CN1548543A - PCR method with unique primer and its application - Google Patents

Abstract

The present invention provides the PCR or RT-PCR method with unique prime and its application, and the unique primer is oligomeric nucleotide chain designed based on the template DNA chain order characteristic and has 3' end with 6-12 bases in complementary order with the template and 5' end flexibly designed. The method is superior to classical PCR method, which has one pair of primers and maybe negative effect on proliferation efficiency and the complementing between 3' end bases in forward and inverse primers. The present invention is suitable for use in the PCR or RT-PCR test of human and pathological microbe gene, in multiple PCR or RT-PCR to reduce the mutual effect between primers and as the inner reference reaction of other PCR or RT-PCR, and is favorable to obtaining quantitative or semi-quantitative results.

Description

Use the polymerase chain reaction method and the application thereof of unique primer

Technical field

The present invention relates to Protocols in Molecular Biology, particularly relate to the method for polymerase chain reaction.

Technical background

PCR is a kind of simple, single-minded, sensitive, gene amplification method fast.The ultimate principle of PCR and method are to design a pair of primer (primer-pair) according to known gene order, wherein, positive primer (forward-primer) is complementary in the upstream and the order of the anti-chain in the template DNA two strands of template order, and anti-primer (reverse-primer) is in the downstream of template order and the positive chain sequence complementation in the template DNA two strands.Template DNA sex change is at high temperature separated and is split into strand, when reducing to annealing region positive primer and anti-primer respectively with the anti-chain of template and positive chain combination, archaeal dna polymerase holds the direction by 5 ' to 3 ' to make chain extension from 3 ' of primer and template bonded position then, thereby finishes a round-robin amplification.Classical PCR all is to design a pair of primer by this principle for over ten years.

According to the design of the ultimate principle of PCR method a pair of have high preciseness, with the complete complementary primer of target gene order be the successful key factor of amplification.But research for over ten years and application practice show that also primer that a pair of and template order mate fully neither finish the sufficient condition of amplification, neither finish the prerequisite of amplification.Template DNA in the PCR reaction system is human gene group DNA or complementary DNA normally, not only its total base sequence is up to tens but also separate after sex change that to be split into strand its three-dimensional arrangement to the process of annealing temperature very complicated, sterically hinderedly may influence archaeal dna polymerase contacting primer and template annealing position.Even under suitable reaction conditions with the primer that mates fully with the template order of a pair of high preciseness, the target product that increases that also differs surely single-mindedly, sometimes even can not get the target amplification product.On the other hand, use and have the primer of mispairing or even serious mispairing also often can finish amplification with gene order even single-minded obtain the target amplification product and single-minded nothing but amplified production disturbs.It should not be the unique pattern of PCR primer that a pair of primer is used in these true promptings.Application with template order upstream and downstream higher coupling is arranged all but unique primer of incomplete coupling might be finished the amplification of target product even obtain single-minded target product.

Once occurred some titles or summary in the past in the document and contain keyword " single primer " research report (single-primer), the original text of following the trail of document shows that these researchs in fact all still adopt a pair of primer rather than a primer.Part in these reports uses so-called " single primer " to emphasize that just they have used " a pair of primer " (single-primer-pair) rather than with application several multiplex PCRs to primer (Multiplex).Other then are to carry out reverse transcription with a single-minded primer (gene-specific-single-primer) of gene earlier, and then with another primer are carried out PCR, still are to use a pair of primer with regard to PCR.Also have some to be called to be the report of single primer PCR to be actually a kind of asymmetric PCR technology, it increases with a pair of primer earlier, the difference of melting temperature(Tm) that utilizes the concentration of positive anti-primer or primer then is after amplification proceeds to certain phase, one of them primer is failed, and another primer (single-primer) thus the continuation effect can obtain the single stranded DNA product.In a word, still not having research report so far attempts and successfully uses a unique primer and one section known nucleic acid of order is increased and obtain single-minded target product.

Unique primer of the present invention uses a primed DNA chain or claims oligonucleotide to finish pcr amplification, and its English meaning is unique primer, and the reaction of being carried out is that unique primer PCR is unique primer PCR.

Summary of the invention

Technical problem to be solved by this invention

The invention provides method and the application thereof of using unique primer to finish PCR or RT-PCR, must use a pair of primer to finish the technology routine of amplification to break through in the classical PCR theory, avoided Standard PC R because positive anti-primer to the different negative impact to amplification efficiency that may cause of the joint efficiency of template DNA chain, has also been avoided the complementation of the end of 3 ' between positive anti-primer base.

Inventive concept

The present invention is based on 2 facts and conception.First, for a primer that is about 20 bases, if its 3 ' terminal number rises preceding 6,7,8,9,10,11,12 or more base and template order are mated fully, and other bases and template order have one, during a plurality of or complete mispairing, the joint efficiency of this primer and template still may reach as a primer or even very high preciseness primer requirement.The second, all may exist to the long successive range of several kilobase a pair of or several at the hundreds of of each gene is 6,7,8,9,10,11,12 or the oligonucleotide fragment of polybase base more to length, and their order is reverse complemental.These 2 explanations are real with the next target product that increases of a primer single-mindedly.

Measurement results with the gene order of selecting at random illustrates above-mentioned conception below, helps to understand design of the present invention.

1. the primer joint efficiency depends primarily on the coupling of 3 ' end base sequence.

The joint efficiency of primer and template just is one of important symbol of primer preciseness height, and the primer joint efficiency depends on the length of whole primer, based composition and arrangement, but depend primarily on composition and the arrangement that 3 ' of primer is held base.For above-mentioned final conclusion there being a quantized understanding, our 5 of picked at random from people actomyosin gene (gene pool numbering BC016045) are long to be the oligonucleotide of 20 bases, with the joint efficiency that primer-design software Oligo6.0 tests calculating primer when the different positions generation at primer has continuous three G and C or A and the mispairing of T metathetical with template, test value and statistics are shown in table 1.

Table 1. base mismatch position is to the influence of primer joint efficiency

The test sequence number	Base mismatch position (from 3 ' terminal number)	The relative joint efficiency of primer *
				Mean value (%)	Standard error
		????1	????18，19，20			????91.8	????1.1
????2	????17，18，19			????90.1	????1.3
		????3	????16，17，18			????87.3	????1.0
????4	????15，16，17			????85.8	????1.4
		????5	????14，15，16			????84.0	????1.9
????6	????13，14，15			????81.7	????1.7
		????7	????12，13，14			????79.4	????2.4
????8	????11，12，13			????74.5	????5.7
		????9	????10，11，12			????70.3	????5.3
????10	????9，10，11			????65.9	????6.7
		????11	????8，9，10			????61.8	????6.3
????12	????7，8，9			????56.2	????6.6
		????13	????6，7，8			????46.6	????3.1
????14	????5，6，7			????37.5	????5.0

????15	????4，5，6	????41.5	????3.2
				????16	????3，4，5	????41.9	????2.5
????17	????2，3，4	????34.6	????4.4
				????18	????1，2，3	????28.7	????3.3

^*Primer joint efficiency with error-free timing is 100%

Table 1 explanation base mismatch has substantial connection to influence and its position of joint efficiency.The mispairing that occurs in 5 ' end is very little to joint efficiency influence, and mispairing begins suddenly to increase to the influence of joint efficiency when mispairing occurs in apart from 10 base left and right sides of 3 ' end, joint efficiency by 70% left and right sides bust to 40% about.

2. length is the joint efficiency of the oligonucleotide of 8,9 and 10 bases.

From people actomyosin gene (gene pool numbering BC016045) picked at random length is each 21 of the oligonucleoside of 8,9 and 10 bases, calculates their joint efficiency with Oligo6.0, and statistics is shown in table 2.

The joint efficiency of table 2. oligonucleotide

	The joint efficiency of 21 different lengths oligonucleotide
				Base is long	????8	????9	????10
Minimum value	????118	????168	????210
				Maximum value	????344	????381	????436
Mean value	????194	????227	????280
					The distribution of joint efficiency (oligonucleotide quantity)

??101-200	????15	????8	????0
				??201-250	????4	????7	????8
??251-300	????1	????4	????7
				??301-350	????1	????1	????4
??＞350	????0	????1	????2

Primer-design software Oligo6.0 just is divided into 6 retainings (table 3) to the primer joint efficiency by the primer preciseness.

The joint efficiency index of the different preciseness primers of table 3.

The primer preciseness	Very high	High	Medium	Can	Low	Very low
							The joint efficiency requirement	＞360	＞340	＞320	＞310	＞300	＞290

Can infer according to table 1 and table 2 and with reference to table 3: if 6-12 or more Nucleotide and the complementation fully of template order of 3 ' end of a primer, and be complete mispairing in other positions, when a plurality of mispairing is arranged or during indivedual mispairing, the joint efficiency of this primer may even surpass 360 above 290, meets the basic demand as unique primer or high preciseness primer.

3. the frequency that anti-phase complementary order occurs.

The frequency of occurrences of seeing each oligonucleotide order from macroscopic view is at random.Any one specified oligonucleotide order is on average every 4 ⁿIndividual Nucleotide occurs once, and n represents the base number of few nuclear Nucleotide.The length of supposing a target P CR amplified production is 1000 bases, and then the chance that repeats in the target amplification product of specified oligonucleotide order is 1000/4 ⁿIf do not specify the order of oligonucleotide, what any one kind repetitive sequence oligonucleotide then occurs in 1000 bases has 1000/ (1000/4 ⁿ) individual.Table 4 has shown the calculation result of the frequency that repeats 6-12 base long oligonucleotide order.

The table 4. oligonucleotide order frequency of occurrences

Oligonucleotide base long number	The specified order oligonucleotide frequency of occurrences	The frequency of occurrences in 1000 base sequences	In 1000 base sequences, duplicate the appropriate number of order
				????6	????1/4096	????1/4	????250
????7	????1/16384	????1/16	????63
				????8	????1/65,536	????1/66	????15
????9	????1/266,144	????1/266	????4
				????10	????1/1,048,576	????1/1048	????1
????11	????1/4,194,304	????1/4194	????1/4
				????12	????1/16,777,216	????1/16777	????1/16

Table 4 presentation of results has chance in various degree the long repetitive sequence of 6-12 base to occur in the nucleic acid sequence of one 1000 left and right sides base.Oligonucleotide repetitive sequence occurring according to principle of randomization in one section nucleic acid sequence is the same with the probability that the anti-phase complementary order of oligonucleotide occurs.In other words, in the nucleic acid sequence of one 1000 left and right sides base, there is chance in various degree the oligonucleotide that 6-12 base length is reverse complemental to occur.If with the 3 ' end of this nucleotide sequence as primer, also adjust individually from the some bases of 5 ' end extension of oligonucleotide according to the template order again, just can obtain a primer has higher coupling in the upstream and downstream of template and anti-and normal chain respectively, with this primer target product that can increase.

Technical scheme

Polymerase chain reaction method of the present invention, its reaction process in turn includes the following steps:

(1) template sex change;

(2) primer and template annealing;

(3) synthetic complementary dna chain is extended in archaeal dna polymerase catalysis down;

(4) set by step (1)-(3) circulation carrying out amplified reaction.

And the primer in entire reaction course is a unique oligonucleotide chain that designs according to template DNA chain sequence feature, increases at the template DNA sequence.6-20 Nucleotide of 3 ' end of said primer is the complementation fully of 8-10 Nucleotide with complementary fully preferred 6-12 Nucleotide of template order and the complementation fully of template order, optimum.

The above-mentioned PCR reaction method of 2-5 is combined into multiplex PCR under the unified condition of reaction conditions, correspondingly, have 2-5 bar primer respectively different dna profiling sequences to be carried out PCR simultaneously in reaction system.

Above-mentioned polymerase chain reaction method is as the internal reference reaction of other PCR reactions, its unique design of primers is in the relative constant housekeeping gene of copy number in genome sequence, unique primer is added amplification simultaneously in other PCR reaction systems, product amount with the internal reference reaction is other PCR product amounts of reference corrected, avoids the different errors that cause with amplification efficiency of template amount because of adding between the tube and tube.

A kind of reverse transcriptase polymerase chain reaction method of the present invention, its reaction process in turn includes the following steps:

(1) dna primer for the guiding, with the RNA chain be template, with reversed transcriptive enzyme catalysis synthetic DNA template;

(2) template sex change;

(3) primer and template annealing;

(4) synthetic complementary dna chain is extended in archaeal dna polymerase catalysis down;

(5) set by step (2)-(4) circulation carrying out amplified reaction.

And the primer in entire reaction course is a unique oligonucleotide chain that designs according to template DNA chain sequence feature, and this oligonucleotide chain is both as the primer of reverse transcription reaction, again as the primer of PCR reaction.The complementation fully of 6-15 Nucleotide of 3 ' end of primer and template order.

The above-mentioned RT-PCR reaction method of 2-5 is combined into multiple RT-PCR under the unified condition of reaction conditions, correspondingly, have 2-5 bar primer respectively the different RNA template sequence to be carried out RT-PCR simultaneously in reaction system.

Above-mentioned reverse transcriptase polymerase chain reaction is as the internal reference reaction of other RT-PCR reactions, its unique design of primers is in the relative constant housekeeping gene of copy number in genome sequence, unique primer is added amplification simultaneously in other RT-PCR reaction systems, product amount with the internal reference reaction is other PCR product amounts of reference corrected, avoids the different errors that cause with amplification efficiency of template amount because of adding between the tube and tube.

Utilize all above-mentioned polymerase chain reaction methods or reverse transcriptase polymerase chain reaction method to detect pathogenic micro-organism, consider two aspects on unique primer design: both required the conserved sequence complementation of unique primer 3 ' terminal sequence and this pathogenic micro-organism, require all or part of base and the various hypotype strains of pathogenic micro-organism or the corresponding base complementrity of variant of 5 ' terminal sequence again, to reduce contingent false negative in the testing process as far as possible.

Be described as follows about unique primer design method:

Use the design of primers of the standard pcr of a pair of primer both can determine with manual method fully, can rely on primer-design software again fully and determine automatically, but also the two is combined in the auxiliary down by manually determining of primer-design software.But it is very difficult or possible hardly to seek unique primer with manual method.On the other hand, though now the primer-design software a multitude of names on market and the network none have the function of automatic searching repetitive sequence or reverse complemental order.Needing nationality software to assist before generation is heard in new functional software exploitation determines with manual method.

The first step is set for example 25 bases of an oligonucleotide length that is used for artificial screening at first artificially.If the short search of the length of setting is quite time-consuming, otherwise the chance of then leaking sieve is higher.Second step was that 25 bases are example with Oligo6.0 and setting testing length, the position of positive primer is fixed on 1-25,26-50,51-75,76-100...... by that analogy, observe the collection of illustrative plates of shown primer of primer software and the false joint efficiency of DNA normal chain one by one, therefrom found out 6-12 base and be reverse complemental order, with it as candidate's oligonucleotide.Because the chance that 6 or 7 bases of appearance are reverse complemental is more, and finally therefrom finds the chance of good unique primer lower, should preferably consider to select 8-10 base to be the order of reverse complemental.The 3rd step held candidate's oligonucleotide as 3 ' of about 20 base long primers, prolong and regulate the 5 ' base of holding according to the template order to make primer approaching or identical as far as possible with the joint efficiency and the base mismatch quantity of upstream template order and downstream template order.When adjusting 5 ' end base, also to consider the complementary and hairpin structure situation of 3 ' end of primer, make it meet the high preciseness requirement of common primer as far as possible.

Beneficial effect

1. use unique primer PCR and only need synthesize and prepare a primer, use the expanding effect that a pair of primer reached but can reach.

2. use unique primer PCR before junior two circulation of amplification promptly forms amplified production, because primer has nuance with incomplete same the making it of joint efficiency of anti-chain just on amplification efficiency.But primer is identical with the joint efficiency of positive anti-chain in the amplification procedure subsequently, when having avoided Standard PC R to use a pair of primer because the positive different negative impact to amplification efficiency that may cause of anti-primer joint efficiency.

3. standard pcr is used a pair of primer and is finished amplification, need consider to avoid the complementation of the end of 3 ' between positive anti-primer base when design of primers, because 3 ' end between positive anti-primer is complementary than the easier formation that causes the primer dipolymer of primer self complementation.The formation of primer dipolymer had both influenced amplification efficiency, also was provided with obstacle for directly detecting the PCR product with fluorescent reagent, had also influenced the exactness and the reliability of SYBRGreen real-time fluorescence quantitative PCR.Use unique primer and then significantly reduce or avoided fully the interference of primer dipolymer.

4. the complementation of primer self also can form dipolymer, but because two sides of the complementary dipolymer that forms of primer self are complementary in proper order, can form strong hairpin structure, thereby in fact amplification seldom or is not fully had influence.The also high order of magnitude of the upper limit of the common the primer concentration range of primer concentration comparable standard PCR when using unique primer amplification target product, this just helps postponing the appearance of PCR flat slope, helping the PCR reaction product accumulation straight line phase increases, and makes the end-point method quantitative PCR and has created prerequisite for using regular-PCR instrument and conventional electrophoretic technique.

5. using unique primer can also be primary template with total RNA or mRNA, finishes the amplification of target gene with single tube single stage method RT-PCR technology, has reduced the chance that non-single-minded product forms.

6. unique primer PCR method also can be used for multiplex PCR, be each target gene all with a primer or one of them gene with a primer, primer quantity total in the multi-PRC reaction system is reduced, reduce or avoided forming between primer the chance of primer dipolymer.

7. need be when the gene quantification of more different samples is expressed with the housekeeping gene conduct by the internal reference of cls gene, use unique primer housekeeping gene that increases, both increased the stable and repeated of housekeeping gene amplification, reduced producing the interferential chance between housekeeping gene primer and tested gene primer again.

8. when using unique primer PCR, primer and template order are mated the most important index that is not design of primers fully.3 ' end base of unique primer is definite by the template order, but 5 ' end of primer is according to what the template upstream and downstream was regulated in proper order higher a piece of wood serving as a brake to halt a carriage flexibility to be arranged in proper order.As virogene, regulating the 5 ' variability that can consider gene when holding base for the higher template of variability order.The annealing temperature of unique primer PCR is lower usually, and false negative result is reduced.

Embodiment

Embodiment 1.

People actomyosin gene (1841 bases, gene pool numbering BC016045) generally is used as housekeeping gene, and conduct is with reference to template in quantitative gene expression analysis and comparison.Present embodiment is that example is tested with this gene, the oligonucleotide sequence that to find tens sequences to be anti-phase complementary length be the 6-12 base, and wherein a part can reach primer or high preciseness primer requirement.

Table 5 and table 6 are listed in the part that finds in the people actomyosin gene can be as the primer order of unique primer PCR method, and analyzes its primer and the joint efficiency of template upstream and downstream bonded position, primer and template and other characteristics of primer.

The order of the unique primer of part that table 5. finds in people actomyosin gene

The primer title	Primer is the position in template	Primer order (5 ' to 3 ') *	With the template joint efficiency
				??A1	?23-52	?AGA?AGC?TCG?CCC?TTG?GGG?ATC?CGT?CGC ?????????????????CCG	??462
?169-140	?AGA?AGC?TCG?CCC?TTG?GGG?ATC?CGT?CGC ?????????????????CCG	??447
			??A2		?54-69	?ATG?GTG?CCG?CCG?CCA?G	??414
?989-974	?ATG?GTG?CCG?CCG?CCA?G	??391
				??A3	?79-100	?GAT?GAT?GCT?ATA?GCC?GCG?CTC?G	??481
?678-657	?GAT?GAT?GCT?ATA?GCC?GCG?CTC?G	??499
			??A4		?81-100	?TGA?AGA?TAT?AGC?CGC?GCT?CG	??473
?676-657	?TGA?AGA?TAT?AGC?CGC?GCT?CG	??487
				??A5	?125-150	?GCC?AGG?CAG?CAT?TTG?CGG?TCG?ACG?AT	??307
?1205-1180	?GCC?AGG?CAG?CAT?TTG?CGG?TCG?ACG?AT	??307
			??A6		?134-153	?GCC?CAG?CGG?GTG?ACG?ATG?CC	??349
?314-295	?GCC?CAG?CGG?GTG?ACG?ATG?CC	??356
				??A7	?497-516	?AAC?TGT?GCG?CAC?CTG?GCC?GT	??385
?825-806	?AAC?TGT?GCG?CAC?CTG?GCC?GT	??393
			??A8		?579-598	?GTA?CAC?CCT?CAG?CCA?TGC?CA	??301
?1294-1275	?GTA?CAC?CCT?CAG?CCA?TGC?CA	??305
				??A9	?598-617	?ATC?CTT?GGT?CTT?GAC?CTG?GC	??300
?828-809	?ATC?CTT?GGT?CTT?GAC?CTG?GC	??293
			??A10		?1063-1084	?ATA?GCT?CGT?CCT?CTG?CGC?AAG?T	??335
?1271-1250	?ATA?GCT?CGT?CCT?CTG?CGC?AAG?T	??348

^*Black face is represented and the mispairing of template order

The characteristic of the unique primer of part that finds in the table 6. people actomyosin gene

The primer title	The base number that 3 ' end and upstream and downstream template are mated fully	Primer length	3 ' end complementary base radix/kilocalorie/mol	3 ' end internal stability degree	Melting temperature(Tm) when mating fully (℃)	Hair clip handle base number/kilocalorie/mol	Amplified production length
								??A1	????6	??30	????2/-3.6	???12.9	??93.0	????3/-2.1	?147
??A2	????8	??16	????3/-5.0	????9.7	??76.6	????3/-0.9	?936
								??A3	????10	??21	????2/-3.6	????9.9	??76.5	????3/0.5	?600
??A4	????10	??20	????2/-3.6	????9.9	??72.0	????3/0.5	?596
								??A5	????6	??26	????2/-1.5	????8.0	??87.1	????3/0.6	?1081
??A6	????9	??20	????2/-3.1	????9.6	??82.9	????3/-1.0	?181
								??A7	????8	??20	????2/-1.3	????11.1	??79.1	????＜2	?329
??A8	????8	??20	????2/-1.9	????10.0	??72.9	????＜2	?716
								??A9	????8	??20	????2/-3.1	????9.7	??69.9	????3/-0.1	?231
??A10	????8	??22	????＜2	????6.7	??73.1	????＜2	?209

Table 6 is presented at and obtains at least 10 oligonucleotide in the gene about long 1800 bases and can be used for unique primer PCR, the upstream and downstream position of they and template DNA joint efficiency all surpass 290, wherein more than halfly surpass 360, reached the desired index of very high preciseness primer.

Embodiment 2.

(1283 bases NM_006959) are carried out unique design of primers to another housekeeping gene glyceraldehyde-3-phosphate dehydrogenase gene with the method identical with embodiment 1.Table 7 and table 8 are listed in the part that finds in this gene can be as the primer order of unique primer PCR method, and analyzes its primer and the joint efficiency of template upstream and downstream bonded position, primer and template and other characteristics of primer.

The order of the unique primer of part that finds in the table 7. people glyceraldehyde-3-phosphate dehydrogenase

The primer title	Primer is the position in template	Primer order (5 ' to 3 ') *	With the template joint efficiency
				G1	?79-100	????GAT?GAT?GCT?ATA?GCC?GCG?CTC?G	?481
?678-657	????GAT?GAT?GCT?ATA?GCC?GCG?CTC?G	?499
			G2		?367-387	????TCC?ACT?GGC?TGC?TTC?ACC?ACC	?318
?875-855	????TCC?ACT?GGC?TGC?TTC?ACC?ACC	?325
				G3	?633-651	????GAC?TGT?GCT?AGG?CCC?CTC?C	?393
?1241- ?1223	????GAC?TGT?GCT?AGG?CCC?CTC?C	?399
			G4		?949-974	????TCC?TCT?ACT?GTT?CTC?GCT?GGG?GCT?GG	?451
?1122- ?1097	????TCC?TCT?ACT?GTT?CTC?GCT?GGG?GCT?GG	?445
				G5	?1088- ?1108	????CGT?GGC?CCA?CCC?TCC?CCA?GCA	?354
?1159- ?1139	????CGT?GGC?CCA?CCC?TCC?CCA?GCA	?343

^*Black face is represented and the mispairing of template order

The characteristic of the unique primer of part that finds in the table 8. people glyceraldehyde-3-phosphate dehydrogenase

The primer title	The base number that 3 ' end and upstream and downstream template are mated fully	Primer length	3 ' end complementary base radix/kilocalorie/mol	3 ' end internal stability degree	Melting temperature(Tm) when mating fully (℃)	Hair clip handle base number/kilocalorie/mol	Amplified production length
								??G1	????10	??22	????2/-3.6	????9.9	??76.5	??3/-0.5	??600
??G2	????10	??21	????2/-3.1	????9.4	??76.5	??3/-0.9	??509
								??G3	????9	??19	????2/-3.1	????9.4	??70.6	??2/1.3	??609
??G4	????11	??26	????2/-3.1	????9.7	??81.3	??＜2	??174
								??G5	????8	??21	????2/-1.9	????8.5	??85.8	??3/-0.7	??72

Table 8 is presented at and obtains at least 5 oligonucleotide in the long 1300 base left and right sides genes and can be used for unique primer PCR, the upstream and downstream position of they and template DNA joint efficiency all surpass 290, wherein more than halfly surpass 360 and reached the desired index of very high preciseness primer.

Embodiment 3.

Embodiment 3 uses listed used primer G2, G3 or the G4 of amplification people glyceraldehyde-3-phosphate dehydrogenase gene in table 7 and the table 8.Every pipe PCR reaction solution volume is 5 microlitres, and primer concentration is 0.5 μ M, and as template DNA, complementary DNA is that reverse transcription primer prepare with the cDNA synthetic agent box of ClonTech company with poly (dT) by people's total tissue RNA with complementary DNA.People's total tissue RNA consumption is per 20 microlitre reaction solutions, 1 microgram during reverse transcription, and it is standby after reverse transcription is finished reaction volume to be diluted to 100 microlitres with deionized water.Per 100 microlitre PCR reaction solutions add above-mentioned diluted complementary DNA 10 microlitres.The PCR reaction Advantage2 test kit of ClonTech company.Reaction conditions is 95 ℃ of sex change first 1 minute, 95 ℃ of 10 second of circulation sex change, anneal 52-72 ℃ 1 minute, extend 72 ℃ 1 minute, ending extend 72 ℃ 3 minutes, reaction totally 28 circulations.Reaction is finished after behind polyacrylamide gel electrophoresis and the ethidium bromide staining, observe and record amplified production band with image analyzer.Test-results is shown in table 9.

The PCR test-results of table 9. embodiment 3

Primer	Amplified production	Different annealing temperature (℃) formation of amplified production down
											????52	??55.2	??57.5	??60.4	??63.8	??66.6	??68.8	??70.4	??72
		????G2	Target	????+++	??+++	??+++	??+++	??+++	??+++	??+										??-	??-
Non-single-minded	????***										??***	??***	??***	??*	??-	??-	??-	??-
			????G3	Target	????+++	??+++	??+++	??+++	??+++	??+++									??-	??-	??-
Non-single-minded	????***	??***									??***	??***	??*	??-	??-	??-	??-

??G4	Target	????-	????-	????+	????++	????++	???+++	?+++	?+++	?+++
											Non-single-minded	????***	????***	????**	????**	????**	???*	?-	?-	?-

Mark+how much be with entry to mark amplified production intensity

Mark ^*How much what represent non-single-minded band

The no band of mark-expression

Three unique primer PCRs that table 9 presentation of results is tried are effective amplification target product in the wide annealing region of tens degree all.Using primer G4 can be under 72 ℃ of left and right sides annealing temperature, uses that G2 and G3 primer can increase target product single-mindedly under 66 ℃ of left and right sides annealing temperatures and single-minded nothing but product forms.

Embodiment 4.

Embodiment 4 uses unique primer A6 of listed amplification people actomyosin gene in table 5 and the table 6.PCR reaction solution composition is identical with embodiment 3 with reaction conditions, and test-results is shown in table 10.

The PCR test-results of table 10. embodiment 4

Amplified production	Different annealing temperature (℃) formation of amplified production down
										??52	??55.2	??57.5	??60.4	??63.8	??66.6	??68.8	??70.4	??72
	Target	??+++	??+++	??+++	??+++	??+++	??+++	??+++	??+++										??+++
Non-single-minded										??***	??***	??**	??*	??-	??-	??-	??-	??-

Table 10 explanation uses that unique primer A6 can increase target gene single-mindedly in the annealing region about 60-72 ℃ and single-minded nothing but product disturbs.

Embodiment 5.

Embodiment 5 uses unique primer A6 and tests the formation of primer dipolymer under the different primer concentrations.Test conditions is identical with embodiment 3, and primer concentration is from 0.5 μ M to 50 μ M, and annealing temperature is 65 ℃ of 30 second.Test-results is shown in table 11.

The PCR test-results of table 11. embodiment 5

Amplified production and primer dipolymer	The final concentration (μ M) of unique primer in the PCR reaction solution
										??0.5	??1.0	??2.0	??5.0	??7.5	??10	??15	??20	??50
	Target product	??++	??+++	??+++	??+++	??+++	??+++	??+++	??+++										??+++
Non-single-minded product										??-	??-	??-	??-	??-	??-	??-	??-	??-
	The primer dipolymer	??-	??-	??-	??-	??-	??-	??-	??-										??+

Table 11 explanation is used unique primer A6 and is carried out PCR, is 20 μ M at primer concentration, promptly under the situation than 20 times of limits for height on the common primer concentration of Standard PC R, still can increase target gene effectively and does not have the primer dipolymer and non-single-minded amplified production forms.

Embodiment 6.

Embodiment 6 uses unique primer A6 and obtains the target amplification product with the single tube single stage method from human total rna.RT-PCR adopts the TITANIUM single stage method RT-PCR test kit of ClonTech company.The reverse transcription temperature is from 35-55 ℃, 1 hour reverse transcription time.Also changed PCR in 3 minutes immediately over to 95 ℃ of insulations after finishing reverse transcription.The PCR program is identical with embodiment 3, and every tube reaction volume is 10 microlitres, is annealed into 65 ℃ of 30 second.Test-results is shown in table 12.

Table 12. embodiment 6 test-results

Amplified production and primer dipolymer	Different reverse transcription temperature (℃) formation of RT-PCR amplified production down
										??35	??38.2	??40.5	??43.4	??46.7	??49.5	??51.8	??53.3	??55
	Target product	??+++	??+++	??+++	??+++	??+++	??+++	??+++	??+++										??+++
Non-single-minded product										??-	??-	??-	??-	??-	??-	??-	??-	??-
	The primer dipolymer	??-	??-	??-	??-	??-	??-	??-	??-										??-

The reverse transcription temperature ranges that various teach book and each company test kits are recommended are 42-50 ℃, and too low or higher specificity and the efficient to reverse transcription and PCR subsequently of temperature has a strong impact on.Unique primer A6 is used in table 12 explanation can finish RT with single stage method in than the much lower and much higher scope of above-mentioned scope ... PCR obtains target P CR amplified production, and without any the interference of non-single-minded product and primer dipolymer.

Embodiment 7

Embodiment 7 is the multiplex PCR of 2 target genes, each target gene is all used unique primer amplification, unique primer G3 of unique primer A6 that two primers are respectively people actomyosin gene (its order and characteristic see Table 5 and table 6) and people's glyceraldehyde-3-phosphate dehydrogenase gene (its in proper order and characteristic see Table 7 and table 8).The complementary order of 3 ' end alkali cardinal sum energy between two primers is respectively 2 bases/every mol-3.1 kilocalorie and 3 a base base/every mol-6.2 kilocalorie.Test conditions is identical with embodiment 3, annealing temperature be 65 ℃ 1 minute.The result obtains 2 target product bands.

Embodiment 8

Embodiment 8 is for proofreading and correct the RT-PCR mensuration of people Beta adrenergic receptor gene (gene pool numbering M15169) as housekeeping gene with unique primer G3 of people's glyceraldehyde-3-phosphate dehydrogenase (its order and characteristic see Table 7 and table 8).Primer order and characteristic are shown in table 13 and table 14.

The order of table 13. embodiment 8 primers

The primer title	Primer is the position in template	Primer order (5 ' to 3 ') *
			BAR2 just	1563-1589	?GCG?CTT?ACC?TGC?CAG?ACT?GCG?CGC ???????????????CAT
BAR2 is anti-	1805-1836	?GCC?CAT?GAC?CAG?ATC?AGC?ACA?GGC ???????????CAG?TGA?AG

The characteristic of table 14. embodiment 8 primers

The primer title	Primer length	3 ' end complementary base radix/kilocalorie/mol		Melting temperature(Tm) (℃)	Close effect with template knot rate	Hair clip handle base number/kilocalorie/mol	The amplified production melting temperature(Tm) (℃)	Amplified production length
									Self	With another primer
		BAR2 just	??27								?2/-1.5	?3/-3.4	?88.8	?630	?4/-4.2	??90.2	??274
BAR2 is anti-	??32			?＜2	?3/-3.5	?88.5	?540	?3/0.5

As 3 ' end complementary base radix and energy kilocalorie/mole number of unique primer G3 of housekeeping gene and positive primer of BAR2 and anti-primer, be respectively 2/-3.1,2/-1.6,2/3.1 and 2/-1.6 after tested.These numerical value illustrate that less than the numerical value between the Beta adrenergic receptor gene primer adding the housekeeping gene primer does not increase primer dimer formation chance, can not bring interference to the amplification of target gene.When the Beta adrenergic receptor gene of more different samples was expressed, each pipe added G3 primer and the positive anti-primer of BAR2, unique primer substrate concentration 0.5 μ M, each 0.25 μ M of target gene primer, anneal 70 ℃ 1 minute, reaction cycle 25-28, other test conditionss are identical with embodiment 3.The result obtains target gene and housekeeping gene amplified band, the target gene band that records with the strong and weak recoverable of housekeeping gene band.

Claims (10)

1. polymerase chain reaction method in turn includes the following steps:

(1) template sex change;

(2) primer and template annealing;

(3) synthetic complementary dna chain is extended in archaeal dna polymerase catalysis down;

(4) set by step (1)-(3) circulation carrying out amplified reaction.

The primer that it is characterized in that entire reaction is a unique oligonucleotide chain.

2. polymerase chain reaction method according to claim 1 is characterized in that 6-20 the Nucleotide and the complementation fully of template order of 3 ' end of primer.

3. polymerase chain reaction method according to claim 1 is characterized in that 6-12 the Nucleotide and the complementation fully of template order of 3 ' end of primer.

4. polymerase chain reaction method according to claim 1 is characterized in that 8-10 the Nucleotide and the complementation fully of template order of 3 ' end of primer.

5. polymerase chain reaction method in turn includes the following steps:

(1) with the dna primer be the guiding, with the RNA chain be template, with reversed transcriptive enzyme catalysis synthetic DNA template;

(2) template sex change;

(3) primer and template annealing;

(4) synthetic complementary dna chain is extended in archaeal dna polymerase catalysis down;

(5) set by step (2)-(4) circulation carrying out amplified reaction.

The primer that it is characterized in that entire reaction is a unique oligonucleotide chain.

6. polymerase chain reaction method according to claim 5 is characterized in that 6-15 the Nucleotide and the complementation fully of template order of 3 ' end of primer.

7. the multiple PCR method that is combined into of the described reaction method of claim 1 is characterized in that having in the reaction system 2-5 bar primer respectively the different templates sequence to be carried out PCR simultaneously.

8. the multiplex RT-PCR method that is combined into of the described reaction method of claim 5 is characterized in that having in the reaction system 2-5 bar primer respectively the different templates sequence to be carried out RT-PCR simultaneously.

9. each described polymerase chain reaction method is characterized in that using unique primer housekeeping gene that increases as the reaction of the internal reference of other reactions among the claim 1-8.

10. utilize each described polymerase chain reaction method detection pathogenic micro-organism among the claim 1-9, it is characterized in that unique primer 3 ' terminal sequence and conserved sequence complementation, the various hypotype strains of all or part of base of 5 ' terminal sequence and pathogenic micro-organism or the corresponding base complementrity of variant.