CN1401783A - Design method for compound amplification of STR primer - Google Patents

Abstract

A process for designing the primer for the complex amplification of STR includes respectively adding a non-human genom sequence to the terminal 5' of the oligonucleotide primer P1 and P2 able to specifically bind with human genom sequence to obtain long primers YPA-P1 and YPB-P2, using them as the primer pair for the first stage of polymerase chain reaction, and directly using said non-human genom sequence as the primer pair for the second stage of PCR.

Description

The primer design method of compound amplification of STR

Technical field

The present invention relates to the segmental primer design method of a plurality of DNA purposes of a kind of external disposable amplification.

Background technology

Deoxyribonucleotide (DNA) in the human nucleus is the carrier of genetic information, and it is the double-spiral structure that is formed by two nucleic acid long-chains.On each bar Nucleotide long-chain deoxynucleotide unit (dNTP) is arranged, A, T, G, C are their bases.The expression of genetic information (as height, body weight, intelligence etc.) has been controlled in the arrangement of above-mentioned base.Article two, the nucleic acid long-chain is with hydrogen bonded, and is principle with the base pairing complementation, i.e. A-T, G-C; The complementary two strands has direction, and one is from 5 ' → 3 ' end, and another chain of complementary is from 3 ' → 5 ' end.When somatic cell division, DNA duplicates in the organism, and double-spiral structure is untied under the effect of some certain enzyme, forms two strands of strands, separately as template, is used for synthetic new complementary strand.Said process needs the effect of archaeal dna polymerase, and it along 5 of fundamental chain ' → 3 ' the new chain of direction catalytic dna molecule synthesis.The dna double chain that daughter cell occurs, wherein one strand is to come from the complete acceptance of parental generation, another strand strand is synthetic fully again, and presses the basepairing rule complementation with fundamental chain.But in external DNA synthetic chemical reaction, archaeal dna polymerase can only be after the section of DNA fragment be attached on the template, could catalytic dna synthetic, can not start anew to duplicate, and this section of DNA fragment is exactly our said primer.So when external DNA is synthetic, need one section primer, archaeal dna polymerase just can carry out replication in vitro DNA.

Some dna fragmentations are purpose fragments that we need in human genome or other biological genome, in order to make its a large amount of amplifications, have adopted a kind of technology that is called polymerase chain reaction (PCR reaction) now, the purpose fragment that amplification in vitro is required.Carrying out PCR when reaction, at first the base sequence of two 3 ' end of the dna fragmentation that should detect according to desire separately the oligonucleotide fragment of synthetic surplus base of the about 17-20 of a segment length as primer (primer).PCR is actually one under the situation that template DNA, primer (known arrays at template fragment two ends) and four kinds of deoxynucleotides etc. exist, the enzymatic reaction that archaeal dna polymerase relies on, and the specificity of amplification depends on the specific combination of primer and template.Whole amplification procedure divided for three steps: (1) sex change: heating makes the two interchain hydrogen bond ruptures of template DNA and forms two strands; Characteristics such as (2) annealing: suddenly alternating temperature rear pattern plate DNA combines by the basepairing rule complementation with primer, also has two combinations between the template strand, but owing to the high density of primer, and is simple in structure, main combination occurs between template and the primer; (3) extend: under the condition that archaeal dna polymerase and magnesium ion etc. exist,,, form and the new DNA chain of template strand complementary in conjunction with mononucleotide from 3 of primer ' end.Above-mentioned three steps are a circulation, and are every through a circulation theoretically, and the DNA amount in the sample should double, and the new chain that forms can become new round round-robin template again, can increase 10 through 25-30 circulation back DNA ⁶-10 ⁹Doubly.

Though the PCR reaction is the effective ways of a kind of amplification in vitro DNA, it typically refers to target DNA fragment of disposable amplification in a reaction system.In real work, people often wish a plurality of target DNA fragments of disposable amplification in a reaction system, have therefore just produced the composite amplification method.From the mechanism of aforementioned PCR reaction as can be seen, in order to realize the composite amplification reaction, it is right just must to design primer at each target DNA fragment in the same reaction system.Method at present commonly used be will the several goal gene seats of compound primer to adding together, directly carry out composite amplification, for example Korean Murim Choi etc. is at " Internetional Journal Legal Medicine " 2000 the 113rd interim " Frequency data on four tetrameric STR loci D18S1270 that deliver, D14S608, D16S3253 and D21S1437 in a Korean population " method introduced in the article.In this kind composite amplification method because each is to the amount of the corresponding amplified production of primer institute and inconsistent, so just must adjustment each to the relative concentration between the primer, also to react and compete between each primer, thereby greatly reduce the efficient of pcr amplification.Simultaneously, also need other reaction composition in the reaction system is adjusted, whole experiment is more loaded down with trivial details, and result's reproducibility is very poor.In addition, some external company has developed the test kit that is used for the composite amplification reaction now, and they are the concrete principle and the method for unexposed test kit also, and most of biology laboratories both domestic and external then utilize these test kits directly to carry out the composite amplification reaction.In experimentation, what they utilized is fluorescent dye primer, uses the dna sequencing instrument, as the direct detected result of ABI 310 sequenators.For example, Xie Xiaodong etc. are at interim " the Tibetan population data on the PCR-typed loci D16S539 that delivers of " Internetional Journal Legal Medicine " calendar year 2001 the 114th, D7S820, D13S317, HUMF13A01, FESFPS, vWA, HUMTH01, TPOX and CSF1PO " what introduced in the article promptly is this method.In addition, people such as Jannine Brownie have mentioned the primer design method that adds one section sequence at 5 of former primer ' end in " Nucleic AcidsResearch " 1997 the 25th volumes 16 interim " Tne elimination of primer-dimeraccumulation in PCR " articles of delivering, be used for the composite amplification reaction, this method is worth using for reference and improvement.

Summary of the invention

The purpose of this invention is to provide a kind of amplification efficiency height, be convenient to laboratory operation and be convenient to detect the primer design method of the composite amplification of expanding effect, especially a kind of STR (short series connection repeats polymorphism) primer design method.

The basic ideas of the inventive method are: choose the genomic short dna fragment of a pair of non-human as short primer to YPA and YPB, and can add this respectively to short primer, thereby obtain long primer YPA-P1 and YPB-P2 with 5 of human genomic sequence specificity bonded Oligonucleolide primers (P1, P2) ' end.Herein, can with human genomic sequence specificity bonded Oligonucleolide primers P1, P2 that is be can with the original primer of human genome bonded of locus to be amplified.Because the composite amplification reaction is the pcr amplification that simultaneously a plurality of locus is carried out, with respect to the different genes seat, can be different with human genomic sequence specificity bonded Oligonucleolide primers P1, the P2 of this locus, corresponding with it, the long primer YPA-P1 of each locus to be amplified and YPB-P2 be different and inequality because of P1, P2 also.Carrying out composite amplification when reaction, in the PCR reaction system, adding above-mentioned short primer and long primer (also need add required heat-resisting polymerase of conventional PCR reaction and mononucleotide etc. certainly) simultaneously at different locus to be amplified.Owing to do not contain the pairing sequence of short primer YPA, YPB in the human genomic sequence of each locus to be amplified, so when this reaction system is carried out first round PCR reaction (experiencing a working cycle of aforementioned sex change, annealing, extension), short primer YPA, YPB can't combine with human genomic sequence, and the dna fragmentation that is increased is still determined P1, P2 by original primer contained among long primer YPA-P1, the YPB-P2.But, after first round reaction finishes, will produce the amplified production that has target gene fragment and non-human genome sequence YPA, YPB simultaneously at locus to be amplified.Carry out second when taking turns PCR reaction (promptly experiencing the working cycle second time of sex change, annealing, extension) in the PCR reaction system, the above-mentioned product that has target gene fragment and non-human genome sequence YPA, YPB is simultaneously further increased, the amplification the primer remains long primer YPA-P1, YPB-P2, the reaction finish after, resulting product have simultaneously target gene fragment and with short primer to YPA, YPB paired non-human genome sequence.In the present invention, we take turns reaction that PCR reaction be referred to as fs, i.e. in the composite amplification process polymerase chain reaction of fs with long primer as the first round and second of reaction primer with above-mentioned.In other words, the polymerase chain reaction of fs has comprised that the above-mentioned first round and second takes turns reaction in the composite amplification process.The resulting amplified production of this elementary reaction have simultaneously target gene fragment and with short primer to (YPA, YPB) paired non-human genome sequence, it is referred to as the product of composite amplification fs herein.React from third round PCR, owing to had the product of above-mentioned fs for each locus, like this, short primer YPA, YPB just can be used as the primer of above-mentioned each amplification gene seat, and the PCR reaction just can be that template increases with the product of above-mentioned fs.Similar therewith, respectively taking turns in the PCR reaction after third round all can be served as the primer of each amplification gene seat with short primer to (YPA, YPB).After the many wheels of experience (taking turns as 30) PCR circulating reaction, the product of above-mentioned fs will be expanded to up to a million times.In the present invention, we react the reaction that be referred to as subordinate phase, i.e. in the composite amplification process polymerase chain reaction of subordinate phase with short primer as the PCR that reacts primer with above-mentioned.Need to prove that in above-mentioned mentality of designing of the present invention, the reaction of fs and subordinate phase is carried out in same reaction system, rather than completely be divided into two secondary responses.In fact, even in the reaction of the PCR after third round and third round, still exist the reaction of above-mentioned fs, just its proportion is extremely low.In addition, when carrying out the pcr amplification reaction of fs, have many to primer participate in simultaneously the reaction, promptly long primer is participated in reaction (as previously mentioned simultaneously with respect to different many of a plurality of locus to be amplified and sequence, the long primer YPA-P1 of each locus to be amplified and YPB-P2 are also inequality), reaction between primer and primer is unavoidable with competition, thereby greatly reduce the efficient of pcr amplification, mentioned when this drawback is discussed prior art in front.But, with regard to the present invention, the purpose of the pcr amplification of fs is not to wish to obtain a large amount of amplified fragments, and only be to make to have target gene fragment simultaneously and produce with the product (product of fs) of YPA, YPB paired non-human genome sequence get final product, therefore above-mentioned drawback is extremely faint to the influence of whole composite amplification reaction process generation.And after the pcr amplification of fs, need 5 ' end of each target gene fragment of amplification all to have and YPA, YPB paired non-human genome sequence, so add a pair of short primer (YPA, YPB) afterwards, just can increase simultaneously to a plurality of locus.Because in the subordinate phase amplification of after this carrying out, the primer that participates in reacting has only a pair of, promptly short primer does not exist competition and reaction between primer to YPA, YPB, need not to adjust the concentration between primer yet, thereby has improved the amplification efficiency of each locus greatly.Therefore we can say that the PCR reaction of fs is only need amplify a small amount of template, and the amplification of subordinate phase to be high-level efficiency increase all locus.With regard to its essence, in the PCR of subordinate phase reaction, be many in the existing composite amplification method to primer a plurality of locus that increase simultaneously, change a pair of primer (YPA, YPB) a plurality of locus that increase simultaneously into; Simultaneously, with respect to each DNA fragment specific that is amplified, YPA, YPB are not to be special primer, therefore, said process also by in the existing composite amplification method by many to special primer a plurality of specific gene fragments that increase simultaneously, be transformed into by a pair of non-special primer a plurality of specific gene fragments that increase simultaneously.

1., it must be non-human genomic sequence in the present invention, the design of above-mentioned short primer YPA, YPB is very crucial, and its design needs to consider the key element of the following aspects:, promptly its sequence can be not identical with human genomic sequence or complementary.2., choose suitable primer length.A large amount of experimental results show that when the design primer, primer length is a very crucial parameter, and the oligonucleotide chain of 18～24 base formations is primer lengths of relatively optimizing; Primer is too short, will cause specific reduction; Primer is long, the template that when annealing amplification is initiated very little, in the index amplification phase, even the little error of each step annealing step all will enlarge, consequently cause the obvious minimizing of amplified production.3., choose the content of bases G C in the primer and the annealing temperature Tm of primer.The primer of PCR reaction should keep rational GC content.Article two, the GC content of primer should be consistent with the Tm value, and amplification efficiency that the primer of poor compatibility is right and specificity are all relatively poor.Lower Tm value can cause specific forfeiture, if adopt too high annealing temperature, the efficient of amplification will be very low; If annealing temperature is too low, primer will produce non-specific annealing, thereby cause the amplification of non-specific dna fragmentation.The temperature that the present invention selects when the Tm value of design primer is 55 ℃, because under this temperature, PCR reactions most in the composite amplification can both be carried out, this temperature will make the back of PCR reaction first a large amount of non-specific assorted bands occur simultaneously, and we just must reduce assorted band by the concentration that reduces long primer.After reducing long primer concentration, the interaction between each primer reduces, and nonspecific product reduces, and meanwhile, we also reduce by required specific product, but this can't influence whole amplification efficiency.Before address, preceding two-wheeled PCR reaction only is the beginning of above-mentioned composite amplification process, this is taken turns the product of PCR reaction and is not required bigger amount, only need to produce the dna fragmentation that has non-human genome sequence and goal gene on a small quantity simultaneously, promptly generate the template of the PCR reaction that is used for subordinate phase on a small quantity, just can high-volume increase by this template of PCR reaction pair thereafter.After the PCR of fs reaction, the primer that participates in reaction just no longer be long primer to (YPA-P1, YPB-P2), but short primer is to (YPA, YPB).

Based on the restriction of above-mentioned condition, the present invention has designed following short primer to (YPA, YPB):

YPA(5′--ATTTAGGTGACACTATAGAATAC—3′)

YPB(5′--TAATACGACTCACTATAGGGAGAC—3′)

By retrieval Human genome database, in human genome, do not exist and above-mentioned primer YPA, the corresponding sequence of YPB, thereby guaranteed that this short primer does not combine with human genomic sequence.

With above-mentioned short primer be added in can with 5 of human genomic sequence specificity bonded Oligonucleolide primers (P1, P2) ' end, just obtain long primer YPA-P1 and YPB-P2.

In sum, the primer design method of compound amplification of STR of the present invention is:

A, respectively can with human genomic sequence specificity bonded Oligonucleolide primers P1,

5 of P2 ' end adds the inhuman genome sequence of the preceding paragraph:

YPA(5′--ATTTAGGTGACACTATAGAATAC—3′)

YPB(5′--TAATACGACTCACTATAGGGAGAC—3′)，

Thereby obtain long primer YPA-P1 and YPB-P2, with this as in the composite amplification process

The primer of one stage polymerase chain reaction is right, and promptly long primer is right;

B, directly with inhuman genome sequence:

YPA(5′--ATTTAGGTGACACTATAGAATAC-3′)

YPB(5′--TAATACGACTCACTATAGGGAGAC-3′)

Primer as composite amplification subordinate phase polymerase chain reaction is right, and promptly short primer is right.

What still need point out emphatically is, in the present invention, above-mentioned can with human genomic sequence specificity bonded Oligonucleolide primers P1, P2 in fact be exactly can with the original primer of Human genome group-specific bonded of locus to be amplified.Also that is to say that the sequence of primer P1, P2 determined by the human genomic sequence of locus to be amplified, and because of the difference of the sequence of locus to be amplified different.

Compare with aforementioned prior art, the present invention designs the primer of composite amplification reaction, thereby can be expeditiously a large amount of amplifying target genes fragments, the reproducibility of experimental result is high, simultaneously, need not in experimentation, to adjust each concentration loaded down with trivial detailsly, simplified whole experiment primer.In addition, the primer that adopts the inventive method to design carries out after the composite amplification reaction, can adopt cma staining mode detected result, cost requirement to experimental installation is not high, and the mode that only can adopt fluorescent dye primer in the aforementioned prior art, detect by automatic sequencer, it is to the requirement height of experimental installation, and therefore to have more advantage with Chinese characteristics for method of the present invention, meets the needs of the most of Molecular Biology Labs of current China.

Content of the present invention further illustrates with the following Examples, but content of the present invention is not limited only to content related among the embodiment.

Embodiment

In this example, designed primer is used for simultaneously the goal gene of four locus D1S1612, D17S2196, D20S161 and D6S477 being carried out composite amplification in same PCR reaction system.

Designed short primer is: YPA:(5 '--ATTTAGGTGACACTATAGAATAC-3 ')

YPB：(5′--TAATACGACTCACTATAGGGAGAC-3′)

Designed long primer YPA-P1, the YPB-P2 of each locus is:

The D1S1612 locus:

YPA-P1：5′--ATTTAGGTGACACTATAGAATAC

TCCCATGCCAAAATTCTTAG-3′

YPB-P2：5′--TAATACGACTCACTATAGGGAGAC

GAAAGAAAGAGAAAGAAGGAAAGG-3′

The D17S2196 locus:

YPA-P1：5′--ATTTAGGTGACACTATAGAATAC

CCAACATCTAGAATTAATCAGAATC-3′

YPB-P2：5′--TAATACGACTCACTATAGGGAGAC

ATATTTCAATATTGTAACCAGTCCC-3′

The D20S161 locus:

YPA-P1：5′--ATTTAGGTGACACTATAGAATAC

CCCCTTCAACTTGTCAGC-3′

YPB-P2：5′--TAATACGACTCACTATAGGGAGAC

TCCTTTCCCAACTGGTATCT-3′

The D6S477 locus:

YPA-P1：5′--ATTTAGGTGACACTATAGAATAC

GATTTGCCATGATAGATGGC-3′

YPB-P2：5′--TAATACGACTCACTATAGGGAGAC

GGGGGATATCTCAAACAACC-3′

Need to prove that above-mentioned four locus human genomic sequence specificity bonded primer P1, P2 are respectively:

The D1S1612 locus:

P1：5′--TCCCATGCCAAAATTCTTAG-3′

P2：5′--GAAAGAAAGAGAAAGAAGGAAAGG-3′

The D17S2196 locus:

P1：5′--CCAACATCTAGAATTAATCAGAATC-3′

P2：5′--ATATTTCAATATTGTAACCAGTCCC-3′

The D20S161 locus:

P1：5′--CCCCTTCAACTTGTCAGC-3′

P2：5′--TCCTTTCCCAACTGGTATCT-3′

The D6S477 locus:

P1：5′--GATTTGCCATGATAGATGGC-3′

P2：5′--GGGGGATATCTCAAACAACC-3′

More than in fact each be exactly can be right with the original primer of human genome bonded of locus to be amplified to primer P1, P2, it is corresponding with the human genomic sequence of each locus D1S1612, D17S2196, D20S161 and D6S477 to be amplified, the pairing P1 of each locus, P2 difference.The short primer of non-human genome sequence that connects design at 5 of above each primer P1, P2 ' end respectively is to (YPA, YPB), promptly obtains long primer with respect to each locus to YPA-P1, YPB-P2.When carrying out composite amplification, right, simultaneously, right as the primer of subordinate phase polymerase chain reaction in the composite amplification process as the primer of fs PCR reaction in the composite amplification process with above-mentioned short primer with above-mentioned long primer.

Be using method and the result of use that further specifies designed primer in this example, go on to say the detailed process when designed primer is used for the composite amplification reaction in the present embodiment below.

Composite amplification is reflected in the PE-9600 amplification instrument and carries out.Composition in the PCR reaction system mainly contains: template DNA (human genomic sequence), long primer to, short primer to, hot resistant DNA polymerase, MgCl ₂, deoxynucleoside triphosphate dNTP, BSA (bovin serum albumin), 1 * Buffer (damping fluid) and distilled water DDH ₂O.Wherein, DNA heat-resisting polymerase, Mgl ₂Directly provide with 10 * Buffer (damping fluid) by commercially available PCR test kit (manufacturer is Invitrogen.USA, and product are called heat-resisting TaqDNA polysaccharase).

The concentration of each composition in the PCR reaction system such as following table:

Reagent	The single hose metering
						??DDH 2O	??????????????????????????????????13.5μl
??dNTP	??????????????????????????????????7.5μl(200μM)
						??10×buffer	??????????????????????????????????3.75μl
Primer	Short primer	The DIS1612 long primer	The D17S2196 long primer	The D20S161 long primer	The D6S477 long primer
						0.3μl(400nM)	0.3μl(40nM)	0.3μl(40nM)	0.3μl(40nM)	0.3μl(40nM)
	0.3μl(400nM)	0.3μl(40nM)	0.3μl(40nM)	0.3μl(40nM)	0.3μl(40nM)
						The Taq enzyme	???????????????????????????????????1μl(3u)
??BSA	???????????????????????????????????3.75μl
						??Mgcl 2	???????????????????????????????????3μl(2.25mM)
??Sample	???????????????????????????????????2μl

Amplification procedure is as follows:

A, the first round and second are taken turns the PCR reaction, i.e. fs amplification (a small amount of amplification), and the primer that reacts is a long primer.

1., sex change: be heated to 94, double-stranded DNA is untied;

2., annealing: be cooled to 55, under this temperature, long primer combines with template DNA;

3., extend: be warming up to 72, this temperature is the most suitable temperature of dna polymerase reaction, and polysaccharase is at Mgcl ₂Prolonging 5 of template ' → 3 ' direction at 5 of long primer ' end and adding dNTP Deng effect down.After the first round and second is taken turns PCR reaction, had simultaneously target gene fragment and with the product (product of fs) of YPA, YPB paired non-human genome sequence.

B, third round and the reaction of PCR afterwards thereof, i.e. subordinate phase amplification, the primer that reacts is a short primer.The PCR reaction mechanism of sex change, annealing and extension but in annealing process, is to combine with template with short primer to former to take turns amplification similar, and 5 ' end of this template has and YPA, YPB paired non-human genome sequence; Simultaneously, in the extension process, be to prolong 5 of above-mentioned template ' → 3 ' direction at 5 of short primer ' end to add dNTP.Whole composite amplification circulates altogether and 36 takes turns, and loop parameter is as follows:

Pre-sex change: 94 ℃ 3 minutes

Sex change: 94 ℃ 50 seconds

Renaturation: 55 ℃ 50 seconds

Extend: 72 ℃ 50 seconds

Cycle index: 36

Extend: 72 ℃ 10 minutes

In addition,, utilize the electrophoresis detection method that it is carried out separation detection especially, so that the check amplification in view of molecular weight size, the fragment length difference of amplified production.When carrying out separation detection, earlier with amplified production and sample-loading buffer mixing in certain proportion, in the polyacrylamide gel hole in the adding electrophoresis chamber, in addition voltage carries out electrophoresis at its two ends.Because the molecular size difference of each amplified production, under the effect of same electrical field force, its distance of being moved in identical medium in the unit time does not wait yet.Therefore, handle after for some time in electrophoresis apparatus, the gap of each amplified production is drawn back gradually, after this takes out gel, to its dye (this example adopts argentation), can see amplification intuitively at last.

The electrophoresis detection raw material, equipment and the condition that are adopted are as follows:

The polyacrylamide gel composition:

30% polyacrylamide (PAG) solution 9.5ml

5 * TBE solution 7ml

DDH ₂O?????????????????????????18.5ml

10% ammonium persulphate (PA), 300 μ l

TEMED??????????????????????????30μl

Electrophoresis chamber: DYY-III electrophoresis chamber (Liuyi Instruments Plant, Beijing)

Electrophoresis apparatus: the multi-functional electrophoresis instrument of pharmacia1000 type

Deposition condition:

1. applied sample amount: sample 3 μ l gel load sample damping fluids: 2 μ l electrode buffers: 1 * TBE solution

2. adopt the current constant mode electrophoretic current: the 80mA time: 1 hour 30 minutes

Dyeing course is as follows:

Adopt argentation:

1,10% ethanol is fixed 10 minutes

2, DDH ₂O washes 2 times

3,1% nitric acid is fixed 3 minutes

4, DDH ₂O washes 2 times

5,1% cma staining is 20 minutes

6, DDH ₂O washes 3 times

7,3%Na ₂CO ₃, the formaldehyde solution colour developing

8, DDH ₂O washes and stops colour developing

The primer that adopts present embodiment to design carries out polygene seat amplification gained result and is compared as follows with the clip size that adopts original Auele Specific Primer (being P1, P2) to carry out the amplification of polygene seat:

The D1S1612 locus: the clip size with original primer amplified is: 93～121bp

With the clip size behind the embodiment primer composite amplification be: 140～168bp

The D17S2196 locus: the clip size with original primer amplified is: 146～174bp

With the clip size behind the embodiment primer composite amplification be: 193～221bp

The D20S161 locus: the clip size with original primer amplified is: 181～205bp

With the clip size behind the embodiment primer composite amplification be: 228～252bp

The D6S477 locus: the clip size with original primer amplified is: 216～248bp

With the clip size behind the embodiment primer composite amplification be: 263～295bp

Can see that after the electrophoresis and the processing of dyeing after the primer that adopts present embodiment to design carried out the amplification of polygene seat, its amplified production growing amount was big, bands of a spectrum were clear after gel was dyed easily distinguishes; Simultaneously, the gained amplified production confirms that the dna sequence dna of amplified production is consistent with the target DNA fragment sequence after checking order through sequenator.And adopt original Auele Specific Primer (P1, P2) to carry out polygene seat when amplification, and owing to be subjected to the reaction and the influence of competition between primer and primer, the growing amount of its amplified production is few, and the dyed bands of a spectrum afterwards of gel are beyond recognition fully, and expanding effect is very poor.

Claims (1)

1, a kind of primer design method of compound amplification of STR is characterized in that carrying out in the following manner:

A, respectively can with human genomic sequence specificity bonded Oligonucleolide primers P1,

5 of P2 ' end adds the inhuman genome sequence of the preceding paragraph:

YPA(5′--ATTTAGGTGACACTATAGAATAC-3′)

YPB(5′--TAATACGACTCACTATAGGGAGAC-3′)，

Thereby obtain long primer YPA-P1 and YPB-P2, with this as in the composite amplification process

The primer of one stage polymerase chain reaction is right, and promptly long primer is right;

B, directly with inhuman genome sequence:

YPA(5′--ATTTAGGTGACACTATAGAATAC-3′)

YPB(5′--TAATACGACTCACTATAGGGAGAC-3′)

Primer as composite amplification subordinate phase polymerase chain reaction is right, and promptly short primer is right.