CN102634510A - Pre-amplification method for trace DNA applied in medicolegal expertise - Google Patents
Pre-amplification method for trace DNA applied in medicolegal expertise Download PDFInfo
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Abstract
The invention discloses a pre-amplification method for trace DNA applied in medicolegal expertise. The method aims at amplifying the trace DNA to obtain high-concentricity DNA template so as to facilitate following medicolegal expertise. The method mainly comprises the steps of exploring the optimum concentration and proportion volume of combined primers; and the amplification conditions are further optimized, the trace DNA aims for being amplified with high efficiency and high fidelity, so that the possibility for the next legal medical expert is provided for determining individuals.
Description
Technical field
The present invention relates to a kind of preparatory amplification method that legal medical expert identifies trace amount DNA that applies to efficiently, belong to fields such as forensic biology, anthropology, criminal investigation.
Background technology
Aspect existing legal medical expert's evaluation; It is ripe that the DNA cloning technology is tending towards, but when the template amount is lower than 100pg, detect through the method for routine with regard to being difficult to, and fingerprint is very important in Criminal Investigation simultaneously; But the incomplete fingerprint that some unique points of crime scene are incomplete tends to cause obstruction to legal medical expert's evaluation; Usually can have few cast-off cells in the fingerprint, because cell number is less, the DNA total amount is not high to be difficult to become the DNA sample.
In real case; Often have the fingerprint that suspect or incident person liable leave in the crime scene; Conventional fingerprint detection is to assert individuality through morphology lines unique point; But some incomplete fingerprint feature points are less often can not be as the means of individual's identification, and the DNA total amount in the fingerprint is lower again, is difficult to directly apply to DNA detection.Along with the development of DNA cloning technology, some trace amount DNAs become another effective way of accusing of the suspect.
Along with to STR (short tandem repeat; STR) research gradually deeply; What particularly the science of STR-DNA typing method and practicality obtained vast judicial department and various circles of society consistently approves and pays much attention to, thus its effect of performance in the investigation of all kinds of cases and incident.Further caused the subsequent DNA recall rate low yet because the DNA residual quantity of crime scene is very little, trace forensic DNA gathers, the recovery is low, the failure of STR somatotype can not be pointed out the individual major cause of crime.
At present; Existing big quantity research and bibliographical information trace amount DNA amplification technique (Cheung VG.Nelson et al.2005, Hanson EK.Ballantyne J et al.2005, Marchion A. et al.2001, Sun G., 2005, Chen Ling et al.2007, Li Y. et al.2008).Its major technology comprises:
1, based on thermal cycling is the WGA technology on basis, like primer extension reaction before the PCR of degeneracy oligonucleotide primer, ligation mediation, the amplification etc. with PCR.
2, based on isothermal reaction be not the WGA technology on basis, like multiple displacement amplification and technological based on the whole genome amplification of primase with PCR.
3, aspect the STR-DNA amplification condition, mainly adopt the 10ul amplification system of Profiler Plus (American AB I company) test kit.
Present technology is in practical application; Sample recall rate for dna content is high is higher, and somatotype is easier to, like the blood stain of leaving over from the scene; Body fluid vestige (Zheng Huifen; Even eat the DNA (Zhang Xiaohong, Tang build new et al.2006) that can detect criminal on the surplus food Dong Yan et al.2006); Yet occur easily allelic unbalance even lose somatotype difficulty (Sun G. et al. 2005, Liu Wei fine jade and gold spring lotus 2007, Hellani A. et al. 2004, Ballantyne KN. et al.2007) for the sample that dna content is lower than 100pg.Incomplete fingerprint then is the comparatively common forensic in crime scene, and the success of this sample DNA is detected the recall rate that can greatly improve forensic DNA.
Early stage of trace amount DNA the STR-DNA somatotype that amplification can effectively improve success ratio, for accusing of the suspect very big help is provided.
Reference
:
[1] Cheung?VG,Nelson?SF.?Whole?genome?amplification?using?a?degenerate?oligonucleotide?primer?allows?hundreds?of?genotypes?to?be?performed?on?less?than?one?nanogram?of?genomic?DNA.?
Proc?Natl?Acad?Sci?U?S?A,?1996,?93(25):?14676-14679.
[2] Hanson?EK,Ballantyne?J.?Whole?genome?amplification?strategy?for?forensic?genetic?analysis?using?single?or?few?cell?equivalents?of?genomic?DNA.?
Anal?Biochem,?2005,?346(2):?246-257.
[3] Marchio?A,?Terris?B,?Meddeb?M,?Pineau?P,?Duverger?A,?Tiollais?P,?Bernheim?A,Dejean?A.?Chromosomal?abnormalities?in?liver?cell?dysplasia?detected?by?comparative?genomic?hybridisation.?
Mol?Pathol,?2001,?54(4):?270-274.
[4] Sun?J,?Bergeron?M,?Barbeau?B,?Boivin?G,Tremblay?MJ.?Influenza?virus?activates?human?immunodeficiency?virus?type-1?gene?expression?in?human?CD4-expressing?T?cells?through?an?NF-kappaB-dependent?pathway.
?Clin?Immunol,?2005,?114(2):?190-198.
[5] Chen Ling, Wang Huijun, Liu Chao. journal, 2007,20 (4): 442-442,444. are given birth in primer extension technology for detection trace dna. medical research before the improvement amplification
[6] Zheng understands fragrant Dong, opens morning. on-the-spot bloodstain DNA inspection case 1 example of scattering. and Journal of Forensic Sciences, 2005,22 (4): 129-129.
[7] Tang builds and begins doing business. the application 2 example of Chelex-100 method in micro-sample DNA check. and Guangdong public security science and technology, 2006,3:69-70,72.
[8] Liu Weiyu, Jin Chunlian. multiple displacement amplification---a kind of new whole genome amplification technology. international genetics magazine, 2007,30 (4): 265-268.
[9] Hellani?A,?Coskun?S,?Benkhalifa?M,?Tbakhi?A,?Sakati?N,?Al-Odaib?A,Ozand?P.?Multiple?displacement?amplification?on?single?cell?and?possible?PGD?applications.?
Mol?Hum?Reprod,?2004,?10(11):?847-852.
[10] Ballantyne?KN,?van?Oorschot?RA,Mitchell?RJ.?Comparison?of?two?whole?genome?amplification?methods?for?STR?genotyping?of?LCN?and?degraded?DNA?samples.?
Forensic?Sci?Int,?2007,?166(1):?35-41.
Summary of the invention
The objective of the invention is to the dna profiling that is unfavorable for the STR somatotype below the 100pg; Improved the condition that increases in advance to the STR somatotype; Combination of primers and amplification system have been optimized; The amplification technique that provides a kind of legal medical expert of applying to identify trace amount DNA can farthest increase to trace amount DNA, for next step STR success somatotype lays the foundation.
Apply to the reagent that legal medical expert identifies the trace amount DNA amplification method among the present invention, mainly by Primer Mix, dNTP, bovine serum albumin (BSA), 10 * reaction buffer (Reaction Buffer), Phi29 archaeal dna polymerase; Wherein, Primer Mix is 36 kinds of primer mixtures (36 kinds of primer sequences are seen table 1) of primer1 to primer36, and dNTP is a DNA synthetic raw material, and the enzyme of amplification is Phi29, and BSA is the adjuvant of assisting amplification, and its content in reaction system is:
Primer Mix (primer1 and primer2 final concentration are 1-3uM, primer3-primer36 final concentration 0.005-0.1uM) Each 2.5mM dNTP 4-8ul
0.5%?(w/v) BSA 2-4ul
10×Reaction?Buffer 7-10ul
5000U Phi29 1-2ul
DdH
2O supplies 70-100ul
Each primer sequence of table 1:primer1-primer36
| The primer title | Sequence (5 '-3 ') | The base number | Modify |
| primer3 | TTCCCTTCCTTCA*C*A | 15 | * represent thio-modification |
| primer4 | TTGCCCAAAGTTA*G*T | 15 | * represent thio-modification |
| primer5 | AGGAGGTGGCAGA*A*G | 15 | * represent thio-modification |
| primer6 | TCTTCCTCATCCC*T*G | 15 | * represent thio-modification |
| primer7 | TGCCTACAGCCAA*T*G | 15 | * represent thio-modification |
| primer8 | CTCCTCCTTCAACT*T*G | 16 | * represent thio-modification |
| primer9 | TGTTCCCACTCTC*A*G | 15 | * represent thio-modification |
| primer10 | GGTGGTAAAACAG*C*C | 15 | * represent thio-modification |
| primer11 | TGAGGCAGGAGGA*G* |
15 | * represent thio-modification |
| primer12 | ATCCTTGTGCGTA*T* |
15 | * represent thio-modification |
| primer13 | GTGTGGTGGCTCA*C*A | 15 | * represent thio-modification |
| primer14 | AATGCTGGGATTA*C*A | 15 | * represent thio-modification |
| primer15 | TGGACAGCCACAC*T*G | 15 | * represent thio-modification |
| primer16 | AGTGTCTGGCACC*C*A | 15 | * represent thio-modification |
| primer17 | TGCTTGAGCCCAG*G*A | 15 | * represent thio-modification |
| primer18 | TTGCCCACTTCTG*C*C | 15 | * represent thio-modification |
| primer19 | GGTGTCCCAGATA*A* |
15 | * represent thio-modification |
| primer20 | GATAGTGGACCTC*A* |
15 | * represent thio-modification |
| primer21 | GGCATGTGCTACT*G*C | 15 | * represent thio-modification |
| primer22 | ACACGATGGAAGG*C*A | 15 | * represent thio-modification |
| primer23 | TGCCTGAGTTTTG*C* |
15 | * represent thio-modification |
| primer24 | TGTTGGGAATGTA*A*A | 15 | * represent thio-modification |
| primer25 | TCATGGAAGGCTG*C*A | 15 | * represent thio-modification |
| primer26 | ACACTCGGGACCA*C*A | 15 | * represent thio-modification |
| primer27 | ATCTTTTGCCCAT*T* |
15 | * represent thio-modification |
| primer28 | TGAGGCTGAAGTA*G*G | 15 | * represent thio-modification |
| primer29 | TCTACTTTGGGCT*T*A | 15 | * represent thio-modification |
| primer30 | GATTGATACATGG*A*A | 15 | * represent thio-modification |
| primer31 | GCAGCAGCTCATG*G* |
15 | * represent thio-modification |
| primer32 | AGGAGGCACCGAA*G*A | 15 | * represent thio-modification |
| primer33 | CCCATGTTCCCAC*T*G | 15 | * represent thio-modification |
| primer34 | TCGTGTTTGCGTC*C*C | 15 | * represent thio-modification |
| primer35 | AGCAGTAGAGAGA*A*C | 15 | * represent thio-modification |
| primer36 | GATAGAGACAGGAT*A*G | 16 | * represent thio-modification |
| primer1 | GGGCAGGA*N*G | 10 | * represent thio-modification |
| primer2 | NNATGT*G*G | 8 | * represent thio- |
| 9N | NNNNNNNNN | 9 | ? |
The amplification method concrete steps of the trace amount DNA of the inventive method before to the STR somatotype are following:
(1) primer1 is put into the PCR single tube to 36 kinds of primers of primer36 and mix, wherein the final concentration of primer1 and primer2 is 1-3uM, and 34 primers of primer3-primer36, each primer final concentration are 0.005-0.1 μ M;
(2) in mixing solutions, add the 10 * reaction buffer of 7-10 μ l and the trace amount DNA template (10-100pg) that need increase, supply distilled water to 58-93ul, mixing;
(3) mixture in the step (2) is placed 98 ℃ of preparatory sex change 5-10min of PCR appearance;
(4) solution after the taking-up sex change; Place 10-20min on ice; Then to the Phi29 that wherein adds dNTP 4-8 μ l, 0.5%BSA 2-4 μ l, 1-2uL 5000U; Put back to behind the mixing in the PCR appearance and increase, wherein every kind of dNTP concentration is 2.5mM, and the pcr amplification condition is 30-35 ℃ of 10-18h; 65 ℃ of 10-30min; 4 ℃ of ∞;
℃ (5)-20 preserve the vestige DNA after product that increases in advance, subsequent use.
36 of primer1-primer36 kinds of primer sequences sequence shown in SEQ ID NO.1-SEQ ID NO.36 among the present invention.
Another purpose of the present invention provides a kind of test kit, and this test kit, is applied to above-mentioned steps and carries out the trace amount DNA somatotype test kit of amplification in advance after the respective concentration and volume proportion according to each reagent for being center reagent with agents useful for same in the preceding method.
Advantage that the inventive method has and technique effect:
1, the present invention adopts many combination of primers, utilizes the amplification technique of Phi29 amplification technique and isothermal PCR; Low to initial dna content; The shortcoming that can't be applied to legal medical expert's identification is improved in the hope of obtaining higher DNA sample, reaches the demand that legal medical expert identifies.
2, use reagent cheap among the present invention, but the large-batch processing sample.
3, the inventive method amplification efficiency is high, as: 10pgDNA can arrive 1000ng/ul after amplification.
4, the DNA fidelity is good after the inventive method DNA cloning, and fidelity is high.
In a word; Show using the inventive method trace amount DNA experimental result that increases in advance; Can be farthest the forensic DNA of trace be increased, for next step STR somatotype successfully provides possibility, this technology all has expanding effect preferably to the DNA amount less than 100pg.Incomplete fingerprint on the forensic can no longer not given up because its unique point is incomplete, utilizes this technology can trace amount DNA wherein be amplified, and for accusing of the suspect possibility is provided.Compare with traditional method, that present technique has is cheap, amplification efficiency is high, the DNA fidelity is good, and fidelity is high, for the trace amount DNA check and analysis provide possibility.
Description of drawings
Fig. 1 is the synoptic diagram as a result of Primer Mix amplification under 4 gradients of the present invention, wherein 1, during Primer Mix concentration 1 to the amplification of 100pg; 2, during Primer Mix concentration 1 to the amplification of 50pg; 3, during Primer Mix concentration 1 to the amplification of 10pg; 4, the blank during Primer Mix concentration 1; 5-8 is Primer Mix concentration 2 amplifications; 9-12 is Primer Mix concentration 3 amplifications; 13-16 is Primer Mix concentration 4 amplifications.
Fig. 2 is the two primers of 9N, primer1 and primer2, the single primer of primer1 and Pmier Mix amplification synoptic diagram, wherein 1,2 DNA amount 100pg; 3,4 DNA amount 50pg; 5,6 DNA amount 10pg; 7 is blank.
Fig. 3 is to be that 1uM and primer3-36 final concentration are to the amplification electrophorogram of 100pg, 50pg and 10pg under the combination of primers of 0.05uM at primer 1, primer2 final concentration; Wherein 1-5 is the electrophorogram after the amplification of 10pgDNA template amount; 6-10 is the electrophorogram after the amplification of 50pgDNA template amount, and 10-15 is the electrophorogram after the amplification of 100pgDNA template amount.
Fig. 4 is to be that 2uM and primer3-36 final concentration are to the amplification electrophorogram of 100pg, 50pg and 10pg under the combination of primers of 0.02uM at primer 1, primer2 final concentration; Wherein 1-5 is the electrophorogram after the amplification of 10pgDNA template amount; 6-10 is the electrophorogram after the amplification of 50pgDNA template amount, and 10-15 is the electrophorogram after the amplification of 100pgDNA template amount.
Embodiment
Below in conjunction with accompanying drawing and embodiment the present invention is done further explain; But protection domain of the present invention is not limited to said content; Method therefor is ordinary method if no special instructions in the embodiment of the invention, and it is commercial conventional reagent or by the reagent of ordinary method preparation that agents useful for same is like no specified otherwise.
The method of Primer Mix amplification 100pg, 50pg and 10pg dna profiling under the embodiment 1:4 gradient, particular content is following:
1, the preparation of primer Mix mixed solution, primer Mix mixed solution are the mixtures of 36 kinds of primers of primer1-primer36, the primer Mix mixed solution of 4 gradients of preparation, and the concentration gradient of mixture is following:
Primer Mix 1:primer1 (final concentration 3uM) and primer2 (final concentration 3uM), primer3-36 (each primer final concentration 0.1uM)
Primer Mix 2:primer1 (final concentration 3uM) and primer2 (final concentration 3uM), primer3-36 (each primer final concentration 0.05uM)
Primer Mix 3:primer1 (final concentration 3uM) and primer2 (final concentration 3uM), primer3-36 (each primer final concentration 0.01uM)
Primer Mix 4:primer1 (final concentration 3uM) and primer2 (final concentration 3uM), primer3-36 (each primer final concentration 0.005uM)
2, in 4 primer mixtures, add following reaction reagent respectively, the reaction system of 4 gradients of preparation is following:
Primer Mix 1 (or 2 or 3 or 4) 36ul
10×Reaction?Buffer 10ul
10 (or 50 or 100) pg/ul DNA 1ul
ddH
2O 39ul
3, after mentioned reagent mixes, place 98 ℃ of preparatory sex change 10 min, take out distortion back solution, place rapidly and place 15min on ice, then to wherein adding following reagent, at last at 33 ℃ of amplification 16h; 65 ℃ of 15min; 4 ℃ of ∞;
Each?2.5mM dNTP 8ul
0.5%?(w/v) BSA 4ul
5000U Phi29 2ul
Embodiment 2: adopt the method for the two primers of random primer 9N, primer1 and primer2, the single primer of primer1 and Pmier Mix amplification 100pg, 50pg and 10pg DNA, particular content is following:
1, random primer 9N amplification 100pg, 50pg and 10pg DNA, amplification system is following:
9N 3ul
10×Reaction?Buffer 2ul
10 (or 50 or 100) pg/ul DNA 1ul
ddH
2O 7ul
At 98 ℃ of preparatory sex change 10 min, take out distortion back solution, place rapidly and place 15min on ice, then to wherein adding following reagent, last 33 ℃ of amplification 16h, 65 ℃ of 15min; 4 ℃ of ∞;
Each?2.5mM dNTP 4ul
0.5%?(w/v) BSA 2ul
5000U Phi29 ?1ul
The original template amount of " 9N " middle 1-2 is that 100pg, 3-4 are that 50pg, 5-6 are 10pg, 7 negative contrasts among Fig. 2; Do not show amplified band among the figure.
2, the single primer amplification 100pg of primer 1 (final concentration 3uM), 50pg and 10pg DNA, amplification system is following:
20uM?primer1 3ul
10 (or 50 or 100) pg/ul DNA 1ul
10 * reaction buffer 2ul
ddH
2O 7ul
At 98 ℃ of preparatory sex change 10 min, take out distortion back solution, place rapidly and place 15min on ice, then to wherein adding following reagent, last 33 ℃ of amplification 16h, 65 ℃ of 15min; 4 ℃ of ∞;
Each?2.5mM dNTP 4ul
0.5%?(w/v) BSA 2ul
5000U Phi29 1ul
Among Fig. 2, the original template amount of 1-2 is that 100pg, 3-4 are that 50pg, 5-6 are 10pg, 7 negative contrasts in " primer 1 ".
3, the two primers of primer 1 (final concentration 3uM) and primer 2 (final concentration 3uM) mix amplification 100pg, 50pg and 10pg DNA, and reaction system is following:
20uM primer1 ?3ul
20uM primer2 ?3ul
10 (or 50 or 100) pg/ul DNA 1ul
10 * reaction buffer 2ul
ddH2O 4ul
At 98 ℃ of preparatory sex change 10 min, take out distortion back solution, place rapidly and place 15min on ice, then to wherein adding following reagent, last 33 ℃ of amplification 16h, 65 ℃ of 15min; 4 ℃ of ∞;
Each?2.5mM?dNTP 4ul
0.5%（w/v）BSA 2ul
5000U Phi29 archaeal dna polymerase 1ul
Among Fig. 2, the original template amount of 1-2 is that 100pg, 3-4 are that 50pg, 5-6 are 10pg, 7 negative contrasts in " primer 1+primer 2 ".
4, primer Mix mix primer (primer1 and primer2 final concentration 3uM, primer3-primer36 final concentration 0.1uM) amplification 100pg, 50pg and 10pg DNA, reaction system is following:
Primer?Mix 36ul
10 * reaction buffer 10ul
10 (or 50 or 100) pg/ul DNA 1ul
ddH
2O 39ul
At 98 ℃ of preparatory sex change 10 min, take out distortion back solution, place rapidly and place 15min on ice, then to wherein adding following reagent, last 33 ℃ of amplification 16h, 65 ℃ of 15min; 4 ℃ of ∞;
Each?2.5mM dNTP 8ul
0.5%?(w/v) BSA 4ul
5000U Phi29 ?2ul
Among Fig. 2, the original template amount of 1-2 is that 100pg, 3-4 are that 50pg, 5-6 are 10pg, 7 negative contrasts in " primer Mix ".The agarose gel electrophoresis result observes, and confirms that Primer Mix is best at the expanding effect to trace amount DNA.
Embodiment 3: apply to legal medical expert and identify the preparatory amplification method of trace amount DNA, adopt the method for Pmier Mix amplification 100pg, 50pg and 10pg DNA, particular content is following:
(1) 36 kinds of primers of primer1-primer36 are put into the PCR single tube and mix, wherein the final concentration of primer1 and primer2 is 1uM, and the final concentration of each primer of primer3-primer36 is 0.05 μ M, divides three groups;
(2) each 1 μ L of 10 * reaction buffer, 42 μ L distilled waters and 10,50 or 100 pg/ul trace amount DNA templates of adding 10 μ L in every group of mixing solutions;
(3) mixture in the step (2) is placed 98 ℃ of preparatory sex change 5min of PCR appearance;
(4) solution after the taking-up sex change; Place 10min on ice, to the Phi29 archaeal dna polymerase that wherein adds dNTP 6 μ l, 0.5%BSA 3 μ l, 2uL 5000U, put back to behind the mixing in the PCR appearance and increase then; Wherein every kind of dNTP concentration is 2.5mM, and the pcr amplification condition is 35 ℃ of amplification 10h; 65 ℃ of 10min; 4 ℃ of ∞; Concrete reagent addition is following:
Primer?1 100uM ?1ul
Primer?2 100uM ?1ul
Each 1ul of Primer 3-36 5uM
10 * reaction buffer 10ul
10 (or 50 or 100) pg/ul DNA 1ul
ddH
2O 42ul
Each?2.5mM dNTP 6ul
0.5%?(w/v) BSA 3ul
5000U Phi29 ?2ul
℃ (5)-20 preserve the vestige DNA after product that increases in advance, subsequent use.
Fig. 3 is the agarose electrophoresis figure of primer1, primer2 (final concentration 1uM) and primer3-36 (final concentration 0.05uM) amplification 100pg, 50pg and 10pg; Wherein " 1-5 " swimming lane is the amplification of 10pg; " 6-10 " swimming lane is the amplification of 50pg, and " 11-15 " swimming lane is the amplification of 100pg.
Embodiment 4: apply to legal medical expert and identify the preparatory amplification method of trace amount DNA, adopt the method for Pmier Mix amplification 100pg, 50pg and 10pg DNA, particular content is following:
(1) 36 kinds of primers of primer1-primer36 are put into the PCR single tube and mix, wherein the final concentration of primer1 and primer2 is 2uM, and the final concentration of each primer of primer3-primer36 is 0.02uM, divides three groups;
(2) 10 * reaction buffer of adding 10 μ L and 46 μ L distilled waters and each 1 μ L of 10,50 or 100 pg/ul trace amount DNA templates in mixing solutions;
(3) mixture in the step (2) is placed 98 ℃ of preparatory sex change 8min of PCR appearance;
(4) solution after the taking-up sex change; Place 20min on ice, to the Phi29 archaeal dna polymerase that wherein adds dNTP4 μ l, 0.5%BSA 2 μ l, 1uL 5000U, put back to behind the mixing in the PCR appearance and increase then; Wherein every kind of dNTP concentration is 2.5mM, and the pcr amplification condition is 30 ℃ of amplification 18h; 65 ℃ of 30min; 4 ℃ of ∞; Concrete reagent addition is following:
Primer?1 200uM ?1ul
Primer?2 200uM ?1ul
Each 1ul of Primer 3-36 2uM
10 * reaction buffer 10ul
10 (or 50 or 100) pg/ul DNA 1ul
ddH
2O 46ul
Each?2.5mM dNTP 4ul
0.5%?(w/v) BSA 2ul
5000U Phi29 ?1ul
℃ (5)-20 preserve the vestige DNA after product that increases in advance, subsequent use.
Fig. 4 is the agarose electrophoresis figure of primer1, primer2 (each 1ul of 110uM) and primer3-36 (each 2ul of 2uM) amplification 100pg, 50pg and 10pg.Wherein " 1-5 " is that amplification, " 6-10 " of 10pg is the amplification of 50pg, and " 11-15 " is the amplification of 100pg.
SEQUENCE?LISTING
< 110>Kunming University of Science and Technology
< 120>a kind ofly apply to the preparatory amplification method that legal medical expert identifies trace amount DNA efficiently
<160> 36
<170> PatentIn?version?3.5
<210> 1
<211> 10
<212> DNA
< 213>artificial sequence
<400> 1
<210> 2
<211> 8
<212> DNA
< 213>artificial sequence
<400> 2
<210>?3
<211>?15
<212> DNA
< 213>artificial sequence
<400> 3
<210> 4
<211> 15
<212> DNA
< 213>artificial sequence
<400> 4
ttgcccaaag ttagt ?15
?
<210> 5
<211> 15
<212> DNA
< 213>artificial sequence
<400> 5
aggaggtggc?agaag 15
?
<210>?6
<211> 15
<212> DNA
< 213>artificial sequence
<400>?6
tcttcctcat?ccctg 15
?
<210>?7
<211> 15
<212> DNA
< 213>artificial sequence
<400>?7
tgcctacagc?caatg 15
<210>?8
<211>?16
<212> DNA
< 213>artificial sequence
<400>?8
ctcctccttc?aacttg 16
<210>?9
<211> 15
<212> DNA
< 213>artificial sequence
<400>?9
tgttcccact?ctcag 15
<210>?10
<211> 15
<212> DNA
< 213>artificial sequence
<400>?10
ggtggtaaaa?cagcc 15
<210>?11
<211> 15
<212> DNA
< 213>artificial sequence
<400>?11
tgaggcagga?ggagt 15
<210>?12
<211> 15
<212> DNA
< 213>artificial sequence
<400>?12
atccttgtgc?gtatt 15
<210> 13
<211> 15
<212> DNA
< 213>artificial sequence
<400>?13
gtgtggtggc?tcaca 15
<210> 14
<211> 15
<212> DNA
< 213>artificial sequence
<400>?14
aatgctggga?ttaca 15
<210> 15
<211> 15
<212> DNA
< 213>artificial sequence
<400>?15
tggacagcca?cactg 15
<210> 16
<211> 15
<212> DNA
< 213>artificial sequence
<400>?16
agtgtctggc?accca 15
<210> 17
<211> 15
<212> DNA
< 213>artificial sequence
<400>?17
tgcttgagcc?cagga 15
<210> 18
<211> 15
<212> DNA
< 213>artificial sequence
<400>?18
ttgcccactt?ctgcc 15
<210> 19
<211> 15
<212> DNA
< 213>artificial sequence
<400>?19
ggtgtcccag?ataat 15
<210> 20
<211> 15
<212> DNA
< 213>artificial sequence
<400>?20
gatagtggac?ctcat 15
<210> 21
<211> 15
<212> DNA
< 213>artificial sequence
<400>?21
ggcatgtgct?actgc 15
<210> 22
<211> 15
<212> DNA
< 213>artificial sequence
<400>?22
acacgatgga?aggca 15
<210> 23
<211> 15
<212> DNA
< 213>artificial sequence
<400>?23
tgcctgagtt?ttgct 15
<210> 24
<211> 15
<212> DNA
< 213>artificial sequence
<400> 24
tgttgggaat?gtaaa 15
<210> 25
<211> 15
<212> DNA
< 213>artificial sequence
<400> 25
tcatggaagg?ctgca 15
<210> 26
<211> 15
<212> DNA
< 213>artificial sequence
<400> 26
acactcggga?ccaca 15
<210> 27
<211> 15
<212> DNA
< 213>artificial sequence
<400> 27
atcttttgcc?cattt 15
<210> 28
<211> 15
<212> DNA
< 213>artificial sequence
<400> 28
tgaggctgaa?gtagg 15
<210> 29
<211> 15
<212> DNA
< 213>artificial sequence
<400> 29
tctactttgg?gctta 15
<210> 30
<211> 15
<212> DNA
< 213>artificial sequence
<400> 30
gattgataca?tggaa 15
<210> 31
<211> 15
<212> DNA
< 213>artificial sequence
<400> 31
gcagcagctc?atggt 15
<210> 32
<211> 15
<212> DNA
< 213>artificial sequence
<400> 32
aggaggcacc?gaaga 15
<210> 33
<211> 15
<212> DNA
< 213>artificial sequence
<400> 33
cccatgttcc?cactg 15
<210> 34
<211> 15
<212> DNA
< 213>artificial sequence
<400> 34
tcgtgtttgc?gtccc 15
<210> 35
<211> 15
<212> DNA
< 213>artificial sequence
<400> 35
agcagtagag?agaac 15
<210> 36
<211> 16
<212> DNA
< 213>artificial sequence
<400> 36
gatagagacag?gatag 16
Claims (3)
1. one kind applies to legal medical expert and identifies the preparatory amplification method of trace amount DNA, it is characterized in that carrying out as follows:
(1) prepares each reaction reagent in the preparatory amplification reaction system; The reaction system TV is 70-100 ul; Reaction system comprises the primer1 of 1 * reaction buffer, 10-100pg trace amount DNA template, 1-3uM, the primer2 of 1-3uM, 34 primers of 0.005-0.1 μ M primer3-primer36, the dNTP of 4-8 μ l, the 0.5%BSA of 2-4 μ l and the Phi29 archaeal dna polymerase of 1-2uL, and surplus is supplied with distilled water;
At first 36 kinds of primers are mixed, in mixing solutions, add 10 * reaction buffer and the 10-100pg trace amount DNA template of 7-10 μ l, supply 58-93ul with distilled water;
(2) mixture in the step (1) is placed 98 ℃ of preparatory sex change 5-10min of PCR appearance;
(3) solution after the taking-up sex change; Place 10-20min on ice; Then to the Phi29 archaeal dna polymerase that wherein adds dNTP4-8 μ l, 0.5%BSA2-4 μ l, 1-2uL 5000U; Put back to behind the mixing in the PCR appearance and increase, wherein every kind of dNTP concentration is 2.5mM, and the pcr amplification condition is 30-35 ℃ of amplification 10-18h; 65 ℃ of 10-30min; 4 ℃ of ∞;
℃ (4)-20 preserve the vestige DNA after product that increases in advance, subsequent use.
2. identify the preparatory amplification method of trace amount DNA according to the said legal medical expert of applying to of claim 1, it is characterized in that: 36 kinds of primer sequences of primer1-primer36 are sequence shown in SEQ ID NO.1-SEQ ID NO.36.
3. the preparatory amplification kit of processing according to agents useful for same in the trace amount DNA amplification method of the said legal medical expert's of the applying to evaluation of claim 1 of trace amount DNA.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105969876A (en) * | 2016-06-15 | 2016-09-28 | 昆明理工大学 | Primer combination for detecting mutation of C-KIT gene in trace tissue and application of primer combination |
| CN105969877A (en) * | 2016-06-15 | 2016-09-28 | 昆明理工大学 | Primer combination for detecting mutation of BRAF gene in trace tissue and application of primer combination |
| CN105969874A (en) * | 2016-06-15 | 2016-09-28 | 昆明理工大学 | Primer combination for detecting mutation of KRAS gene in trace tissue and application of primer combination |
| CN105969875A (en) * | 2016-06-15 | 2016-09-28 | 昆明理工大学 | Primer combination for detecting mutation of PIK3CA gene in trace tissue and application of primer combination |
| CN106011253A (en) * | 2016-06-15 | 2016-10-12 | 昆明理工大学 | Primer combination for detecting gene PDGFRA mutation in microtissue and application thereof |
| CN106048021A (en) * | 2016-06-15 | 2016-10-26 | 昆明理工大学 | Primer combination for detecting mutation of genes P53 in micro-tissues and application of primer combination |
| CN106086169A (en) * | 2016-06-15 | 2016-11-09 | 昆明理工大学 | Combine and application for detecting the primer of EGFR genetic mutation in microcomponent |
| CN107937494A (en) * | 2017-12-22 | 2018-04-20 | 上海美吉生物医药科技有限公司 | It is a kind of to be used for trace sample individual identification and the method for paternity test |
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| CN105969874B (en) * | 2016-06-15 | 2019-11-08 | 昆明理工大学 | Primer for detecting KRAS gene mutation in microcomponent combines and its application |
| CN105969874A (en) * | 2016-06-15 | 2016-09-28 | 昆明理工大学 | Primer combination for detecting mutation of KRAS gene in trace tissue and application of primer combination |
| CN105969875A (en) * | 2016-06-15 | 2016-09-28 | 昆明理工大学 | Primer combination for detecting mutation of PIK3CA gene in trace tissue and application of primer combination |
| CN106011253A (en) * | 2016-06-15 | 2016-10-12 | 昆明理工大学 | Primer combination for detecting gene PDGFRA mutation in microtissue and application thereof |
| CN106048021A (en) * | 2016-06-15 | 2016-10-26 | 昆明理工大学 | Primer combination for detecting mutation of genes P53 in micro-tissues and application of primer combination |
| CN105969877A (en) * | 2016-06-15 | 2016-09-28 | 昆明理工大学 | Primer combination for detecting mutation of BRAF gene in trace tissue and application of primer combination |
| CN105969877B (en) * | 2016-06-15 | 2019-12-03 | 昆明理工大学 | Primer for detecting BRAF gene mutation in microcomponent combines and its application |
| CN105969876A (en) * | 2016-06-15 | 2016-09-28 | 昆明理工大学 | Primer combination for detecting mutation of C-KIT gene in trace tissue and application of primer combination |
| CN105969876B (en) * | 2016-06-15 | 2019-11-08 | 昆明理工大学 | Primer for detecting C-KIT gene mutation in microcomponent combines and its application |
| CN106048021B (en) * | 2016-06-15 | 2019-11-08 | 昆明理工大学 | Primer for detecting P53 gene mutation in microcomponent combines and its application |
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| CN106011253B (en) * | 2016-06-15 | 2019-11-08 | 昆明理工大学 | Primer for detecting PDGFRA gene mutation in microcomponent combines and its application |
| CN105969875B (en) * | 2016-06-15 | 2019-11-08 | 昆明理工大学 | Primer for detecting PIK3CA gene mutation in microcomponent combines and its application |
| CN107937494A (en) * | 2017-12-22 | 2018-04-20 | 上海美吉生物医药科技有限公司 | It is a kind of to be used for trace sample individual identification and the method for paternity test |
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