, it is preferred that
The amplimer group in described 11 CpG sites is the nucleotide sequence of SEQ ID No.1 to SEQ ID No.22 in sequence table；This
The various primer sequences that invention provides are synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
2.2, multi-PRC reaction
The present embodiment uses the ABI 9700 type DNA cloning instrument with 384 hole PCR plate to carry out PCR reaction.
(1) configuration PCR reactant liquor (5 μ L reaction system)
PCR reactant liquor in upper table is added in 384 hole PCR plate reacting holes, at the ABI 9700 enterprising performing PCR of amplification instrument
Amplification.
Owing to, in the primer for 11 CpG sites of the application, the primer sequence in the CpG site of serial number 1,2,3,4 is
Identical；Sequence number 5,6CpG site primer sequence the same；Sequence number 7,8CpG site primer sequence the same；Sequence number 9,10,
The primer sequence in 11CpG site is the same.Therefore, above-mentioned PCR reactant liquor can be divided into 4 groups according to the difference of primer sequence, i.e. has
Four single PCR system, every individual system configures according to upper table.
(2) amplification program
2.3, SAP reaction: 2 μ l SAP MIX (0.3 μ l SAP Enzyme, 1.7 μ l will be added in 5 μ l amplified productions
Nuclease-free water), reaction condition is: 37 DEG C of 20min, 85 DEG C of 5min, 4 DEG C of preservations.
Taking 2.5g agarose, 250mlTBE electrophoretic buffer, EB dyestuff configures 1% agarose gel.Every pair of primer is random
Inspect 8 samples by random samples, take 1.5ul PCR/SAP product, Loading Buffer 2ul loading, 200V electrophoresis 20min.
T cleavage Transcription/RNase reactant liquor is configured according to form 3.
Form 3T cleavage Transcription/RNase reactant liquor
| Single reaction volume (μ l) | |
| Water without nuclease | 3.15 |
| 5*T7 polymerase buffer | 0.89 |
| T cleavage Mix | 0.24 |
| DTT,100mM | 0.22 |
| T7RNA&DNA polymerase | 0.44 |
| RNase A | 0.06 |
| Cumulative volume | 5.00 |
The T cleavage Transcription/RNase reactant liquor and the 2 μ lPCR that add 5 μ l in each reacting hole produce
Thing, hatches 3 hours for 37 DEG C, 4 DEG C of preservations.
Adding resin, after rocking 15min purification DNA, 3200g is centrifuged 5min, utilizes MassARRAY Nanodispenser
After RS1000 point sample, MassARRAY Compact System is utilized to carry out Mass Spectrometer Method methylation level.
4, the methyl rate result in 11 CpG sites of described female individual
| CpG site | Methyl rate |
| X21 | 0.02 |
| X25 | 0.13 |
| X27 | 0.02 |
| X28 | 0.61 |
| X68 | 0.78 |
| X70 | 0.93 |
| X76 | 0.82 |
| X77 | 0.68 |
| X92 | 0.22 |
| X93 | 0.18 |
| X95 | 0.027 |
5, the methyl rate in described each CpG site utilize recurrence mould obtain the age of female individual.
Utilize regression model Y (regression models in 11 CpG sites)=73.518-45.008X21 '-27.493X25 '+
59.265X27′-16.307X28′-19.231X68′-6.554X70′-13.057X76′-26.705X77′+2.790X92′+
60.252X93 '+39.703X95 ', it is thus achieved that the age (Y) of this women is 22.8 years old；
Utilize regression model (regression models in 8 CpG sites) Y=68.40-46.562X21 '-27.991X25 '+
59.545X27 '-15.985X28 '-26.465X68 '-35.773X77 '+65.322X93 '+42.313X95 ' obtains this women's
Age (Y) is 23.2 years old；
The age of this women known is 20 years old, and this female age and its actual age utilizing the application scheme to obtain is described
Closely.
Embodiment 2
Utilize the method and system that embodiment 1 is identical, use unrelated to 49 whole all ages and classes of identical step
The age of Chinese population female individual infers.Checking the accuracy of said system, result is as shown in the table:
The age range of error of the female individual obtained by the present embodiment method, at about 6.4 years old, illustrates by side of the present invention
Method and system can be to 20 to 80 years old, and the individuality of even more than 80 years old carries out Age Estimation, and has preferable accuracy, can be case
Part detection provides data support.
Claims (6)
1. the method obtaining the Chinese population female individual age, it is characterised in that including:
1) DNA of described female individual is extracted；
2) obtaining the methyl rate in described 11 CpG sites of below DNA, described 11 CpG sites are: X21, X25, X27, X28,
X68, X70, X76, X77, X92, X93 and X95；
3) utilize the methyl rate in described 11 CpG sites, use SPSS17 software, 11 CpG sites were carried out with the age
Regression analysis, builds regression model, to obtain the described Chinese population female individual age；
Step 2) include that it is expanded to obtain expansion by employing and described 11 CpG sites 11 pairs of amplimers one to one
The step of volume increase thing；
Described amplimer is the nucleotide sequence of SEQ ID No.1 to SEQ ID No.22 in sequence table.
Method the most according to claim 1, described regression model is:
Y=73.518-45.008 X21 '-27.493 X25 '+59.265 X27 '-16.307 X28 '-19.231 X68 '-
6.554 X70 '-13.057 X76 '-26.705 X77 '+2.790 X92 '+60.252 X93 '+39.703 X95 ', wherein
X21 ', X25 ', X27 ', X28 ', X68 ', X70 ', X76 ', X77 ', X92 ', X93 ', X95 ' are respectively described 11 CpG sites
Methyl rate；Or
Y=68.40-46.562 X21 '-27.991 X25 '+59.545 X27 '-15.985 X28 '-26.465X68 '-
35.773 X77 '+65.322 X93 '+42.313 X95 ', wherein X21 ', X25 ', X27 ', X28 ', X68 ', X77 ', X93 ' and
X95 ' is respectively the methyl rate of 8 CpG sites X21, X25, X27, X28, X68, X77, X93 and X95.
Method the most according to claim 1, it is characterised in that step 2) be additionally included in acquisition amplified production after, use Jiao
Phosphoric acid sequencing or mass spectroscopic assays obtain the step of the methyl rate in described 11 CpG sites.
4. the system obtaining the Chinese population female individual age, it is characterised in that described system includes DNA extraction system,
Composite amplification detection system, data acquisition system；
Described DNA extraction system is for extracting the DNA of described female individual；
Described composite amplification detection system is for expanding 11 CpG sites of described DNA, and utilizes amplified production to obtain
The methyl rate in described CpG site, described 11 CpG sites are: X21, X25, X27, X28, X68, X70, X76, X77, X92,
X93 and X95；
Described data acquisition system, for by the methyl rate in described each CpG site, uses SPSS17 software, to 11 CpG positions
Point and age carry out regression analysis, build regression model, to obtain the described Chinese population female individual age；
Described composite amplification detection system is for using to enter it with described 11 CpG sites 11 pairs of amplimers one to one
Row amplification is to obtain amplified production, and utilizes described amplified production to obtain described 11 by Manganic pyrophosphate complex initiation method or mass spectroscopic assays
The methyl rate in individual CpG site；
Described amplimer is the nucleotide sequence of SEQ ID No.1 to SEQ ID No.22 in sequence table.
System the most according to claim 4, described regression model is:
Y=73.518-45.008 X21 '-27.493 X25 '+59.265 X27 '-16.307 X28 '-19.231 X68 '-
6.554 X70 '-13.057 X76 '-26.705 X77 '+2.790 X92 '+60.252 X93 '+39.703 X95 ', wherein
X21 ', X25 ', X27 ', X28 ', X68 ', X70 ', X76 ', X77 ', X92 ', X93 ', X95 ' are respectively described 11 CpG sites
Methyl rate；Or
Y=68.40-46.562 X21 '-27.991 X25 '+59.545 X27 '-15.985 X28 '-26.465 X68 '-
35.773 X77 '+65.322 X93 '+42.313 X95 ', wherein X21 ', X25 ', X27 ', X28 ', X68 ', X77 ', X93 ' and
X95 ' is respectively the methyl rate of 8 CpG sites X21, X25, X27, X28, X68, X77, X93 and X95.
6. a composite amplification detection system, it is characterised in that described system includes female individual DNA, 11 CpG sites, with
And amplimer,
Described composite amplification detection system for utilize amplimer expand described female individual DNA 11 CpG sites with
Obtain amplified production, and utilize described amplified production to obtain described 11 CpG positions by Manganic pyrophosphate complex initiation method or mass spectroscopic assays
The methyl rate of point；
Described 11 CpG sites are: X21, X25, X27, X28, X68, X70, X76, X77, X92, X93 and X95；
Described amplimer is formed by with described 11 CpG sites 11 pairs of amplimers one to one, and described amplimer is
The nucleotide sequence of SEQ ID No.1 to SEQ ID No.22 in sequence table.
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| CN110392740A (en) * | 2017-01-25 | 2019-10-29 | 深圳华大生命科学研究院 | The method and its application for determining crowd's sample Biological indicators collection, predicting biological age |
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| WO2018229032A1 (en) * | 2017-06-12 | 2018-12-20 | Beiersdorf Aktiengesellschaft | Age determination of human individual |
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| CN104357561A (en) | 2015-02-18 |
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