CN1506470A - Human STR genotype testing kit - Google Patents
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Abstract
The present invention relates to biological technology product, and is one kind of human STR genotype testing kit. The testing kit consists of mainly DNA extracting liquid, HS-Taq, sample buffering solution, reaction mixture, primer mixture, Ladder, reference template, etc. The present invention is especially suitable for the test of Chinese people and has test result of PM less than 10E(-18) and RCP greater than 99.9999%. The present invention may be used widely in medical jurisprudence, archaeology, genetics, oncology and other fields.
Description
Technical field
The present invention relates to a kind of biological technology products, particularly relate to a kind of human str locus type detection kit.
Background technology
The human genome DNA has 3 * 10
9Bp, wherein 10% is tandem repetitive sequence, is called satellite DNA.Length by repeating unit can be divided into large satellite, middle satellite, moonlet and little satellite again.Wherein little satellite only is by what 2-7mer formed in repeating unit, claims that again it is STR (Short Tandem Repeat.STR).The number of different human body genome satellite DNA repeating unit is variable, therefore, has formed extremely complicated allelotrope fragment length polymorphism.According to this rule, set up a kind of brand-new str locus typing method in recent years, this technology can differentiate Different Individual, and can carry out the genetic analysis between individuality, thus give fields such as medical jurisprudence, archeology, genetics and oncology development band fork-like farm tool used in ancient China new change.Up to the present, have only the PE and Promega two companies of the U.S. successively to release a kind of str locus type detection kit, but such str locus type detection kit emphasis is to design at white race crowd's STR site, and it is poor to suitability that yellow's str locus type detects.
Summary of the invention
The purpose of this invention is to provide a kind of fast, accurately and be more suitable for yellow's human str locus type detection kit.
The present invention is that the technical scheme that addresses the above problem is: this test kit mainly is made up of DNA extraction liquid, HS-Taq, sample-loading buffer, reaction mixture, primer mixture, Ladder, contrast template.
Described DNA extraction liquid, HS-Taq, sample-loading buffer, reaction mixture, primer mixture, Ladder component are:
DNA extraction liquid: 1.20ml * 2
HS-Taq:200 μ l * 1
11 * sample-loading buffer: 900 μ l * 1
5 * reaction mixture: 1.64ml * 1
10 * primer mixture:
A group: D3S1358+TH01+D13S317 210 μ l * 1
B group .TPOX+vWA+D7S820 210 μ l * 1
C group .XY+D8S1179+FGA 210 μ l * 1
D group .D5S818+CSF1PO+D16S539 210 μ l * 1
E group .D6S1043+D2S1772+D7S3048 210 μ l * 1
Ladder：
A group .D3S1358+TH01+D13S317 110 μ l * 1
B group .TPOX+vWA+D7S820 110 μ l * 1
C group .XY+D8S1179+FGA 110 μ l * 1
D group .D5S818+CSF1PO+D1 6S,539 110 μ l * 1
E group .D6S1043+D2S1772+D7S3048 110 μ l * 1
Described test kit also comprises interior mark, and described DNA extraction liquid, HS-Taq, sample-loading buffer, reaction mixture, primer mixture, Ladder component are:
DNA extraction liquid: 1.20ml * 2
HS-Taq:50 μ l * 1
11 * sample-loading buffer: 220 μ l * 1
5 * reaction mixture: 410 μ l * 1
10 * primer mixture: 210 μ l * 1
Ladder：
The A group. the fluorescein of mark is TAMRA
D3S1358+TH01+D13S317+D16S539+D6S1043 110 μ l * 1
The B group. the fluorescein of mark is HEX
TP0X+vWA+D7S820+D5S818+D2S1772 110 μ l * 1
The C group. the fluorescein of mark is FAM
XY+D8S1179+CSF1PO+FGA+D7S3048 110 μ l * 1
Interior mark:
Be made up of 100～400bp, differ 20bp between per two fragments, the fluorescein of mark is ROX 110 μ l * 1
The present invention is to the detection of human str locus type, particularly the detection to yellow's human str locus type has following positively effect: (1) selected STR site major part can be general in international criminal's gene pool, increase by three site (D6S1043 that are more suitable for Chinese population simultaneously, D2S1772, D7S3048), thus have more suitability; (2) DNA extraction method, simple, quick, low price, and to human body and environmental nonpollution; (3) result of detection China Han is: PM＜10
-18RCP＞99.9999%.(international standard is: PM＜10 to have surpassed domestic and international specified standards
-11RCP＞99.73%).
Embodiment
Embodiment one
In the present embodiment, the present invention adopts silver-colored dyeing technique to detect human str locus type.
This test kit mainly is made up of DNA extraction liquid, HS-Taq, sample-loading buffer, reaction mixture, primer mixture, Ladder, contrast template, and its component is:
DNA extraction liquid: 1.20ml * 2
HS-Taq:200 μ l * 1
11 * sample-loading buffer: 900 μ l * 1
5 * reaction mixture: 1.64ml * 1
10 * primer mixture:
A group .D3S1358+TH01+D13S317 210 μ l * 1
B group .TPOX+vWA+D7S820 210 μ l * 1
C group .XY+D8S1179+FGA 210 μ l * 1
D group .D5S818+CSF1PO+D16S539 210 μ l * 1
E group .D6S1043+D2S1772+D7S3048 210 μ l * 1
Ladder：
A group .D3S1358+TH01+D13S317 110 μ l * 1
B group .TPOX+vWA+D7S820 110 μ l * 1
C group .XY+D8S1179+FGA 110 μ l * 1
D group .D5S818+CSF1PO+D16S539 110 μ l * 1
E group .D6S1043+D2S1772+D7S3048 110 μ l * 1
The preparation of HS-Taq:
The archaeal dna polymerase that uses in this patent (HS-Taq) is the warm start enzyme, this enzyme is on the basis of preparation general T aq enzyme, be closed with the active group of antienzyme antibody enzyme, make it conventional or do not have the activity of enzyme slightly under the high-temperature, has only pyroprocessing through more than 90 degree, after the antibody protein sex change, the active of enzyme just recovered.
The preparation of 11X sample-loading buffer:
55% bromjophenol blue, 55% dimethylbenzene orchid, 10mM NaOH, 95% formyl waist amine.
The preparation of 5 * reaction mixture:
Tower in the reaction mixture between the each component join with pH value with direct shadow noon to what of the height of amplified production amount and non-specific amplification band.Particularly in composite amplification system, magnesium ion concentration is wanted most.Its adjustment program is as follows:
1, adjust each primer in the compound system between optimum concn.
2, primer mixture and the magnesium ion concentration of adjusting (generally being located between 1～5mM) carried out the chessboard titration.
3, according to titration results, special taking out of under the existing prerequisite nothing but, selecting the highest magnesium ion concentration of amplification output and primer concentration be the concentration of selecting at last.
The tower of concentration is joined between each primer:
In order to ensure the amplified production amount in each site in same amplification system about equally, and special nothing but band is amplified, thus between each primer the tower of concentration be assigned to close important.To join program as follows for the tower of concentration between each primer:
1, with ultrapure water or TE the synthetic primer is dissolved into 100～200PM earlier.
2, after paired primer equal-volume mixes, be diluted to 0.2 respectively; 0.1; 0.05; 0.025PM/100 μ l reaction volume increases in same system.
3, select special nothing but band and special band more clearly primer concentration be primary dcreening operation concentration.
4, in same composite amplification system, carry out composite amplification with the primary dcreening operation concentration of each site primer.
5, according to the product amount that increases between each site, and the non-special existing situation of taking out of, increase and decrease at primer concentration corresponding site.
6, to be transferred to the amplified production amount of all sites in every group equal substantially always, and do not have obviously non-special band, and the side is qualified.
The preparation contrast template:
With gene clone technology with a certain individual in the present invention all allelotrope fragment clonings in 15 sites come out, then the segmental recombinant plasmid equivalent of each allelotrope is merged back packing, template in contrast.
The screening of loop parameter:
1, used archaeal dna polymerase is warm start enzyme (HS-Taq) in this amplification system, therefore must could recover the activity of enzyme through comparatively high temps with after the long period, so selected pre-sex change condition is 96 ℃ in this patent, and 5 minutes.This also is the human huge and complicated thorough sex change conditions needed of karyomit(e).
2,, determine that 60 ℃ are annealing temperature according to the TM value of selected each primer in this patent.
3, according to mentioned above principle and conventional procedure, the loop parameter of setting this patent is: 96 ℃ of pre-sex change 5 minutes, and 95 ℃ of sex change are 44 seconds then; Annealed 40 seconds for 60 ℃; 72 ℃ were extended 1 minute, and circulated 5 times.And then 92 ℃ of sex change are 30 seconds; Annealed 40 seconds for 60 ℃; 72 ℃ were extended recirculation 25 times 1 minute.Last 72 ℃ of insulations 10 minutes.
The preparation of Ladder:
1, extracts DNA.
2, the sample of collecting no any genetic connection from Han nationality is more than 200 parts, and the primer with unit point increases respectively, therefrom filters out the amplification segment of desired length in all gene fragments in each site and the interior mark.
3, by the program of chapter 8 in " round pcr experiment guide ", the PCR product is cloned.
4, extract plasmid DNA by the program on the 19th page of " molecular cloning " Chinese edition second edition, and carry out purity and quantitative assay.
5, prepare each allelotrope fragment by the gene amplification technology, carry out purifying and quantitative assay to various then.
6, each allelotrope fragment is exceptionally carried out nucleic acid sequence analysis, to measure the multiplicity of each segmental size and series connection unit.On this basis, each allelotrope fragment is named by international rule.
7, after the equipotential substrate section balanced mix with each site, add stablizer, and carry out packing by appropriate amount.
Below the detection step of this enforcement:
One, the extraction of DNA
Different samples have different extracting method, and its extracting method is as follows:
1, from whole blood, extract DNA:
Get 20 μ l anticoagulations in the 0.5ml sterile tube, add the ultrapure water about 500 μ l, behind the mixing, centrifugal 2 minutes of 12000rpm abandons supernatant, (if still have red corpuscle in the precipitation, need to repeat this operation 1-2 time) adding DNA extraction liquid 20 μ l, mixing, 100 ℃ were boiled 5 minutes (can carry out on the instrument in amplification) back 12000rpm centrifugal 5 minutes, and got 2 μ l supernatants and increase in 20 μ l reaction volumes.
2, from other karyocyte, extract DNA:
Can directly add an amount of sample extracting solution in the pipe that karyocytes such as root of hair, dandruff, marrow, seminal stain, blood stain and fragment of tissue are housed, 100 ℃ boil 5 minutes (can carry out on the instrument) in amplification after, centrifugal 5 minutes of 12000rpm gets supernatant and increases.
3, extract DNA from attached material:
After the sample such as cloth, paper scrap, silt that will have human karyocyte with scissors or other sharp device is divided into the millet size, put into the 0.5ml plastic centrifuge tube, the DNA extraction liquid that provides in this reagent is provided again all floods it, put 100 ℃ again and boiled 5 minutes, get attached material that small pieces (or granule) boiled then and directly put into the PCR pipe and increase
Two, prepare amplification mixture by the following method, and increase:
Get a centrifuge tube, add (reaction tubes number+1) * [(HS-Taq 0.4 μ l (must spare with the finger bullet)+5 * reaction mixture, 4 μ l+10 * primer mixtures, 2 μ l+ ultrapure waters, 11.6 μ l)] with preceding, behind the mixing, each reaction tubes packing 18 μ l, each reaction tubes adds the DNA 2 μ l of extraction respectively and a dropstone wax oil (if template DNA concentration is too low, can suitably increases its volume, but will reduce the ultrapure water volume simultaneously then, to keep total reaction volume is 20 μ l), increase after centrifugal slightly.
Three, add 2 μ l sample-loading buffers in amplified production, prepare electrophoresis behind the mixing.
Four, with 95% ethanol-0.5% acetic acid solution with 100 times of glue paste dilutions, with absorbent cotton it is coated on the clean sheet glass then, the room temperature leeward in after just can be used for glue.
Five, two edge strips are placed on two both sides between the glass respectively, glass and edge strip all align at lower edge, each is fixed on edge strip between the sheet glass with two long-tail folders, the impetus of clip is just in time on edge strip. in case glass deformation, then will long glass down, and level is placed on the support preparation encapsulating.
Six, add 3 TEMED and 6 10%AP in the small beaker that 15ml 6%PAG is housed, the limit edged shakes (can not exert oneself very much, in order to avoid produce bubble), pours into behind the mixing in the laminated glass and (uses finger tapping glass while irritating, in case produce bubble), and put comb as early as possible well.Comb inserts in the glue and gets final product about 0.5cm.
Seven, treat that gelling is solid after one hour, unclamp four long-tail folders earlier, and respectively put a plastic foam to fill short sheet glass that part shorter than long sheet glass on short glass upper limb both sides, press from both sides with four long-tails then they are fixed on the electrophoresis chamber in (short glass inwardly, long glass is outwardly).Used the 300V prerunning then 20 minutes.
Eight, upward slowly take off comb before the sample, and wash the electrophoresis road with electrophoresis liquid.Get 2 μ l, 11 * sample-loading buffer in 20 μ l amplified productions, fully behind the mixing, sample 3 μ l on the per pass, every kind of Ladder are 3 μ l on each also.
Nine, the 600V electrophoresis when promptly blue indicator is gone to apart from the lower edge 3cm left and right sides of glue, can dye by silver about 1 hour behind the last sample.
Ten, the sheet glass that takes off band glue shook 10 minutes in stationary liquid, outwelled stationary liquid, with ddH2O rinsing twice, added staining fluid again and (with preceding 2 gram AgNO3 was dissolved in 1000ml ddH
2Among the O, put room temperature and keep in Dark Place standby) shake 15 minutes after, in 10 seconds, use ddH
2The NaOH rinsing one time of 0.75M is used in O rinsing one time again, add immediately then colour developing liquid (in the NaOH of 100ml0.75M, adding formaldehyde 800 μ l, now with the current) shake to nucleic acid belt clear till (about about 15 minutes).Outwell colour developing liquid, with drying after the abundant rinsing of tap water, and observations.
11, from the gene frequency cartogram, (see China Han gene frequency cartogram) and search corresponding gene frequency, and calculate corresponding RCP value by the method that the method for embodiment three is calculated corresponding PM value and embodiment four.
Embodiment two
In the present embodiment, the present invention adopts fluorescent method to detect human str locus type.
This test kit mainly is made up of DNA extraction liquid, HS-Taq, sample-loading buffer, reaction mixture, primer mixture, Ladder, interior mark, contrast template, and its component is:
DNA extraction liquid: 1.20ml * 2
HS-Taq:50 μ l * 1
11 * sample-loading buffer: 220 μ l * 1
5 * reaction mixture: 410 μ l * 1
10 * primer mixture: 210 μ l * 1
Ladder：
The A group. the fluorescein of mark is TAMRA
D3S1358+TH01+D13S317+D16S539+D6S1043 110 μ l * 1
The B group. the fluorescein of mark is HEX
TP0X+vWA+D7S820+D5S818+D2S1772 110 μ l * 1
The C group. the fluorescein of mark is FAM
XY+D8S1179+CSF1PO+FGA+D7S3048 110 μ l * 1
Interior mark:
Be made up of 100～400bp, differ 20bp between per two fragments, the fluorescein of mark is ROX 110 μ l * 1
The tower of concentration is joined between the preparation of the preparation of HS-Taq, 11X sample-loading buffer, the preparation of 5 * reaction mixture, each primer, the preparation of Ladder is identical with embodiment one, and interior target preparation is identical with the preparation of Ladder.
Below the detection step of this enforcement:
One, the extraction of DNA:
Different samples have different extracting method, and its extracting method is as follows:
1, from whole blood, extracts DNA: get 20 μ l anticoagulations in the 0.5ml sterile tube, add the ultrapure water about 500 μ l, behind the mixing, centrifugal 2 minutes of 12000rpm abandons supernatant, (if still have red corpuscle in the precipitation, need to repeat this operation 1-2 time) adding DNA extraction liquid 20 μ l, mixing, 100 ℃ were boiled 5 minutes (can carry out on the instrument in amplification) back 12000rpm centrifugal 5 minutes, and got 2 μ l supernatants and increase in 20 μ l reaction volumes.
2, from other karyocyte, extract DNA: can directly add an amount of sample extracting solution in the pipe that karyocytes such as root of hair, dandruff, marrow, seminal stain, blood stain and fragment of tissue are housed, 100 ℃ boil 5 minutes (can carry out on the instrument) in amplification after, centrifugal 5 minutes of 12000rpm gets supernatant and increases.
3, extract DNA from attached material: after the sample such as cloth, paper scrap, silt that will have human karyocyte with scissors or other sharp device is divided into the millet size, put into the 0.5ml plastic centrifuge tube, the DNA extraction liquid that provides in this reagent is provided again all floods it, put 100 ℃ again and boiled 5 minutes, get attached material that small pieces (or granule) boiled then and directly put into the PCR pipe and increase
Two, following method preparation amplification mixture, and increase:
Get a centrifuge tube, add (reaction tubes number+1) * [(HS-Taq 0.4 μ l (must spare with the finger bullet)+5 * reaction mixture, 4 μ l+10 * primer mixtures, 2 μ l+ ultrapure waters, 11.6 μ l)] with preceding, behind the mixing, each reaction tubes packing 18 μ l, each reaction tubes adds the DNA 2 μ l of extraction and a dropstone wax oil respectively (if template DNA concentration is too low then, can suitably increase its volume, but to reduce the ultrapure water volume simultaneously, to keep total reaction volume is 20 μ l), increase by following condition in centrifugal slightly back: 96 ℃ of pre-sex change 5 minutes, and 95 ℃ of sex change are 45 seconds then; Annealed 40 seconds for 60 ℃; 72 ℃ were extended 1 minute, and circulated 5 times.And then 92 ℃ of sex change are 30 seconds; Annealed 40 seconds for 60 ℃; 72 ℃ were extended recirculation 25 times 1 minute.Last 72 ℃ of insulations 10 minutes.
Three, respectively with four kinds of fluoresceins that use among the present invention (TAMRA, HEX, FAM, ROX) marker is done matrix analysis in the full-automatic fluorescent core fine horse sequential analysis of PE 310,377 or its model, to adjust the parameter of instrument.
Four, carry out electrophoresis after three kinds of fluorescein-labeled Ladder mix by suitable proportion respectively with interior mark, then the fluorescence data with laser scanning behind the electrophoresis is recorded in the computer.
Five, with interior mark respectively with after the amplified production of every part of sample mixes by a certain percentage, carry out electrophoresis under the same conditions, then in computer by interior mark, the electrophoresis result and the Ladder of amplified production compared, to obtain the allelotrope under every part of sample.
Six, from the gene frequency cartogram, (see China Han gene frequency cartogram) and search corresponding gene frequency, and calculate corresponding RCP value by the method that the method for embodiment three is calculated corresponding PM value and embodiment four.
Embodiment three (personnel's individual recognition)
On the electrophoretogram of STR-PCR gene type, homozygote shows as a characteristic strip, and heterozygote shows as two characteristic strips.According to the H-W law, be in the colony of genetic equilibrium, the homozygote genotype frequency equal gene frequency square, the heterozygote genotype frequency equals 2 times of two gene frequency products.Discern in the evaluation the individual, when the str locus type of two parts of material evidence materials not simultaneously, can get rid of two parts of material sources in same individuality.If the str locus type of two parts of materials is identical, then calculate the coupling probability as stated above.Unite a plurality of STR of amplification site, the type of two samples is all identical, and coupling probability P M is the product of a plurality of str locus type frequencies.Following table is the calculation result (" n " in the table represents the tandem sequence multiplicity) of a real case.
By the calculation specifications of following table, these the two parts of samples possibility (being coupling probability P M) inequality that is used for comparison is 7.5689 * 10
-18This presentation of results can not have second multiple possibility in whole world total population (6,000,000,000 people), thereby can assert that these two parts of samples are from same individuality.
| The site | Genotype | Gene frequency | Genotype frequency |
| ????D3S1358 | ????n 16/n 16 | ????n 16＝0.2895 | ????P 1＝n 16 2＝0.0838 |
| ????TH01 | ????n 7/n 9 | ????n 7＝0.2996 ????n 9＝0.4612 | ????P 2＝2*n 7*n 9＝0.2763 |
| ????D13S317 | ????n 11/n 12 | ????n 11＝0.2303 ????n 12＝0.1382 | ????P 3＝2*n 11*n 12＝0.0636 |
| ????TPOX | ????n 8/n 8 | ????n 8＝0.5216 | ????P 4＝n 8 2＝0.2720 |
| ????VWA | ????n 14/n 18 | ????n 14＝0.2215 ????n 18＝0.1908 | ????P 5＝2*n 14*n 18＝0.0845 |
| ????D7S820 | ????n 9/n 11 | ????n 9＝0.0759 ????n 11＝0.3840 | ????P 6＝2*n 9*n 11＝0.0211 |
| ????D8S1179 | ????n 10/n 10 | ????n 10＝0.1266 | ????P 7＝n 10 2＝0.0160 |
| ????FGA | ????n 20/n 21 | ????n 20＝0.0760 ????n 21＝0.1005 | ????P 8＝2*n 20*n 21＝0.0152 |
| ????D5S818 | ????n 10/n 12 | ????n 10＝0.2179 ????n 12＝0.2222 | ????P 9＝2*n 10*n 12＝0.0968 |
| ????CSFIPO | ????n 11/n 11 | ????n 11＝0.2690 | ????P 10＝n 11 2＝0.0723 |
| ????D16S539 | ????n 10/n 12 | ????n 10＝0.1690 ????n 12＝0.2405 | ????P 11＝2*n 10*n 12＝0.0812 |
| ????D6S1043 | ????n 15/n 20 | ????n 15-＝0.0117 ????n 20＝0.0374 | ????P 12＝2*n 15*n 20＝0.0008 |
| ????D2S1772 | ????n 20/n 24 | ????n 20＝0.1154 ????n 24＝0.2620 | ????P 13＝2*n 5*n 6＝0.0604 |
| ????D7S3048 | ????n 18/n 19 | ????n 18＝0.1352 ????n 19＝0.0893 | ????P 14＝2*n 18*n 19＝0.0241 |
| Coupling probability (PM)=P 1×P 2×……×P 14＝7.5689×10 -18 | |||
Embodiment four (paternity test)
1, principle: the theoretical foundation of judging one's own relation is the law of segregation of Mendelian inheritance.According to this ` one rule, when gametid [cell formed, paired allelotrope was separated from one another, entered gametid [cell separately respectively.Smart, ovum fertilization forms filial generation, and one of two genome of child are from mother, and one from father; Therefore with right allelotrope just one from mother, one from father.If the result who identifies meets this rule, then do not get rid of one's own relation, if do not meet, then get rid of one's own relation (except the variation situation).
In most of the cases, female, subrelation is known, requires to identify whether one's own relation of hypothesis father and child.This moment at first from female, sub genotypic contrast, can determine in child's gene may from father's gene (own father's gene, OG).Observe hypothesis father's genotype then,, then can get rid of hypothesis father and child's one's own relation if do not have own father's gene.If the hypothesis father also has own father's gene, the result just can not get rid of the one's own relation of supposing the father, supposes that mother is the vWA-16/17 type in certain case, and child is 16/18 type, can determine that from comparison own father's gene is vWA-18.Hypothesis father 1 is the vWA-14/20 type in this case; Suppose that father 2 is the vWA-14/18 type.Suppose that wherein father 1 does not possess own father's gene vWA-18, so can get rid of he and child's one's own relation; By contrast, suppose father 2 because of having vWA-18, not getting rid of with child has one's own relation.
2, the calculating of paternity index: go up that hypothesis father 2 can not get rid of the one's own relation of father and son in the example, but his own father of child not necessarily, because the vWA-18 gene frequency is 0.1908 among the crowd.Though the own father must have own father's gene, the man that the vWA-18 gene is arranged not necessarily is exactly child's own father.According to probability theory, the hypothesis father with own father's gene has the possibility that becomes the child own father with the man at random with own father's gene.Suppose the father provide own father's gene become child own father's possibility and at random man's ratio of providing own father's gene to become child own father's chance be called paternity index (paternity index, PI).Preceding a kind of possibility is assumed to be X; A kind of possibility in back is assumed to be Y.Hypothesis father's 2 genotype in the last example are the vWA-14/18 heterozygote, and the possibility that he provides own father's gene vWA-18 is 1/2, i.e. X=1/2.The man provides the frequency of the chance of own father's gene vWA-18 for this gene, i.e. Y=0.1908 at random.Therefore, this routine PI value is 0.5/0.1908=2.6208.If suppose father 2 child's really own father, no matter then detect how many sites, the one's own relation that all can not get rid of he and child, site in all detections, each site can calculate a PI value, the accumulative total PI value in a plurality of sites equals the product of each site PI value, but precondition is not have the genetic linkage relation between each site of being detected.
Triplet genotype combination reduce three principles: (1) is as hypothesis father during for homozygote, X=1; Suppose the father when the heterozygote, X=1/2, but 2 genes of heterozygote hypothesis father be all may be own father's gene the time, X=1.When (2) only relating to 1 own father's gene, the Y value equals the frequency of own father's gene.(3) if when relating to 2 own father's genes, the Y value is 2 own father's gene frequency sums.
The PI value Simple calculation method of paternity test can be with reference to " paternity test dna typing PI value rapid design method ".
For example certain paternity test case detected result is as follows:
| ??THD1 | ??D13S317 | ?TPOK | ??VWA | ??D7S820 | ??D8S1179 | ??FGA | ??D5S818 | ??CSFIPO | ??D16S539 | ??D6S1043 | ??D2D1772 | ??D7S3048 | |
| ????AF | ??7/9 | ??11/12 | ??8/8 | ??14/18 | ??9/11 | ??10/10 | ??20/21 | ??10/12 | ??11/11 | ??10/12 | ??15/20 | ??20/24 | ??18/19 |
| ????C | ??9/9 | ??11/12 | ??8/8 | ??16/18 | ??9/10 | ??10/14 | ??20/25 | ??10/11 | ??11/12 | ??11/12 | ??15/17 | ??24/27 | ??19/23 |
| ????M | ??9/9 | ??10/12 | ??8/11 | ??16/17 | ??10/11 | ??14/15 | ??22/25 | ??10/11 | ??11/12 | ??11/11 | ??14/17 | ??27/27 | ??17/23 |
| ????PI | ??1.0841 | ??2.1710 | ??1.9171 | ??2.6205 | ??6.5876 | ??7.8988 | ??6.5789 | ??0.9750 | ??1.5790 | ??2.0790 | ??42.7350 | ??1.9083 | ??5.5991 |
Detect through 14 locus, all not getting rid of child and AF has one's own relation, can calculate the PI value of these 14 locus.Each value accumulative total PI value that obtains this case that multiplies each other is 9657145.0995 then.
2, the relative chance of set membership (relative chance of paternity, RCP): the PI value that aforementioned calculation goes out is an absolute value, for making the qualification result can be with the relative chance of probability formal representation set membership, the PI value must convert a kind of relative value RCP to.After calculating the PI value, RCP=[PI/PI+1] * 100%.According to the convention of domestic and international paternity test, when the RCP value greater than 99.73% the time, can think that then hypothesis father and child have one's own relation.If the RCP value does not reach 99.73%, should increase detecting position and count till RCP is greater than 99.73%.Nine locus that this test kit provides all are high polymorphisms, and detecting these 9 str locus seats in the last example all can not paternity exclusion, and the RCP value is 99.9999%, and this value has substantially exceeded 99.73%.Can assert that AF-C is one's own relation.Paternity test dna typing PI value rapid design method
Table 1.AF-C-M triplet PI value
The PI value of table 2. diad AF-G
Principle:
1. triplet AF-C-M identifies example:
(1) when AF is homozygote, X=1; When AF is heterozygote, X=1/2; But when 2 genes of heterozygote AF all may be own father's gene, X=1.
When (2) only relating to own father's gene, Y=own father gene frequency.
When (3) relating to 2 own father's genes, Y=2 own father's gene frequency sum.
2. diad AF-C identifies example: mother is transmitted the indispensable gene chance handle by random individual, that is: Xm=(AF provides the p chance) * q+ (AF provides the q chance) * p
Ym＝2pq
China Han gene frequency cartogram
| The A group | ??????????????????D3S1358 | ????????????????????TH01 | ?????????????????????D13S317 | ||||||
| Sampling total number of persons: 228 | Sampling number: 232 | Sampling number: 228 | |||||||
| Homozygote number: 48 | Homozygote number: 69 | Homozygote number: 39 | |||||||
| Heterozygote number: 180 | Heterozygote number: 163 | Heterozygote number: 189 | |||||||
| Allelotrope | Fragment length | Gene frequency | Allelotrope | Fragment length | Gene frequency | Allelotrope | Fragment length | Gene frequency | |
| ????13 | ????119 | ????0.0000 | ????5 | ????168 | ????0.0022 | ????8 | ????222 | ????0.2741 | |
| ????14 | ????123 | ????0.0351 | ????6 | ????172 | ????0.0970 | ????9 | ????226 | ????0.1316 | |
| ????15 | ????127 | ????0.3531 | ????7 | ????176 | ????0.2996 | ????10 | ????230 | ????0.1842 | |
| ????16 | ????131 | ????0.2895 | ????8 | ????180 | ????0.0517 | ????11 | ????234 | ????0.2303 | |
| ????17 | ????135 | ????0.2478 | ????9 | ????184 | ????0.4612 | ????12 | ????237 | ????0.1382 | |
| ????18 | ????139 | ????0.0658 | ????10 | ????188 | ????0.0884 | ????13 | ????242 | ????0.0329 | |
| ????19 | ????143 | ????0.0088 | ????11 | ????192 | ????0.0000 | ????14 | ????246 | ????0.0088 | |
| ????20 | ????147 | ????0.0000 | ????15 | ????250 | ????0.0000 | ||||
| The B group | ?????????????????????TPOX | ???????????????????????VWA | ????????????????????????D7S820 | ||||||
| Sampling number: 232 | Sampling number: 228 | Sampling number: 237 | |||||||
| Homozygote number: 90 | Homozygote number: 43 | Homozygote number: 51 | |||||||
| Heterozygote number: 142 | Heterozygote number: 185 | Heterozygote number: 186 | |||||||
| Allelotrope | Fragment length | Gene frequency | Allelotrope | Fragment length | Gene frequency | Allelotrope | Fragment length | Gene frequency | |
| ????6 | ????112 | ????0.0000 | ????13 | ????155 | ????0.0022 | ????7 | ????199 | ????0.0021 | |
| ????7 | ????116 | ????0.0022 | ????14 | ????159 | ????0.2215 | ????8 | ????203 | ????0.1203 | |
| ????8 | ????120 | ????0.5216 | ????15 | ????163 | ????0.043 | ????9 | ????207 | ????0.0759 | |
| ????9 | ????124 | ????0.1056 | ????16 | ????167 | ????0.1711 | ????10 | ????211 | ????0.1392 | |
| ????10 | ????128 | ????0.0366 | ????17 | ????171 | ????0.2522 | ????11 | ????215 | ????0.384 | |
| ????11 | ????132 | ????0.3168 | ????18 | ????175 | ????0.1908 | ????12 | ????219 | ????0.2025 | |
| ????12 | ????136 | ????0.0172 | ????19 | ????179 | ????0.0943 | ????13 | ????223 | ????0.0696 | |
| ????13 | ????140 | ????0.0000 | ????20 | ????183 | ????0.0241 | ????14 | ????227 | ????0.0063 | |
| ??????????Amelogenin | ????????????????????D8S1179 | ?????????????????????FGA | |||||||
| Sampling number: 237 | Sampling number: 204 | ||||||||
| Homozygote number: 48 | Homozygote number: 26 | ||||||||
| Heterozygote number: 189 | Heterozygote number: 178 | ||||||||
| Allelotrope | Fragment length | Allelotrope | Fragment length | Gene frequency | Allelotrope | Fragment length | Gene frequency | ||
| The C group | ????X | ????112 | ????10 | ????133 | ????0.1266 | ????15 | ????187 | ????0.0000 | |
| ????Y | ????116 | ????11 | ????137 | ????0.1329 | ????16 | ????191 | ????0.0490 | ||
| ????12 | ????141 | ????0.1371 | ????17 | ????195 | ????0.0980 | ||||
| ????13 | ????145 | ????0.1857 | ????18 | ????199 | ????0.0319 | ||||
| ????14 | ????149 | ????0.1941 | ????19 | ????203 | ????0.0539 | ||||
| ????15 | ????153 | ????0.154 | ????20 | ????207 | ????0.0760 | ||||
| ????16 | ????157 | ????0.0612 | ????21 | ????211 | ????0.1005 | ||||
| ????17 | ????161 | ????0.0084 | ????21.2 | ????213 | ????0.0049 | ||||
| ????22 | ????215 | ????0.2132 | |||||||
| ????22.2 | ????217 | ????0.0049 | |||||||
| ????23 | ????219 | ????0.1912 | |||||||
| ????23.2 | ????221 | ????0.0074 | |||||||
| ????24 | ????223 | ????0.1593 | |||||||
| ????24.2 | ????225 | ????0.0049 | |||||||
| ????25 | ????227 | ????0.0882 | |||||||
| ????25.2 | ????229 | ????0.0025 | |||||||
| ????26 | ????231 | ????0.0319 | |||||||
| ????27 | ????235 | ????0.0147 | |||||||
| ????28 | ????239 | ????0.0000 | |||||||
| The D group | ??????????????????D5S818 | ?????????????????CSFIPO | ????????????????D16S539 | ||||||
| Sampling number: 234 | Sampling number: 210 | Sampling number: 210 | |||||||
| Homozygote number: 40 | Homozygote number: 55 | Homozygote number: 37 | |||||||
| Heterozygote number: 194 | Heterozygote number: 155 | Heterozygote number: 173 | |||||||
| Allelotrope | Fragment length | Gene frequency | Allelotrope | Fragment length | Gene frequency | Allelotrope | Fragment length | Gene frequency | |
| ????7 | ????112 | ??0.0278 | ????6 | ????167 | ????0.0000 | ????8 | ????221 | ????0.0048 | |
| ????8 | ????116 | ??0.0043 | ????7 | ????171 | ????0.0095 | ????9 | ????225 | ????0.2333 | |
| ????9 | ????120 | ??0.094 | ????8 | ????175 | ????0.0024 | ????10 | ????229 | ????0.1690 | |
| ????10 | ????124 | ??0.2179 | ????9 | ????179 | ????0.0548 | ????11 | ????233 | ????0.1952 | |
| ????11 | ????128 | ??0.2949 | ????10 | ????183 | ????0.1929 | ????12 | ????237 | ????0.2405 | |
| ????12 | ????132 | ??0.2222 | ????11 | ????187 | ????0.2690 | ????13 | ????241 | ????0.1571 | |
| ????13 | ????136 | ??0.1325 | ????12 | ????191 | ????0.3643 | ????14 | ????245 | ????0.0000 | |
| ????14 | ????140 | ??0.0043 | ????13 | ????195 | ????0.0929 | ????15 | ????249 | ????0.0000 | |
| ????15 | ????144 | ??0.0021 | ????14 | ????199 | ????0.0119 | ||||
| ????15 | ????203 | ????0.0024 | |||||||
| The E group | ????????????????????D6S1043 | ????????????????????D2S1172 | ????????????????????D7S3048 | ||||||
| Sampling total number of persons: 214 | Sampling total number of persons: 208 | Sampling total number of persons: 96 | |||||||
| Homozygote number: 27 | Homozygote number: 28 | Homozygote number: 26 | |||||||
| Heterozygote number: 187 | Heterozygote number: 180 | The heterozygote number: | |||||||
| Allelotrope | Fragment length | Gene frequency | Allelotrope | Fragment length | Gene frequency | Allelotrope | Fragment length | Gene frequency | |
| ????21 | ????157 | ??0.0047 | ????30 | ????227 | ????0.0024 | ????24 | ????290 | ????0.0102 | |
| ????20 | ????153 | ??0.0374 | ????29 | ????223 | ????0.024 | ????23 | ????284 | ????0.0281 | |
| ????19 | ????149 | ??0.1425 | ????28 | ????219 | ????0.0865 | ????22 | ????280 | ????0.0638 | |
| ????18 | ????145 | ??0.1776 | ????27 | ????215 | ????0.1202 | ????21 | ????276 | ????0.1454 | |
| ????19 | ????141 | ??0.0327 | ????26 | ????211 | ????0.0337 | ????20 | ????272 | ????0.1633 | |
| ????16 | ????137 | ??0.007 | ????25 | ????207 | ????0.0529 | ????19 | ????268 | ????0.0893 | |
| ????15 | ????133 | ??0.0117 | ????24 | ????203 | ????0.262 | ????18 | ????264 | ????0.1352 | |
| ????14 | ????129 | ??0.1729 | ????23 | ????199 | ????0.0409 | ????17 | ????260 | ????0.1684 | |
| ????13 | ????125 | ??0.1121 | ????22 | ????195 | ????0.0601 | ????16 | ????256 | ????0.1046 | |
| ????12 | ????121 | ??0.1636 | ????21 | ????191 | ????0.1418 | ????15 | ????252 | ????0.0842 | |
| ????11 | ????117 | ??0.1028 | ????20 | ????187 | ????0.1154 | ????14 | ????248 | ????0.0077 | |
| ????10 | ????113 | ??0.0327 | ????19 | ????183 | ????0.0433 | ||||
| ????9 | ????109 | ??0.0023 | ????18 | ????179 | ????0.0096 | ||||
| ????17 | ????175 | ????0.0072 | |||||||
Claims (3)
1, a kind of human str locus type detection kit is characterized in that this test kit mainly is made up of DNA extraction liquid, HS-Taq, sample-loading buffer, reaction mixture, primer mixture, Ladder, contrast template.
2, human str locus type detection kit according to claim 1 is characterized in that described DNA extraction liquid, HS-Taq, sample-loading buffer, reaction mixture, primer mixture, Ladder component are:
DNA extraction liquid: 1.20ml * 2
HS-Taq:200 μ l * 1
11 * sample-loading buffer: 900 μ l * 1
5 * reaction mixture: 1.64ml * 1
10 * primer mixture:
The A group.
1 * 1 of D3S1358+TH01+D13S317 210 μ
The B group.
TPOX+vWA+D7S820 210 μ l * 1
The C group.
1 * 1 of XY+D8S1179+FGA 210 μ
The D group.
D5S818+CSF1PO+D16S539 210 μ l * 1
The E group.
D6S1043+D2S1772+D7S3048 210 μ l * 1
Ladder：
The A group.
D3S1358+TH01+D13S317 110 μ l * 1
The B group.
TPOX+vWA+D7S820 110 μ l * 1
The C group.
XY+D8S1179+FGA 110 μ l * 1
The D group.
D5S818+CSF1PO+D16S539 110 μ l * 1
The E group.
D6S1043+D2S1772+D7S3048 110 μ l * 1
3, human str locus type detection kit according to claim 1 is characterized in that it also comprises interior mark, and described DNA extraction liquid, HS-Taq, sample-loading buffer, reaction mixture, primer mixture, Ladder component are:
DNA extraction liquid: 1.20m1 * 2
HS-Taq:50 μ l * 1
11 * sample-loading buffer: 220 μ l * 1
5 * reaction mixture: 410 μ l * 1
10 * primer mixture: 210 μ l * 1
Ladder：
The A group. the fluorescein of mark is TAMRA
D3S1358+TH01+D13S317+D16S539+D6S1043 110 μ l * 1
The B group. the fluorescein of mark is HEX
TP0X+vWA+D7S820+D5S818+D2S1772 110 μ l * 1
The C group. the fluorescein of mark is FAM
XY+D8S1179+CSF1PO+FGA+D7S3048 110 μ l * 1
Interior mark:
Form by 100～400bp, differ 20bp between per two fragments,
The fluorescein of mark is ROX 110 μ l * 1
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA021521670A CN1506470A (en) | 2002-12-09 | 2002-12-09 | Human STR genotype testing kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA021521670A CN1506470A (en) | 2002-12-09 | 2002-12-09 | Human STR genotype testing kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1506470A true CN1506470A (en) | 2004-06-23 |
Family
ID=34234658
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA021521670A Pending CN1506470A (en) | 2002-12-09 | 2002-12-09 | Human STR genotype testing kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1506470A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101691607A (en) * | 2009-08-14 | 2010-04-07 | 河北医科大学 | Method and reagent kit for testing human gene typing by labeling mini-STR (short tandem repeats) with fluorescence |
| CN101144774B (en) * | 2007-08-24 | 2010-05-19 | 张兹钧 | Human STRtyper PCR amplification fluorescence detection reagent kit |
| CN107012225A (en) * | 2017-04-20 | 2017-08-04 | 司法部司法鉴定科学技术研究所 | A kind of detection kit and detection method of the str locus seat based on high-flux sequence |
-
2002
- 2002-12-09 CN CNA021521670A patent/CN1506470A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101144774B (en) * | 2007-08-24 | 2010-05-19 | 张兹钧 | Human STRtyper PCR amplification fluorescence detection reagent kit |
| CN101691607A (en) * | 2009-08-14 | 2010-04-07 | 河北医科大学 | Method and reagent kit for testing human gene typing by labeling mini-STR (short tandem repeats) with fluorescence |
| CN101691607B (en) * | 2009-08-14 | 2013-07-17 | 河北医科大学 | Method and reagent kit for testing human gene typing by labeling mini-STR (short tandem repeats) with fluorescence |
| CN107012225A (en) * | 2017-04-20 | 2017-08-04 | 司法部司法鉴定科学技术研究所 | A kind of detection kit and detection method of the str locus seat based on high-flux sequence |
| CN107012225B (en) * | 2017-04-20 | 2020-10-09 | 司法鉴定科学研究院 | STR locus detection kit and detection method based on high-throughput sequencing |
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