CN1312371A - Molecular cloning prepn of short tandom human gene repeated sequence typing reference material - Google Patents
Molecular cloning prepn of short tandom human gene repeated sequence typing reference material Download PDFInfo
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Abstract
The present invention discloses a method using molecular clone technique to prepare human short tandem repeated sequence locus (STR) allelic typing standard material. It is characterized by that STR allelic segment obtained by RCR amplification is recombined with plasmid vector, mass allelic segments are prepared by bacteria to make mass propagation of recombinant plasmid charged with STR allelic segment, or through RCR amplification by using recombinant plasmid as templete, finally it utilizes simple purification and mixing processes to obtain mass high-quality STR allelic typing standard materials. Said invented standard materials possess permanent identity and international unified standard names.
Description
The invention belongs to the recombinant DNA utilisation technology.Specifically, the present invention relates to a kind of recombinant plasmid that utilizes and prepare human DNA STR locus (short tandem repeat, the method of allelic ladder abbreviation STR), it can solve STR long-standing accuracy and standardization issue on somatotype.
Human DNA STR (STR) extensively is present in the human genome, and every 6-10 Kb base just has one, is made of 2-6bp its core sequence, and its difference of multiplicity between individuality forms abundant fragment length polymorphism.The size of this fragment length is pressed mendelian inheritance heredity.The allelotrope fragment of str locus seat is little, and the specificity height is very easily by polymerase chain reaction (polymerase chainreaction is called for short PCR) amplification, electropherotyping; Comprising prolific hereditary information in human genome, is important contents in the dna fingerprint of new generation, begins from the beginning of the nineties, be widely used in genetics, clinical medicine, biology, and in the field such as the identification of the individual in the forensic science, paternity test.But in the research and application process of these more than ten years, scholars also notice and have quality control and the quality-guarantee of carrying out DNA analysis only, set up standardized DNA analysis technology, just can guarantee forensic dna check and analysis result's accuracy, fairness and reliability.The key of problem is the name of allelotrope unified standard and the somatotype problem of str locus seat, promptly to same str locus seat detection can (English be called Ladder at same allelic ladder, below be referred to as Ladder) reference under detect, name according to identity principle, make between the different experiments chamber, same laboratory can accurately be compared between the experimental result that different time obtains.For this reason, the method taked for the somatotype problem of scholars has two:
1. use from external import and the test kit that comes comprising whole reagent of pcr amplification and electropherotyping, also has supporting with it corresponding gene seat Ladder.To a certain extent, it can solve typical problem, but has following wretched insufficiency simultaneously:
(1) locus is defined: the detection of certain str locus seat is determined by its specific primer, that is to say, exists that str locus seat primer to be amplified, to amplify with regard to the dna fragmentation of having determined that locus place in the PCR reaction system.In the import reagent box, str locus seat primer is and the supporting existence of other reagent that this just defines the str locus seat that can detect.Although the str locus seat all has higher polymorphism in the crowd, promptly higher individual recognition ability (height of recognition capability is by the height decision of polymorphism).But same str locus seat is in different ethnic groups, and this polymorphism has evident difference.Though in most str locus seats some colonies abroad that the import reagent box can detect comparatively ideal polymorphism is arranged, unsatisfactory in Chinese population.For these locus that can not effectively distinguish Different Individual in the test kit, remedial measure has only two selections: or add and do extra locus, locus with high recognition capability replaces it, and the consequent is exactly following 1-(3) that will mention and the problem among the 2-(2).
(2) waste is uncontrollable, and the characteristics of test kit are simple and easy to do, but must be strictly operates according to its condition and the step of specification sheets defined, to the change of any link with operation can the whole check of influence accidentally, even causes the failure of whole test.On the one hand, test kit generally carries out entire package with all ingredients, and the operator has no way of answering particular requirement that single reagent is adjusted, and causes originally the result who can successful test obtains failing; On the other hand, test kit generally is to design at some samples that are easy to detect, and it usually is difficult to finish detection varied, that preservation condition differs sample, to usually being subjected to the to a certain degree corrupt medical jurisprudence sample that influences all the more so.So, utilize the test kit detection can bring unpredictable, unmanageable waste.
(3) detect the cost costliness: the test kit that uses mostly is greatly from external import at present, the disposable in addition characteristic at angle that makes makes test kit detection cost can not be in any more, add and do extra locus if add, or cause the waste of whole reagent because of single problem described in the 1-(2) to remedying the low recognition capability locus described in the 1-(1).Can imagine, use the import reagent box to detect routinely, will bring huge economic pressures for real work.
2. at the above-mentioned wretched insufficiency of import reagent box, domestic scholars has taked the another one scheme to finish the detection of STR: promptly the reagent in PCR-polyacrylate hydrogel electrophoresis-Yin dyeing and finishing testing process is all by the single as required order in laboratory, the preparation of locus Ladder laboratory oneself, or the Ladder of the corresponding gene seat in the employing import reagent box.
(1) advantage of this scheme:
1. can select locus and quantity thereof according to different needs.Especially limited at sample, in the time of need being chosen at the higher locus of recognition capability in China colony, it is of crucial importance that this point seems.
2. the expense of finishing the same detection reagent that consumes descends significantly.Can be from 4 of three locus of per hundred person-portions of import reagent, 000-5,000 Renminbi 1 of three locus of per hundred person-portions that descend, 000-2000 Renminbi.
3. can be as required, the arbitrary link in the testing process is made amendment and optimized, and do not influence whole detection efficiency.As to the degraded sample, be subjected to the sample of pollution, under the situations such as the individual's identification in the gang-rape case, can usually determine that to the suitable adjustment of individual links can whole detection successful.
(2) problem of this scheme existence:
This scheme has solved the existing locus of import reagent box really and has been defined, wastes uncontrollable and detect problem such as cost costliness, and the problem of unified standard is not resolved:
Although 1. oneself orders other reagent, the method of using the locus Ladder that provides in the test kit to finish PCR and whole testing process simultaneously both can be reduced expenses, can take into account the problem of unified standard again, the businessman that do not have that can regret can provide STR-Ladder separately, it is always with other reagent systems selling, so the Ladder in the test kit is limited.
2. the scholar is exploring new str locus seat constantly, many str locus seats that in China's population analysis, shows higher recognition capability have also been found really, detection to these locus, also do not have businessman that its reagent corresponding box is provided at present, its commercial Ladder certainly more is in no position to take possession of.The way that domestic scholars are taked is the Ladder that oneself makes up these locus.Method and problem are as follows:
3. present domestic scholars prepares str locus seat Ladder method and is:
The genomic dna of A, mixing different genotype is a template with them directly, and pcr amplification, product are made Ladder and used.
B, mix different genotype PCR product, do certain processing, as dilution, purifying etc., be template, pcr amplification is made Ladder usefulness after product mixes once more.
C, reclaim the allelotrope fragment in the gel, be template, pcr amplification is made Ladder after product mixes and is used once more.
Above method prepares the method for str locus seat Ladder, and there are the following problems:
Because the different titles of same locus can appear in the naming method difference that follow in each laboratory, the phenomenon of the different titles of same allelotrope of same locus.A this major reason of not naming according to the international uniform standard guidelines is can not finish its sequential structure analysis with existing dna sequencing method less than the allelotrope fragment of the small segment of 150 bases (bp).And this principles and requirements is named the equipotential gene fragment according to the multiplicity of core sequence.The difference name result who has occurred the order-checking of multiple process thus and named by the international uniform standard, this relatively makes troubles for the result between the laboratory.
This homemade Ladder lacks continuity, no matter be the Ladder that obtains in above-mentioned any mode, the segmental pcr amplification of its allelotrope that comprises obtains, or is template with the genomic dna of different genotype, or is template with the allelotrope fragment of some small segments.Yet it is limited obtaining genomic dna from the individual there of several fixed, is difficult to satisfy the secular needs in a plurality of laboratories; The allelotrope of small segment can not prolonged preservation, and degraded loses template action easily, can not keep a plurality of breadboard long-term needs.So, can lose its continuity because obtaining the change of the segmental pcr template of allelotrope with the Ladder of these method preparations.
Owing to be subjected to the restriction of template source and amount and pcr amplification efficient, the not only amount of using the Ladder that this method can obtain is very limited, and lacks continuity, is difficult to satisfy a plurality of breadboard long-term needs, can not solve standardization issue.
The purpose of this invention is to provide a kind of method of utilizing molecule clone technology to prepare the allelic ladder of human DNA STR locus (STR), it can solve STR long-standing accuracy and standard problem on somatotype.
Realize that the above-mentioned purpose technical scheme is that it comprises the following steps:
(1) obtains each different allelotrope fragment of human DNA STR locus (STR) length to be cloned with polymerase chain reaction (PCR) amplification;
(2) with the allelotrope fragment of above-mentioned pcr amplification and plasmid vector reorganization, will be mounted with the segmental recombinant plasmid of STR allelotrope again transfection to the competence bacterium, cultivate and breeding, select then to have contained and insert the correct clone of fragment;
(3) dna sequencing is analyzed the STR allelotrope fragment sequence in the recombinant plasmid of above-mentioned acquisition and is named by the international uniform standard;
(4) use and confirm to contain correct allele through order-checking to insert segmental recombinant plasmid be template, carry out pcr amplification, its product is purified, promptly obtain corresponding STR allelic ladder (being Ladder) after mixing.
Obtaining the segmental technology of STR allelotrope to be cloned in the above-mentioned the first step can be:
1., the colony's dna sample that extracts is handled through methods such as CHELEX-100, obtain exposed dna molecular;
2., the above-mentioned dna molecular of obtaining is through pcr amplification and detected through gel electrophoresis, finds and reclaim the allelotrope fragment of the different big or small slice segment length of str locus seat from gel, is prepared into the amplification template again of PCR;
3., be template with above-mentioned STR allelotrope fragment, carry out pcr amplification once again and amplify, improve the allelotrope copy number separately of its different big or small slice segment length.
In aforementioned second step, the allelotrope fragment of pcr amplification can be passed through T/A cloning, flush end interpolation or sticky end connection method and realize and the plasmid vector reorganization.The recombinant plasmid transfection is adopted PCR method to select and is contained the segmental clone of correct insertion to the competence microbial culture.
The technical scheme of the allelic ladder (Ladder) of the preparation STR of design of the present invention has following advantage and meaning:
1. prepare str locus seat allelic gene typing standard (reference) thing with molecule clone technology, use more simple pcr amplification method relatively at present, be characterized in preserving by recombinant plasmid the allelotrope of str locus seat, the foundation of the segmental recombinant plasmid of allelotrope has realized the permanent identity of somatotype standard (reference) thing.Make the comparison of test-results not be subjected to the restriction of time and region.
2. can forever preserve by recombinant plasmid with the str locus seat allelic gene typing marker of molecule clone technology preparation.The cyclic plasmid DNA of purifying has the stability of height, but in prolonged preservation below-20 ℃; It can be by the transfection bacterium simultaneously, and enlarged culturing is bred, and this vegetative propagation in bacterium can keep the continuity of its height, finally realizes the identity of Ladder.
3. being mounted with the segmental recombinant plasmid of STR allelotrope and not only can breeding in a large number by bacterium, can be template with the recombinant plasmid of minute quantity also, prepares a large amount of allelotrope fragments by PCR.Because the DNA of recombinant plasmid in the form of a ring, so the allelotrope that is carried on the plasmid duplicates in the mode of " roll and climb " in the pcr amplification process, show high amplification efficiency,, can produce the PCR product that has more several times than common dna profiling promptly in the pcr amplification process of similarity condition.The STR allelotrope fragment that the pcr amplification of these high densitys obtains just can obtain allelic gene typing standard (reference) thing of a large amount of fine str locus seats by simple purifying, mixing.
4. after the allelotrope fragment of small segment is loaded on the plasmid, can finish analysis, exempt the shortcoming that small segment STR allelotrope fragment can not directly be carried out sequential analysis with the consensus primer of plasmid order-checking to its sequential structure.So the allelotrope fragment in str locus seat allelic gene typing standard (reference) thing of this method preparation all can be suitable for standard (reference) thing of any breadboard detection corresponding gene seat according to the name of international uniform standard.
5. the present invention can set up the allelic recombinant plasmid storehouse that is mounted with high recognition capability str locus seat.This not only can reduce the cost of detection greatly, can improve the efficient of detection greatly simultaneously.On the one hand, need not adopt Ladder in the test kit for solving the unified standard problem, but must purchase other reagent that can order at a low price separately in the commercially available reagent box with high price simultaneously; On the other hand, the str locus seat and the corresponding allelic gene typing marker thereof of the high recognition capability of tool arranged, the chance that just reaches individual identifying purpose in one-time detection just therefore rises, and has avoided adding the expense that extra locus produces of doing.
6. str locus seat allelic gene typing standard (reference) thing that obtains with present method replaces the somatotype Ladder in the commercially available reagent box, can exempt fully simultaneously use the import reagent box with and the great deficiency that exists, and demonstrate fully whole advantages by the single as required order detection reagent in laboratory.
7. str locus seat allelic gene typing standard (reference) thing that obtains with the molecule clone technology scheme is because of having permanent identity, but unlimited reproductive ability, productivity that can be a large amount of is named according to the international uniform standard again, so it can satisfy the needs of numerous STR-DNA assay laboratory to str locus seat Ladder, can solve the standardization issue of STR-DNA somatotype, and the dependence fully to more external reagent companies is finally broken away from the production domesticization that will advance standard str locus seat to detect.
In a word, what the allelic gene typing marker for preparing the str locus seat with this programme can advance standard str locus seat to detect to use in China's forensic science popularizes, they have established solid basis for correctly setting up China's STR-DNA fingerprint database, will be for guaranteeing that forensic dna check and analysis technology is accurate, just and giving a clue, try for law and produce evidence to bring into play important meaning for the detection of case reliably.
Further specify the present invention below in conjunction with accompanying drawing and example.
Fig. 1 is the allelotrope gel electrophoresis separating resulting figure of accurate somatotype thing of str locus coordinate and different fragments size.
Among the figure: 1-9 is for being template with the recombinant plasmid, by each allelotrope fragment of pcr amplification acquisition; M is a DYS385 locus allelic ladder.
The operation steps of allelic ladder that the designed technical scheme of the present invention prepares STR is as follows:
1, obtains STR allelotrope fragment to be cloned with pcr amplification, claim the segmental preparation of allelotrope to be cloned again
(1) extracts dna molecular, the allelotrope fragment of promptly collecting the different lengths of str locus seat
Any tissue or cell that contains human DNA passes through under the multiple factors such as enzyme, high temperature and CHELEX, dna molecular can separate with its protein that combines under state of nature and other impurity, open ground is present in the aqueous solution, for good condition is created in next step reaction.
(2) for the first time pcr amplification, detected through gel electrophoresis: purpose is to find the allelotrope fragment of different lengths, as the pcr amplification template second time
The colony's dna sample that extracts is carried out pcr amplification, with 7% non-denaturing polyacrylamide gel (degree of crosslinking is 5%) electrophoretic separation pcr amplified fragment, silver dyes colour developing or gene gel scanning system is judged the sample gene type, selects the allelic individual specimen and the amplified production that comprise different big or small slice segment length.
Its ultimate principle is as follows:
Polymerase chain reaction (polymerase chain reaction is called for short PCR)
A, PCR reaction: mix the required reagent of PCR reaction: comprise target gene seat primer (fluorescent mark is arranged or do not have), dNTP, 1X standard Taq damping fluid, MgCl2, TaqDNA polysaccharase, add sample template DNA 1 μ l at last, reaction system 20-100 μ l.
B, PCR reaction conditions: 95 ℃ of 3min, 94 ℃ of 30s then, 56-62 ℃ of 30s, 72 ℃ of 1min, 30 circulations, 7min is extended in last circulation.
C, PCR ultimate principle: DNA are when having mentioned reagent to exist, when temperature at continuous circulation time between the 94 ℃-56-62 ℃-72 ℃, its two strands can be followed temperature and constantly be repeated to open-circulation of the new copy of polymerization-formation, the copy number of final multiplication, amplification locus place specific DNA fragments, thus as seen small dna fragmentation can become naked eyes by common detection method.
D, electrophoretic separation: dna molecular has negative charge, when voltage exists, its can be from the negative pole of gel to anodal swimming, but because the clip size difference of dna molecular, its mobility speed is inconsistent, after stable voltage kept certain hour, the dna molecular of different sizes was with the different positions of swimming to gel.Concrete operations are: the sample P CR amplified production and the allelic ladder of (1-5 μ l) load on the polyacrylamide gel synchronous voltage stabilizing 500-700V electrophoresis 60-100min in right amount.Dye development process or genescan software reading judgement sample gene type by silver.
(3) pcr amplification for the second time: the allelotrope fragment of the different lengths that purpose is found for pcr amplification, detected through gel electrophoresis for the first time, double, amplify, promptly improve the allelotrope copy number separately of its different big or small slice segment length.The template of pcr amplification can be following three kinds for the second time:
The genomic dna of A, not homoallelic homozygote individuality.The homozygote individuality is that his genotype is made of two identical allelotrope of fragment length.
B, not homoallelic homozygous purified, the dilution 5-1000 pcr amplification product first time doubly.
The allelotrope fragment of C, the different big or small slice segment length that from gel, reclaim.Method contains the segmental gel of each allelotrope for cutting out, and incites somebody to action wherein corresponding allelotrope fragment wash-out in the sterilization distilled water, is prepared into the amplification template again of PCR.
2, the segmental clone of the allelotrope of pcr amplification, the allelotrope fragment that is about to the different fragments size is recombinated on the plasmid vector, transfection is carried out enlarged culturing and breeding in the competence bacterium then, and uses the pcr amplification method, selects to contain and inserts the correct clone of fragment.
(1) the plasmid reorganization can realize by following three kinds of modes:
A, flush end interpolation, the allelotrope fragment of capacity can through the catalysis of T4 ligase enzyme, can terminally with two of plasmid DNA be connected under the condition of certain temperature and the existence of necessary reagent.
B, sticky end connection method, primer to the str locus seat carries out special design, make the point of application that comprises a certain restriction endonuclease in the fragment behind the pcr amplification, after this restriction endonuclease effect, discharge the allelotrope fragment that has certain outstanding base (as the C base), then under the catalysis of T4 ligase enzyme, react with having the plasmid vector that the base of being given prominence to the allelotrope fragment can complementary base (as the G base), thereby realize that the allelotrope fragment is connected with two ends of plasmid DNA.
C, T/A cloning, with adding the A test kit, add an A base to the allelotrope fragment behind the pcr amplification, then under the catalysis of T4 ligase enzyme, react with having the plasmid vector (as the pUC plasmid) that the A base of being given prominence to the allelotrope fragment can complementary 1 base, thereby realize that the allelotrope fragment is connected with two ends of plasmid DNA.
(2) with recombinant plasmid 42 ℃ of transfections in the competence bacterium, bacterium shaking culture about 2 hours in 37 ℃ of constant temperature; After utilizing that selectivity is dull and stereotyped and cultivating, select to contain with PCR method and insert the correct bacterial clone of clip size, therefrom extract plasmid DNA.
3, the dna sequencing of recombinant plasmid and standard name
Adopt fluorescent mark dideoxy nucleotide (ddNTP) sequencing kit, the full-automatic sequenator of ABI310 carries out sequencing analysis from inserting segmental two ends to recombinant plasmid with the public primer of plasmid.By the nomenclature mo of blood genetics association of the International Court of Justice (International Society of Forensic Haemogenetics is called for short ISFH) regulation, the allelotrope fragment of inserting is named.This principle is with the core tumor-necrosis factor glycoproteins cycle index of str locus seat corresponding allelotrope fragment to be named.For example: the core tumor-necrosis factor glycoproteins of DYS385 locus is GAAA, when the GAAA that comprises in a certain fragment circulation 11 times, and this fragment called after 11.So analogize.
4, with the recombinant plasmid be template, carry out pcr amplification, its product is purified, promptly obtain the Ladder of corresponding str locus seat after mixing.
(1) is template with the recombinant plasmid, carries out pcr amplification, obtain the segmental amplified production of each allelotrope of high density;
(2) with each allelotrope pcr amplified fragment of PCR product purification test kit purifying;
(3) each allelotrope fragment of purifying being crossed is mixed, and forming ladder shape standard somatotype thing band spectrum behind its mixture electrophoresis is Ladder, and different standard allelotrope fragments are in different positions in this band spectrum.The genotypic judgement of testing sample can draw according to its bands of a spectrum and the corresponding position of this Ladder and number.
Allelic ladder below in conjunction with DYS385 str locus seat illustrates:
1, the segmental preparation of allelotrope to be cloned
(1) pcr amplification, the allelotrope fragment of preparation DYS385 locus is amplification template again
Preparation standard somatotype object of reference template DNA carries out pcr amplification with the colony's dna sample that extracts, and with 7% non-denaturing polyacrylamide gel (degree of crosslinking is 5%) electrophoretic separation pcr amplified fragment, silver dyes colour developing.From population sample, select the allelotrope of 9 different big or small slice segment length, cut out the gel that contains respective segments, reclaim wash-out in the sterilization distilled water, be prepared into the amplification template again of PCR.
(2) PCR increases again, and product is identified and purifying
9 parts of template DNAs that prepare are carried out pcr amplification, and reaction system 100 μ l include 0.2 μ mol/L DYS385 primer, 200 μ mol/L dNTP, 50 μ mol/L KCl, 10mmol/L Tris-HCl (pH8.3), 1.5mmol/L MgCl2,1U Taq enzyme, template DNA 1 μ l.The PCR reaction conditions is 95 ℃ of 3min, 94 ℃ of 30s then, and 59 ℃ of 30s, 72 ℃ of 1min, 30 circulations, 7min is extended in last circulation.Get 1 μ l amplified production and carry out electrophoresis, after silver dyes, determine its clip size, with the PCR purification kit pcr amplified fragment product purification that size is correct.
2, the clone of pcr amplified fragment
Adopt flush end to insert clone's test kit, the position, Sma I enzyme point of contact of 9 PCR fragments behind the purifying directly being inserted the pUC18/19 plasmid.Plasmid transfection after the reorganization is arrived competence bacterium DH5 α
TMIn, after selecting to cultivate screening, select to contain with PCR method again and insert the correct clone of fragment.
3, the dna sequencing of recombinant plasmid
Adopt fluorescent mark dideoxy nucleotide (ddNTP) sequencing kit, the full-automatic sequenator of ABI310, from inserting segmental two ends 9 recombinant plasmids are carried out sequencing analysis with the public primer of pUC plasmid, the result proves that 9 fragments of its insertion are different lengths fragments that is repeated respectively to form for 11-19 time by GAAA tetranucleotide core sequence respectively, and its length is 252bp-288bp.By the nomenclature mo of blood genetics association of the International Court of Justice (ISFH) regulation, the allelotrope fragment of insertion is DYS385 allelotrope 11-19 (referring to a 1-9 swimming lane among Fig. 1).
4, with the recombinant plasmid be template, carry out pcr amplification, purified, be mixed with out required Ladder.
(1) enlarged culturing of recombinant plasmid and purifying
Insert segmental plasmid and carry out enlarged culturing confirm to contain correct allele through order-checking, behind plasmid purification test kit purifying, obtain the DYS385 allelic gene typing marker recombinant plasmid of standard name.
(2) allelic ladder stagewise preparation
To insert segmental recombinant plasmid dna be template with containing DYS385 allelotrope, carry out pcr amplification, the purifying amplified production mixes the allelic PCR purifying of each DYS385 thing at last, is visible DYS385 locus Ladder (participating in the M swimming lane among Fig. 1) after the electrophoretic separation colour developing.
Claims (6)
1, a kind of molecular cloning preparation of short tandom human gene repeated sequence typing reference material is characterized in that it comprises the following steps:
(1) obtains each allelotrope fragment that varies in size of human DNA STR locus to be cloned with polymerase chain reaction (PCR) amplification;
(2) with the allelotrope fragment and the plasmid vector reorganization of above-mentioned amplification, recombinant plasmid transfection is again cultivated and breeding to the competence bacterium, selects then to have contained and inserts the correct clone of fragment;
(3) dna sequencing is analyzed the allelotrope fragments sequence in the recombinant plasmid of above-mentioned acquisition and is named by the international uniform standard;
(4) use and confirm to contain correct allele through order-checking to insert segmental recombinant plasmid be template, carry out polymerase chain reaction (PCR) amplification, its product is purified, promptly obtain corresponding allelic ladder after mixing.
2, the method for claim 1 is characterized in that obtaining the segmental technology of allelotrope to be cloned and is:
(1) the colony's dna sample that extracts is handled through methods such as CHELEX-100, obtained exposed genomic dna molecule;
(2) the above-mentioned dna molecular of obtaining finds and reclaims the allelotrope fragment of different big or small slice segment length through polymerase chain reaction (PCR) amplification and detected through gel electrophoresis from gel, is prepared into amplification template again;
(3) be template with above-mentioned allelotrope fragment, carry out polymerase chain reaction (PCR) amplification once again,, improve each allelotrope copy number separately to amplify.
3, method as claimed in claim 1 or 2, it is characterized in that the allelotrope fragment that increases can realize and the plasmid vector reorganization by the T/A cloning, promptly adopt and add the A test kit, add an A base for the allelotrope fragment of amplification, then under the catalysis of T4 ligase enzyme with the plasmid vector reaction of the outstanding base of band T, realize that allelotrope fragment and plasmid DNA two hold to be connected.
4, method as claimed in claim 1 or 2 is characterized in that the allelotrope fragment that increases can realize and the plasmid vector reorganization by the flush end interpolation, and promptly the allelotrope fragment is through the catalysis of T4 ligase enzyme, with two terminal connections of plasmid DNA.
5, method as claimed in claim 1 or 2, it is characterized in that the allelotrope fragment that increases can realize and the plasmid vector reorganization by the sticky end connection method, promptly Kuo Zeng allelotrope fragment is through the restriction endonuclease effect, make it discharge the allelotrope fragment that has certain outstanding base, by base complementrity and the plasmid vector reaction that has the respective complementary base, realize that under the catalysis of T4 ligase enzyme the allelotrope fragment is connected with two ends of plasmid DNA then.
6, method according to claim 1 is characterized in that the recombinant plasmid transfection to the competence microbial culture, adopts polymerase chain reaction method to select to contain to insert the correct clone of clip size.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102251032A (en) * | 2011-07-01 | 2011-11-23 | 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) |
| CN102286477A (en) * | 2011-07-12 | 2011-12-21 | 公安部物证鉴定中心 | Method for preparing short tandem repeat (STR) parting standard substances |
| CN107122625A (en) * | 2016-02-24 | 2017-09-01 | 北京爱普益生物科技有限公司 | The processing method of mankind's Short tandem repeats sequence high-flux sequence information |
| CN107326075A (en) * | 2017-07-05 | 2017-11-07 | 公安部第研究所 | Target preparation method in a kind of fluorescence molecule amount |
| CN116004764A (en) * | 2022-10-12 | 2023-04-25 | 苏州阅微基因技术有限公司 | Method for preparing allele parting standard of two-base repeated STR |
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2001
- 2001-03-05 CN CN 01107235 patent/CN1312371A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102251032A (en) * | 2011-07-01 | 2011-11-23 | 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) |
| 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) |
| CN102286477A (en) * | 2011-07-12 | 2011-12-21 | 公安部物证鉴定中心 | Method for preparing short tandem repeat (STR) parting standard substances |
| CN107122625A (en) * | 2016-02-24 | 2017-09-01 | 北京爱普益生物科技有限公司 | The processing method of mankind's Short tandem repeats sequence high-flux sequence information |
| CN107122625B (en) * | 2016-02-24 | 2020-10-09 | 北京爱普益生物科技有限公司 | Method for processing high-throughput sequencing information of human short segment tandem repeat sequence |
| CN107326075A (en) * | 2017-07-05 | 2017-11-07 | 公安部第研究所 | Target preparation method in a kind of fluorescence molecule amount |
| CN116004764A (en) * | 2022-10-12 | 2023-04-25 | 苏州阅微基因技术有限公司 | Method for preparing allele parting standard of two-base repeated STR |
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