CN102424834A - Rapid multiplex-PCR amplification fluorescence detection kit for 16 gene loci, and application thereof - Google Patents
Rapid multiplex-PCR amplification fluorescence detection kit for 16 gene loci, and application thereof Download PDFInfo
- Publication number
- CN102424834A CN102424834A CN2011102437349A CN201110243734A CN102424834A CN 102424834 A CN102424834 A CN 102424834A CN 2011102437349 A CN2011102437349 A CN 2011102437349A CN 201110243734 A CN201110243734 A CN 201110243734A CN 102424834 A CN102424834 A CN 102424834A
- Authority
- CN
- China
- Prior art keywords
- mark
- locus
- pcr amplification
- dna
- amplification
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention discloses a rapid multiplex-PCR amplification fluorescence detection kit for detection of 16 gene loci of the human genome. The kit comprises a pre-amplification reagent and a post-amplification reagent, wherein the pre-amplification reagent comprises: a reaction mixture comprising a PCR buffer, MgCl2, dNTPs and bovine serum albumin, heat start fast Taq polymerase, a mixture comprising 16 pairs of primers, and ultrapure water, the post-amplification reagent comprises: a allele mixture for genotyping, and an internal standard. According to the present invention, the human genome DNA sample is only subjected to nucleic acid amplification to carry out STR genotyping detection, wherein the time for the nucleic acid amplification is about 40-50 minutes; with the single-tube amplification detection, the genotyping results of 15 STR loci and 1 sex identification locus can be concurrently obtained so as to substantially reduce the time for obtaining the final genotyping result of the sample.
Description
Technical field
The present invention relates to the test kit of a plurality of str locus seats among a kind of human genome DNA of detection, be specifically related to the quick composite PCR amplification fluorescence detection reagent kit of a kind of 15 str locus seats of detection and 1 sex identified gene seat, belong to technical field of biological.
Background technology
STR locus (STR) is the genetic marker of present widespread usage.The beginning of the nineties str locus seat polymorphum discovery; Particularly because little, the easy amplification of fragment of str locus seat; Be suitable for check trace and degraded sample, and the amplification condition of each locus is similar and can composite amplification, thus have sensitivity, accurately, fast, advantage such as contain much information.Especially setting up aspect the DNA DB, STR composite amplification technology has great meliority, so legal medical expert educational circles and company that some are big all drop into a large amount of funds it has been carried out explorative research.The middle and later periods nineties is that the research and development merchant of representative has set up the fluorescent composite amplification system with American AB I company.The most representative at present is ABI (Applied Biosystems, USA) Identifiler of company
TMAnd the PowerPlex-16 fluorescence detection reagent kit of Promega (USA) company.
The forensic dna analysis is exactly to utilize the dna typing technology, confirms the DNA genotype of on-the-spot forensic, and the definite suspect's of the same technology of application DNA genotype, and through both comparisons, thereby asserting crime is true or the eliminating suspicion of crime.Development along with the forensic dna typing method; Can carry out dna typing at present to similar blood stain, seminal stain, salivary stain, hair, bone etc.; Reach the identification level; Become one of method that scientific and technological content is the highest in the criminal technique, practicality is the strongest, assay has obtained particularly generally accepting and believing of judicial organ of juridical authorities, and the dna typing technology becomes one of most important foundation of criminal investigation and court judgment.
Present STR typing method often comprises that DNA extraction, pcr amplification and the somatotype of sample sample detects three main operational steps, and the final somatotype result who obtains a sample often needs more than 8 hours.Shorten sample DNA proving time; In actual inspection case, will help the quick lock in suspect, or get rid of innocent people, significant in the cracking of cases process; Having become a new emphasis of scientific research, is the inevitable direction of forensic DNA profiling typing method development.
Summary of the invention
The purpose of the embodiment of the invention is the defective to above-mentioned prior art, provides a kind of can the shortening to obtain the final somatotype of the sample quick composite PCR amplification fluorescence detection reagent kit of 16 locus of time as a result.
In order to realize above-mentioned order, the technical scheme of taking:
The quick composite PCR amplification fluorescence detection reagent kit of a kind of 16 locus is characterized in that this test kit comprises: PCR damping fluid, MgCl
2, dNTPs and bovine serum albumin mixture, the quick Taq enzyme of warm start, the 16 pairs of primer mixtures and ultrapure water;
Wherein 16 pairs of primer mixtures are:
, whenever be centering to rare one 5 ' end in above-mentioned 16 pairs of primers by fluorescent mark.
Described 16 locus are divided into 4 groups, and its corresponding primer is to carrying out mark with the optical dye of 4 kinds of distinct colors respectively, and described four kinds of optical dyes are blue fluorescent dyes 6-FAM, green fluorescence dyestuff HEX; Yellow fluorochrome TAMRA, red fluorescence dyestuff mark ROX; Interior mark is selected fluorescent orange SIZ for use.
Described 16 locus are divided into 4 groups; And its corresponding primer carries out mark with the optical dye of distinct colors in 4 respectively; Packet mode is: Amelogenin, D3S1358, D13S317, D7S820, D16S539 and D6S1043 are one group; Adopt the blue fluorescent dyes mark, fluorescent marker is 6-FAM; TPOX, TH01, D2S1338 and CSF1PO are one group, adopt the green fluorescence dye marker, and fluorescent marker is HEX; VWA, D5S818 and FGA are one group, adopt the Yellow fluorochrome mark, and fluorescent marker is TAMRA; D8S1179, D21S11 and D18S51 are one group, adopt the red fluorescence dyestuff mark, and fluorescent marker is ROX; It is SIZ that interior mark is selected fluorescent orange mark, fluorescent marker for use.
Use described test kit to be 8.0-9.0 as the pH value of PCR damping fluid; Magnesium ion concentration is 1.5-3.5mM; The final concentration of 4 kinds of dNTP respectively is 200-300 μ M; The final concentration of bovine serum albumin in said pcr amplification system is 0.1-1mg/ml, and the consumption of the quick Taq enzyme of warm start is 0.1-0.4U/ μ L, and the list in the 16 pairs of primer mixtures to the primer final concentration does
A kind of application of 16 quick composite PCR amplification fluorescence detection reagent kits of locus is characterized in that it is used for dna typing.
Specifically:
Described 16 locus are: Amelogenin, D2S1338, D3S1358, D5S818, D6S1043, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, TPOX, TH01, CSF1PO, vWA, FGA.The invention provides the locus unitized design scheme of fluorescence labeling composite amplification system.The present invention has selected blue, green, yellow, red, orange 5 look fluorescence assembled schemes respectively for use.This locus array mode makes and only to need four kinds of fluorescence of mark just can realize that these 16 locus increase in same reaction, simultaneously full gene seat amplified production is controlled at 400bp with interior and realization highly sensitive.
Confirm on the basis of 5 look fluorescence assembled schemes; Through experiment repeatedly in a large number; Design the locus array mode voluntarily, below be three kinds of product mix modes wherein, first kind: one group of Amelogenin, D3S1358, D13S317, D7S820 and D16S539; Adopt the blue fluorescent dyes mark, fluorescent marker is 6-FAM; TPOX, TH01, D2S1338, CSF1PO are one group, adopt the green fluorescence dye marker, and fluorescent marker is HEX; VWA, D5S818, FGA, D6S1043 are one group, adopt the Yellow fluorochrome mark, and fluorescent marker is TAMRA; D8S1179, D21S11 and D18S51 are one group, adopt the red fluorescence dyestuff mark, and fluorescent marker is ROX; It is SIZ that interior mark is selected fluorescent orange mark, fluorescent marker for use.Second kind: Amelogenin, D3S1358, D13S317, D7S820, D16S539 and D6S1043 are one group, adopt the blue fluorescent dyes mark, and fluorescent marker is 6-FAM; TPOX, TH01, D2S1338 and CSF1PO are one group, adopt the green fluorescence dye marker, and fluorescent marker is HEX; VWA, D5S818 and FGA are one group, adopt the Yellow fluorochrome mark, and fluorescent marker is TAMRA; D8S1179, D21S11 and D18S51 are one group, adopt the red fluorescence dyestuff mark, and fluorescent marker is ROX.The third: one group of Amelogenin, D3S1358, D13S317, D7S820, D16S539 and CSF1PO, adopt the blue fluorescent dyes mark, fluorescent marker is 6-FAM; TPOX, TH01, D2S1338 and D6S1043 are one group, adopt the green fluorescence dye marker, and fluorescent marker is HEX; VWA, D5S818 and FGA are one group, adopt the Yellow fluorochrome mark, and fluorescent marker is TAMRA; D8S1179, D21S11 and D18S51 are one group, adopt the red fluorescence dyestuff mark, and fluorescent marker is ROX; It is SIZ that interior mark is selected fluorescent orange mark, fluorescent marker for use.
Further use described test kit to carry out the condition of PCR composite amplification reaction: the pH value of pcr amplification system is 8.0-9.0; Magnesium ion concentration is 1.5-3.5mM; The final concentration of 4 kinds of dNTP respectively is 200-300 μ M; The final concentration of bovine serum albumin in said pcr amplification system is 0.1-1mg/ml, and the consumption of the quick Taq enzyme of warm start (precious biotechnology (Dalian) ltd) is 0.1-0.4U/ μ L.
Further, when using said test kit to carry out pcr amplification, amplification elementary reaction in a composite amplification reaction system, increase simultaneously 15 str locus seats and 1 sex identified gene seat.
Described 16 pairs of primers are to lay respectively at said 16 locus both sides and the primer of this locus that is used to increase; Wherein there is 5 ' end of a primer to carry out fluorochrome label in every pair of primer; Primer sequence and primer concentration see the following form, and are configured to the primer mixture of 5X by primer concentration in the table.
The composite amplification of said 16 locus adopts the polymerase chain reaction to realize, adopts multiple tracks or single track capillary electrophoresis to detect.
Further, the human gene group DNA who is wherein detected is: use Chelex method, magnetic bead extraction method or phenol/chloroform extraction method that the source sample is handled the DNA that obtains; Described source sample is: derive from human: filter paper blood/buccal swab sample, FTA card blood/buccal swab sample, buccal swab sample, blood/trace, tissue, seminal stain, salivary stain, hair or bone sample.
Further, when using described test kit to carry out pcr amplification, the amplification elementary reaction carries out amplification program on the PCR of any model appearance: 94-98 ℃ of 30-120s; 26-32 round-robin 94-98 ℃ 1-15s, 55-65 ℃ of 5-30s, 72 ℃ of 5-30s; 72 ℃ of 1-5min, amplification takes 40-50min approximately.
The beneficial effect of the embodiment of the invention is: using existing convenience goods test kit to carry out pcr amplification often needs the time about 3h; And use test kit of the present invention, and carrying out the pcr amplification of 40-50min, can further carry out genetic analyzer to detect, accomplish the STR somatotype to detect, shortened the time that obtains the final somatotype result of sample greatly.
Description of drawings
Fig. 1 is the test kit locus of the present invention somatotype result after the DNA cloning of 1 pair of blood cake source that arranges;
Fig. 2 is the test kit locus of the present invention somatotype result after the DNA cloning of 1 pair of seminal stain source that arranges;
Fig. 3 is the test kit locus of the present invention somatotype result after the DNA cloning of 1 pair of saliva source that arranges;
Fig. 4 is the test kit locus of the present invention somatotype result after the DNA cloning of 1 pair of cast-off cells source that arranges;
Fig. 5 is the test kit locus of the present invention somatotype result after the DNA cloning of 1 pair of muscle tissue source that arranges;
Fig. 6 is the test kit locus of the present invention somatotype result after the DNA cloning of 1 pair of hair source that arranges;
Fig. 7 is a contrast-conventional fluorescence labeling STR multiplex amplification testing system to the somatotype result after the DNA cloning of blood cake source;
Fig. 8 is a contrast-conventional fluorescence labeling STR multiplex amplification testing system to the somatotype result after the DNA cloning of seminal stain source;
Somatotype result after 3 pairs of muscle derived DNA cloning of Fig. 9 embodiment;
Somatotype result after the DNA cloning of 4 pairs of hair sources of Figure 10 embodiment;
Figure 11 embodiment 5 paternity test somatotype results.
Embodiment
Below in conjunction with accompanying drawing and specific embodiment the present invention is described further, but not as to qualification of the present invention.
Embodiment 1
The dna sample of 16 the quick composite PCR amplification fluorescence detection reagent kit of locus Detection and Extraction purifying, locus is arranged and is arranged by first kind.Be that arrangement mode is: one group of Amelogenin, D3S1358, D13S317, D7S820 and D16S539, adopt the blue fluorescent dyes mark, fluorescent marker is 6-FAM; TPOX, TH01, D2S1338 and CSF1PO are one group, adopt the green fluorescence dye marker, and fluorescent marker is HEX; VWA, D5S818, FGA and D6S1043 are one group, adopt the Yellow fluorochrome mark, and fluorescent marker is TAMRA; D8S1179, D21S11 and D18S51 are one group, adopt the red fluorescence dyestuff mark, and fluorescent marker is ROX; It is SIZ that interior mark is selected fluorescent orange mark, fluorescent marker for use.
1, various sample samples by * * public security bureau provides.
2, the extracting genome DNA of various samples
The extraction of various sample genomic dnas is carried out with reference to " GA/T 383-2002 forensic DNA profiling laboratory inspection specification ".
3, the check and analysis of amplification and amplified production
3.1 16 locus fast PCR fluorescence detection reagent kits
3.1.1 pcr amplification system:
3.1.2 pcr amplification program:
3.2 contrast technology/routine techniques:
In the present embodiment, routine techniques reagent uses AGCU 17+1 STR fluorescence detection reagent kit (patent publication No.: CN 101440410A).
3.2.1 pcr amplification system:
| Component | Volume |
| Template | Obtain through step 2 processing |
| Reaction Mix | 10.0 μL |
| Primers 17+1 | 5.0 μL |
| Warm start Taq enzyme (5U/ μ L) | 0.5 μL |
| sdH 2O | Complement to 25.0 μ L |
3.2.2 pcr amplification program:
4, amplified production fluoroscopic examination on genetic analyzer
Form appearance mixture ((mark in the 0.5 μ L molecular weight) * (sample introduction number)+(12 μ L deionized formamide) * (sample introduction number)) by mark in deionized formamide and the molecular weight.Appearance mixture on the 12.5 μ L is mixed with 1 μ L amplified production or allelotrope analytical standard, avoid producing bubble.95 ℃ of sex change 3min, ice bath 3min, and electrophoresis as early as possible.Use the genetic analyzer check and analysis.
5, conclusion
The present invention all can obtain complete somatotype to the DNA that above various conventional sample extractings obtain; The result is shown in Fig. 1-8; Wherein the DNA in blood cake and seminal stain source detects with the present invention and conventional test kit respectively simultaneously; The result shows, two kinds of test kits are consistent to the homologous genes seat somatotype result of the sample in same source.
2.5 * PCR damping fluid of used different pH among the above embodiment, with the Tris-HCl damping fluid preparation of different pH values, the Tris-HCl concentration in 1 * PCR damping fluid is 10mM, KCl concentration is 50mM; Bovine serum albumin, Taq archaeal dna polymerase and other reagent and material used among the present invention are the commercially available prod.
Annotate: the PCR appearance that embodiment uses is rich day scientific and technological Lifepro type pcr amplification appearance.
Embodiment 2
The application of 16 quick composite PCR amplification fluorescence detection reagent kits of locus on the PCR of different model appearance
Use is collected in the filter paper blood cake of same individuality as detected object; Sample and practical implementation process and embodiment 1 are in full accord; Use multiple pc R appearance to carry out parallel laboratory test in amplification step, investigate and use 16 locus fast PCR fluorescence detection reagent kit reagent to carry out the time of pcr amplification at different PCR appearance.The same Fig. 1 of amplification.
Result such as following table:
| PCR appearance title/model | PCR appearance producer | Proliferation time |
| Veriti | American AB I | 35min |
| 9700 | American AB I | 50min |
| Life Express TC-96/T/H(a) | Rich day of Hangzhou | 41min |
| Lifepro | Rich day of Hangzhou | 40min |
| DNA Engine | U.S. Bio-Rad | 41min |
| Piko? Thermal Cycler | Finland Finnzymes Instruments | 41min |
| T-Grandient | Germany Biometra | 50 min |
The result is identical with embodiment 1 blood filter paper amplification, and 16 locus all obtain complete somatotype result and consistent.
Embodiment 3
The dna sample of 16 the quick composite PCR amplification fluorescence detection reagent kit of locus Detection and Extraction purifying, locus is arranged and is arranged by second kind.Be that arrangement mode is: Amelogenin, D3S1358, D13S317, D7S820, D16S539 and D6S1043 are one group, adopt the blue fluorescent dyes mark, and fluorescent marker is 6-FAM; TPOX, TH01, D2S1338 and CSF1PO are one group, adopt the green fluorescence dye marker, and fluorescent marker is HEX; VWA, D5S818 and FGA are one group, adopt the Yellow fluorochrome mark, and fluorescent marker is TAMRA; D8S1179, D21S11 and D18S51 are one group, adopt the red fluorescence dyestuff mark, and fluorescent marker is ROX; It is SIZ that interior mark is selected fluorescent orange mark, fluorescent marker for use.
Hair muscle DNA with among the embodiment 1 is a masterplate, detects.
The practical implementation process is with embodiment 1. detected results such as Fig. 9. and the muscle detected result is consistent among result and the embodiment 1.
Embodiment 4
The dna sample of 16 the quick composite PCR amplification fluorescence detection reagent kit of locus Detection and Extraction purifying, locus is arranged and is arranged by the third.Be that arrangement mode is: Amelogenin, D3S1358, D13S317, D7S820, D16S539 and CSF1PO are one group, adopt the blue fluorescent dyes mark, and fluorescent marker is 6-FAM; TPOX, TH01, D2S1338 and D6S1043 are one group, adopt the green fluorescence dye marker, and fluorescent marker is HEX; VWA, D5S818 and FGA are one group, adopt the Yellow fluorochrome mark, and fluorescent marker is TAMRA; D8S1179, D21S11 and D18S51 are one group, adopt the red fluorescence dyestuff mark, and fluorescent marker is ROX; It is SIZ that interior mark is selected fluorescent orange mark, fluorescent marker for use.
Coming source DNA with the hair among the embodiment 1 is masterplate, detects.
The practical implementation process is with embodiment 1, and detected result is shown in figure 10, and the hair detection result is consistent among result and the embodiment 1.
Embodiment 5
The quick composite PCR amplification fluorescence detection reagent kit of 6 locus of application 1 carries out paternity test
1, collect blood cake in the paternity test case: the paternity test sample by * * public security bureau provides.
2, the extracting genome DNA of various samples
The extraction of various sample genomic dnas is carried out with reference to " GA/T 383-2002 forensic DNA profiling laboratory inspection specification ".
3, augmentation detection
Carry out pcr amplification, genetic analyzer detection and the final somatotype result that obtains according to embodiment 1.
The ultimate principle of paternity test is: according to mendel's law, parental generation genotype determiner is for genotype.Under the prerequisite that does not have transgenation, somatotype mistake: 1. child's pair of alleles must be one from father, one from mother; 2. child can not have the allelotrope that parents all do not have.
Detect a routine father-woman-female triplet parent with embodiment 1 method, its result sees accompanying drawing 11.
In accompanying drawing 11, each row is followed successively by child, father, mother's respective detection result in each chromatic graph.
Obtain the paternity test father-son-female gene type such as the following table 1 of this experiment by accompanying drawing 11:
Table 1:
PI is a paternity index; Claim the parent index again; Be meant the ratio that the hypothesis father provides the possibility (X) of own father's gene and man at random that the possibility (Y) of own father's gene is provided, expression hypothesis father be child own father's possibility what times, i.e. PI=X/Y greatly than man at random for the child own father.
PI=X/Y＝∑f×c/∑f×p
F representes that breeder mother gives child essential allelotrope chance;
C representes that controlled father gives child essential allelotrope chance;
P representes that the man gives child essential allelotrope chance at random, equals essential gene frequency;
Associating paternity index CPI is each product of linked gene seat PI not;
Relative parental right probability (RCP)=(CPI/ (CPI+1)) * 100%;
According to aforementioned calculation, father-son in this experiment-female triplet paternity test RCP value is 0.99999999936 %, assert parent.
Detection system of the present invention is applicable to the amplification through leaching process gained dna sample simultaneously, like the amplification of samples such as blood/trace, buccal swab, tissue, seminal stain, salivary stain, hair, bone source dna sample.
The human gene group DNA that embodiment of the invention extraction capable of using obtains is a parent material, and nucleic acid amplification that only need about 40-50min can carry out the STR somatotype and detect, and has shortened the final somatotype result's of acquisition sample time greatly.
Above-described embodiment, the present invention embodiment a kind of more preferably just, common variation that those skilled in the art carries out in technical scheme scope of the present invention and replacement all should be included in protection scope of the present invention.
SEQUENCE?LISTING
<110>Zhongde Meilian Biotech Co., Ltd. Wuxi,
Shanghai Municipal Public Security Bureau material evidence evaluating center
< 120>16 quick composite PCR amplification fluorescence detection reagent kits of locus and application thereof
<130>
<160> 32
<170> PatentIn?version?3.3
<210> 1
<211> 23
<212> DNA
< 213>artificial sequence
<400> 1
gttctcctgc?tcttgaacat?aag 23
<210> 2
<211> 22
<212> DNA
< 213>artificial sequence
<400> 2
agaagcaatg?tgtgcatgga?gg 22
<210> 3
<211> 19
<212> DNA
< 213>artificial sequence
<400> 3
gggtcaagag?cttgtaaag 19
<210> 4
<211> 19
<212> DNA
< 213>artificial sequence
<400> 4
ttgtgtgtgc?atctgtaag 19
<210> 5
<211> 23
<212> DNA
< 213>artificial sequence
<400> 5
taacgattcc?acatttatct?cat 23
<210> 6
<211> 22
<212> DNA
< 213>artificial sequence
<400> 6
tcagctgact?atggattatt?tt 22
<210> 7
<211> 22
<212> DNA
< 213>artificial sequence
<400> 7
acagaagtct?gggatgtgga?tg 22
<210> 8
<211> 19
<212> DNA
< 213>artificial sequence
<400> 8
gcccaaaaga?cagaaagac 19
<210> 9
<211> 21
<212> DNA
< 213>artificial sequence
<400> 9
gccccactgc?agtccaatct?g 21
<210> 10
<211> 21
<212> DNA
< 213>artificial sequence
<400> 10
atgaaatcaa?cagaggcttg?c 21
<210> 11
<211> 23
<212> DNA
< 213>artificial sequence
<400> 11
acccctgggc?tctgtaaaga?tag 23
<210> 12
<211> 22
<212> DNA
< 213>artificial sequence
<400> 12
gtaagcttaa?actgggaagc?tg 22
<210> 13
<211> 23
<212> DNA
< 213>artificial sequence
<400> 13
aacctgagtc?tccaaggact?agc 23
<210> 14
<211> 23
<212> DNA
< 213>artificial sequence
<400> 14
ttccacacac?cctggccatc?ttc 23
<210> 15
<211> 21
<212> DNA
< 213>artificial sequence
<400> 15
tgctacatcc?ctacctaggt?a 21
<210> 16
<211> 23
<212> DNA
< 213>artificial sequence
<400> 16
gaaatggaat?tcctactggc?taa 23
<210> 17
<211> 21
<212> DNA
< 213>artificial sequence
<400> 17
attcaaaggt?agcttggttg?g 21
<210> 18
<211> 19
<212> DNA
< 213>artificial sequence
<400> 18
gtggctgaaa?agctcccat 19
<210> 19
<211> 22
<212> DNA
< 213>artificial sequence
<400> 19
aacaggactt?agggaccctc?ca 22
<210> 20
<211> 22
<212> DNA
< 213>artificial sequence
<400> 20
ggctgtggct?ggtcttaacc?tc 22
<210> 21
<211> 22
<212> DNA
< 213>artificial sequence
<400> 21
gggcataaca?ttatccaaag?tc 22
<210> 22
<211> 20
<212> DNA
< 213>artificial sequence
<400> 22
tcacggtctg?aaatcgaaat 20
<210> 23
<211> 21
<212> DNA
< 213>artificial sequence
<400> 23
tgtgacaagg?tgattttcca?t 21
<210> 24
<211> 21
<212> DNA
< 213>artificial sequence
<400> 24
tctcagagga?tgctttagtg?c 21
<210> 25
<211> 22
<212> DNA
< 213>artificial sequence
<400> 25
gaataatcag?tatggacttg?ca 22
<210> 26
<211> 22
<212> DNA
< 213>artificial sequence
<400> 26
ggacagatga?taatacatag?ga 22
<210> 27
<211> 21
<212> DNA
< 213>artificial sequence
<400> 27
tcagcctaag?gtggacatgt?t 21
<210> 28
<211> 21
<212> DNA
< 213>artificial sequence
<400> 28
gctgaggcag?gaggagttcg?g 21
<210> 29
<211> 20
<212> DNA
< 213>artificial sequence
<400> 29
cccaagtgaa?tgccttctat 20
<210> 30
<211> 22
<212> DNA
< 213>artificial sequence
<400> 30
gttgtattgt?caatgttctc?ca 22
<210> 31
<211> 18
<212> DNA
< 213>artificial sequence
<400> 31
ctttctgcca?cacggcct 18
<210> 32
<211> 21
<212> DNA
< 213>artificial sequence
<400> 32
tgtagattat?ttttgtggga?a 21
Claims (5)
1. the quick composite PCR amplification fluorescence detection reagent kit of 16 locus is characterized in that this test kit comprises: PCR damping fluid, MgCl
2, dNTPs and bovine serum albumin mixture, the quick Taq enzyme of warm start, the 16 pairs of primer mixtures and ultrapure water;
Wherein 16 pairs of primer mixtures are:
, whenever be centering to rare one 5 ' end in above-mentioned 16 pairs of primers by fluorescent mark.
2. the quick composite PCR amplification fluorescence detection reagent kit of a kind of 16 locus according to claim 1; It is characterized in that: described 16 locus are divided into 4 groups; And its corresponding primer is to carrying out mark with the optical dye of 4 kinds of distinct colors respectively, and described four kinds of optical dyes are blue fluorescent dyes 6-FAM, green fluorescence dyestuff HEX; Yellow fluorochrome TAMRA, red fluorescence dyestuff mark ROX; Interior mark is selected fluorescent orange SIZ for use.
3. the quick composite PCR amplification fluorescence detection reagent kit of a kind of 16 locus according to claim 2; It is characterized in that: described 16 locus are divided into 4 groups; Its corresponding primer carries out mark with the optical dye of distinct colors in 4 respectively; Packet mode is: Amelogenin, D3S1358, D13S317, D7S820, D16S539 and D6S1043 are one group, adopt the blue fluorescent dyes mark, and fluorescent marker is 6-FAM; TPOX, TH01, D2S1338 and CSF1PO are one group, adopt the green fluorescence dye marker, and fluorescent marker is HEX; VWA, D5S818 and FGA are one group, adopt the Yellow fluorochrome mark, and fluorescent marker is TAMRA; D8S1179, D21S11 and D18S51 are one group, adopt the red fluorescence dyestuff mark, and fluorescent marker is ROX; It is SIZ that interior mark is selected fluorescent orange mark, fluorescent marker for use.
4. according to the quick composite PCR amplification fluorescence detection reagent kit of each described a kind of 16 locus of claim 1-3; It is characterized in that: use the pH value of the pcr amplification system of said test kit to be 8.0-9.0; Magnesium ion concentration is 1.5-3.5mM; The final concentration of 4 kinds of dNTP respectively is 200-300 μ M; The final concentration of bovine serum albumin in said pcr amplification system is 0.1-1mg/ml, and the consumption of the quick Taq enzyme of warm start is 0.1-0.4U/ μ L, and the list in the 16 pairs of primer mixtures is as shown in the table to the primer final concentration:
。
5. the application of one kind 16 the quick composite PCR amplification fluorescence detection reagent kits of locus is characterized in that it is used for dna typing.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110243734 CN102424834B (en) | 2010-09-10 | 2011-08-24 | Rapid multiplex-PCR amplification fluorescence detection kit for 16 gene loci, and application thereof |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010282414.X | 2010-09-10 | ||
| CN201010282414XA CN101956008A (en) | 2010-09-10 | 2010-09-10 | Rapid compound PCR (Polymerase Chain Reaction) amplification fluorescence assay kit for 16 gene loca |
| CN 201110243734 CN102424834B (en) | 2010-09-10 | 2011-08-24 | Rapid multiplex-PCR amplification fluorescence detection kit for 16 gene loci, and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102424834A true CN102424834A (en) | 2012-04-25 |
| CN102424834B CN102424834B (en) | 2013-04-03 |
Family
ID=43483593
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201010282414XA Withdrawn CN101956008A (en) | 2010-09-10 | 2010-09-10 | Rapid compound PCR (Polymerase Chain Reaction) amplification fluorescence assay kit for 16 gene loca |
| CN 201110243734 Active CN102424834B (en) | 2010-09-10 | 2011-08-24 | Rapid multiplex-PCR amplification fluorescence detection kit for 16 gene loci, and application thereof |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201010282414XA Withdrawn CN101956008A (en) | 2010-09-10 | 2010-09-10 | Rapid compound PCR (Polymerase Chain Reaction) amplification fluorescence assay kit for 16 gene loca |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN101956008A (en) |
Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102251032B (en) * | 2011-07-01 | 2013-07-24 | 公安部物证鉴定中心 | Preparation method of reference material (RM) for detecting human DNA (deoxyribonucleic acid) STR (short tandem repeat) |
| CN102286477B (en) * | 2011-07-12 | 2013-06-05 | 公安部物证鉴定中心 | Method for preparing short tandem repeat (STR) parting standard substances |
| CN102321748A (en) * | 2011-08-11 | 2012-01-18 | 无锡中德美联生物技术有限公司 | Test kit and the method for use and the application of the fluorescence labeling composite amplification of 22 locus of a kind of while analyst genomic dna |
| CN102816853A (en) * | 2012-08-30 | 2012-12-12 | 山东百福基因科技有限公司 | Kinesiological related gene EPO (erythropoietin) fluorescent detection reagent kit and detection method |
| CN103966207B (en) * | 2013-02-01 | 2017-10-13 | 天昊生物医药科技（苏州）有限公司 | New short nucleotides tandem repetitive sequence site and its application |
| CN104099324B (en) * | 2013-04-02 | 2018-01-19 | 天昊生物医药科技（苏州）有限公司 | New short nucleotides tandem repetitive sequence site and its application |
| CN104099326B (en) * | 2013-04-02 | 2018-02-02 | 天昊生物医药科技（苏州）有限公司 | New short nucleotides tandem repetitive sequence site and its application |
| CN104099328B (en) * | 2013-04-02 | 2018-07-06 | 天昊生物医药科技（苏州）有限公司 | New short nucleotide tandem repetitive sequence site and its application |
| CN104099327B (en) * | 2013-04-02 | 2018-06-05 | 天昊生物医药科技（苏州）有限公司 | New short nucleotide tandem repetitive sequence site and its application |
| CN104099325B (en) * | 2013-04-02 | 2018-06-26 | 天昊生物医药科技（苏州）有限公司 | New short nucleotide tandem repetitive sequence site and its application |
| CN104031989B (en) * | 2014-05-16 | 2016-01-20 | 无锡中德美联生物技术有限公司 | The test kit of the composite amplification of a kind of human gene group DNA 26 locus |
| CN105648100B (en) * | 2016-03-25 | 2019-01-11 | 中国食品药品检定研究院 | The composite amplification system and detection kit of mouse short tandem repeat |
| CN107385064B (en) * | 2017-08-16 | 2020-12-15 | 广东华美众源生物科技有限公司 | Fluorescence labeling composite amplification kit for simultaneously amplifying human autosomal SNP and STR loci and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101440410A (en) * | 2008-12-29 | 2009-05-27 | 无锡中德美联生物技术有限公司 | Fluorescence labeling composite amplification detection system with 18 loci |
-
2010
- 2010-09-10 CN CN201010282414XA patent/CN101956008A/en not_active Withdrawn
-
2011
- 2011-08-24 CN CN 201110243734 patent/CN102424834B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101440410A (en) * | 2008-12-29 | 2009-05-27 | 无锡中德美联生物技术有限公司 | Fluorescence labeling composite amplification detection system with 18 loci |
Non-Patent Citations (1)
| Title |
|---|
| 王琳等: "D21S11、PentaD基因座的遗传多态性及其在唐氏综合征基因诊断中的初步应用研究", 《中国计划生育学杂志》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101956008A (en) | 2011-01-26 |
| CN102424834B (en) | 2013-04-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102424834B (en) | Rapid multiplex-PCR amplification fluorescence detection kit for 16 gene loci, and application thereof | |
| CN109880911B (en) | Composite amplification kit for 25 human chromosome loci and application thereof | |
| CA3106132C (en) | Methods and compositions for rapid multiplex amplification of str loci | |
| CN108441565B (en) | Fluorescence labeling multiplex amplification kit for 37 STR loci of human Y chromosome and application thereof | |
| CN109468384B (en) | Composite amplification detection kit for simultaneously detecting 45Y loci | |
| CN107988353B (en) | A kind of mankind MTHFR and/or MTRR genetic polymorphism detection kit | |
| CN104131072B (en) | A kind of method and system unknown sample being carried out to individual recognition and paternity identification | |
| CN101440410A (en) | Fluorescence labeling composite amplification detection system with 18 loci | |
| CN107904317A (en) | Mankind's euchromosome STR polymorphic site composite amplification reagent kit and its application | |
| CN112322733A (en) | Nucleic acid composition and kit for detecting EGFR gene mutation and method for detecting EGFR gene mutation | |
| CN110564861B (en) | Fluorescent marker composite amplification kit for human Y chromosome STR locus and InDel locus and application thereof | |
| CN102321748A (en) | Test kit and the method for use and the application of the fluorescence labeling composite amplification of 22 locus of a kind of while analyst genomic dna | |
| CN107254516A (en) | A kind of six color fluorescence STR classifying methods and system | |
| CN109929936B (en) | Fluorescence labeling multiplex amplification kit for detecting human Y chromosome rapid mutation STR locus and application | |
| CN101845496A (en) | Fluorescence labeling STR multiplex amplification testing system free from DNA extraction | |
| CN102286476B (en) | Short tandem repeat (STR) parting standard substance | |
| CN108251537B (en) | Fluorescence labeling composite amplification kit for simultaneously amplifying STR loci of human autosome and Y chromosome and application thereof | |
| CN102286477B (en) | Method for preparing short tandem repeat (STR) parting standard substances | |
| CN112266952B (en) | Supplementary STR locus composite amplification kit for refractory materials and application thereof | |
| CN112852973A (en) | Primer group and kit for simultaneously amplifying 13 STR loci of human and application thereof | |
| CN114703291B (en) | Eight-color fluorescent composite amplification kit for detecting micro-degradation DNA and application thereof | |
| CN105543388B (en) | Medical jurisprudence quick detection kit based on 21 mitochondria SNP sites | |
| Zhang et al. | Mitochondrial DNA typing of laser‐captured single sperm cells to differentiate individuals in a mixed semen stain | |
| CN113403406B (en) | Multiplex amplification system and kit for X chromosome STR locus | |
| CN116751872A (en) | STR composite amplification detection kit for pigeon identity identification and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |