CN1204261C - Polymerase chain reaction method using ultra-long primer to improve gene amplification efficacy - Google Patents

Abstract

The present invention relates to a molecular biology technology, particularly to an improved method of polymerase chain reaction PCR for synthesizing a specific DNA segment in a promotion mode by invitro enzyme. The present invention is characterized in that the general perception and the artificial constraint of the existing PCR for the design of the length of a primer are renovated; a pair of ultralong primers whose length is 31 to 50 basic groups are used for replacing a primer which has the existing length basic group; a PCR circulation step is correspondingly simplified to comprise a high temperature denaturation step and an annealing extension step. The annealing temperature and the extension temperature are respectively from 72 to 82 DEG C. The ultralong primer of the present invention has the high stringency, the combination property is obviously better than that of the existing general primer, the chain fusion temperature is enhanced, the intensity of specifically combining a standard target and being mismatched with the standard target is increased, and the mismatched combining intensity is not increased. The present invention is suitable for standard PCR and reverse transcription PCR. The present invention is also used for competition quantitative PCR and real time fluorescent quantitative PCR in order to reduce formation of non-specificity products. The present invention can also be used for multiplex PCR nested PCR and other various kinds of improved PCR methods derived from the standard PCR. The present invention has the wide application prospect.

Description

Use ultra-long primer instead to improve the polymerase chain reaction method of gene amplification efficacy

Technical field

The present invention relates to Protocols in Molecular Biology, the polymerase chain reaction method of the synthetic specific DNA fragment of promptly a kind of external enzymatic.

Background technology

The polymerase chain reaction is that PCR is a kind of simple, single-minded, sensitive gene amplification method, as described in " molecular biology theory and technology " (Beijing science tech publishing house version in 2002) book, is one of the most frequently used Protocols in Molecular Biology.Its primary process is according to the known array synthetic primer, is the synthetic gene fragment that contains intron or do not contain intron of template with genomic dna, library DNA or cDNA.Existing typical PCR by 1, high-temperature denatured template; 2, primer and template annealing; 3, primer and template are extended three-step reaction and are formed a circulation, by circulating reaction repeatedly, make target DNA be able to rapid amplification.Its key step is: template DNA to be amplified is put (be generally 93-94 ℃) under the high temperature and make its sex change separate into strand; Two Oligonucleolide primers of synthetic combine with two strands of goal gene both sides are complementary respectively under its suitable renaturation temperature, two primers on template the bonded determining positions length of amplified fragments; Heat-stable archaeal dna polymerase (Taq enzyme) begins mononucleotide to mix from 3 of primer ' end at 72 ℃, is that template is extended the new complementary strand of synthetic DNA from 5 ' → 3 ' direction with the goal gene.The necessary composition of PCR method has hot resistant DNA polymerase, four kinds of deoxyribonucleotides, magnesium ion, damping fluid, target DNA and primer.Wherein design of primers is the decisive factor of PCR success.Every characteristic of primer is by primer length, the based composition and the decision that puts in order.Wherein, primer length is a key parameter.The primer length scope is subjected to the restriction of two aspects, and primer is short to meet the demands aspect secondary structure and the primer complementary characteristic easily, but it is not enough often to react specificity; Primer is longer, though the reaction specificity reaches standard, often can not use too by force because of complementation between primer secondary structure or primer.Generally believe that now it is the 15-30 base that Standard PC R fills the primer length scope of being permitted usually, optimum primer length scope is the 18-25 base.If the long then cost of primer increases, with the hybridization speed reduction of template DNA, specificity reduces.

Summary of the invention

Of the present invention is exactly to the common cognition of design of primers length and artificially qualification in the existing PCR method of Gonna breakthrough, provide a kind of non-single-minded amplified production and primer dipolymer of making to reduce or elimination, make the target amplification product stronger, purer, reaction times can be shortened, can reduce the used main agents amount of reaction, the PCR method that reaction stability and repeatability are improved.

The necessary composition of polymerase chain reaction method PCR of the present invention has: hot resistant DNA polymerase, four kinds of deoxyribonucleotides, magnesium ion, damping fluid, target DNA and a pair of positive and negative primers.Wherein, the length of a pair of positive and negative primer is the 31-50 base, is preferably the 35-45 base, the ultra-long primer of meaning.Should be complementary fully on the principle of temporal sequence of ultra-long primer with the template order, but as required the discrete mispairing can be arranged.The chain melting temperatur of positive and negative primer is that the scope of Tm is 82-92 ℃, is preferably 85-90 ℃.The difference that the chain melting temperatur of PCR product subtracts positive and negative primer strand melting temperatur is-4-10 ℃.Positive and negative primer all has higher preciseness.Polymerase chain reaction PCR of the present invention is owing to adopt ultra-long primer to substitute general length primer, its PCR circulation step is by sex change-annealing-three steps of extension are reduced to high-temperature denatured and annealing extended for two steps, annealing and extension unite two into one, and can finish the PCR reaction under the condition of annealing of Standard PC R head and shoulders above and the ultimate value of extending.Annealing temperature is 72-82 ℃, and elongating temperature also is 72 ℃-82 ℃, is preferably 75-80 ℃.The present invention is applicable to Standard PC R and reverse transcription PCR, also can be used to compete quantitative PCR and real-time fluorescence quantitative PCR reduces non-single-minded product and forms.Can also be used for multiplex PCR, nested PCR and other are had wide practical use by the various improvement PCR method of Standard PC R deutero-.

For understanding essence of the present invention and advantage, following ten genes of picked at random carry out test analysis:

Table one

	The gene title	The gene pool numbering	Sequence length (bp)
				A	People's glycerol-3-phosphate dehydrogenase mRNA	XM-006959.3	1282
B	The full mRNA of people beta-actomyosin	BC016045.1	1841
				C	The full mRNA of people Theta-protein kinase C	L07032.1	2705
D	Human serine-full the mRNA of threonine kinase CLIKI	AF195023.1	1806
				E	The full mRNA of people's neuroglobulin	AF422797.1	1909
F	People's fibroblast growth factor acceptor 3 full mRNA	AF245114.1	2184
				G	Human tumor inhibiting protein p53 gene extron 2-9	AF136270.1	3407
H	People Cyclin D1	NM-05306	4306
				I	People's embryo brain adenylate cyclase 3 ' end mRNA	L05500.1	2731
J	People gamma-changes glutaminase 1 full mRNA	BC025927	2347

Ten mrna length from 1282 to 4306 alkali bases, mean length is 2451 alkali bases, adopt widely used commercialization PCR primer-design software Oligo6 to carry out test analysis, this software with the primer preciseness be divided into very high, high, medium, can, low and very low six classes, preciseness is high more good more.Wherein glycero-3-phosphate deoxygenase gene (1282 alkali base) test result sees the following form:

Table two has the frequency of occurrences of the general primer and the ultra-long primer of different preciseness

Primer	The preciseness classification	Set PCR product length range
											150-250	150-300	150-400	150-500	150-600	150-800	150-1000
General primer (20 alkali bases)	Very high	3	7	13	14	23	31	37
									High	324	527	899	1257	1459	2084	2353
	Medium	10,245	14,944	23,809	30,950	37,900	49,025	More than 50,000
									Can	20,397	29,627	46,356	More than 50,000	More than 50,000	More than 50,000	More than 50,000
	Low	30,005	43,940	More than 50,000	More than 50,000	More than 50,000	More than 50,000	More than 50,000
									Very low	39,728	More than 50,000	More than 50,000	More than 50,000	More than 50,000	More than 50,000	More than 50,000
Ultra-long primer (40 alkali bases)									Very high	0	0	0	0	0	0	0
	High	0	0	4	14	14	44	44
									Medium	176	284	509	558	661	991	1465
	Can	757	1,201	2,001	2,729	3,266	4,295	5,226
									Low	2,129	3,382	5,449	7,292	8,712	11,176	13,671
	Very low	4,760	7,082	11,171	15,192	18,281	23,758	27,503

Obviously, the primer frequency of occurrences increases with preciseness and significantly descends, and the frequency of occurrences of the logical primer of 20 alkali kip sharply descends when forwarding very high preciseness to by high preciseness, and its quantity is in being convenient to the scope that actually operating selects; The frequency of occurrences of 40 alkali base ultra-long primers sharply descends when then forwarding high preciseness to by medium preciseness.Though the frequency of occurrences with ultra-long primer of very high preciseness is zero.But the ultra-long primer with high preciseness has some amount.And press the actomyosin that randomly assigne is selected, the statistics of the all-cis preface test of listed ten mRNA of tables one such as protein kinase C sees the following form.

Table three, has the frequency of occurrences of the primer of very high preciseness and high preciseness

(the PCR product is set at 150-800 alkali base)

The primer preciseness	Primer length	Minimum value	Maximum value	Mean value	Standard error
						Very high	15	291	1640	665	467
18	71	560	212	183
					20		24	472	123	144
22	1	332	64	103
					25		0	152	23	46
30	0	26	3	8
					High		15	5672	24564	13975	6199
20	1284	13651	6383	3718
						25	591	4920	2283	1575
30	97	1563	771	584
						35	6	615	245	215
40	0	314	79	96
						45	0	132	25	41
50	0	58	7	18

The frequency of occurrences of the very high preciseness primer of test shows descends rapidly when 15 are increased to 30 alkali bases.Average each gene can search has more than 100 of the long primers of very high preciseness 20 alkali bases.Except that a gene, all search less than having the long primer of very high preciseness 30 alkali bases.Though it is very high to have the frequency of occurrences of the long primer of very high preciseness 15 alkali bases, the Tm of these primers is on the low side, and specificity is not enough; Have the long primer of very high preciseness 18-25 alkali base, existing certain quantity is available higher Tm and specificity again, thereby now is acknowledged as best primer length scope.And the present invention finds when primer length is set at 31-50 alkali base, though be difficult to search the primer with very high preciseness, almost each tested person gene can both obtain the primer that some amount has high preciseness.Average each gene can search has 79 of the long primers of high preciseness 40 alkali bases.Preciseness standard with PCR primer software Oligo6 judges that these have the seemingly too late general primer with very high preciseness of ultra-long primers of high preciseness, thereby have been left in the basket always since the round pcr invention.The present invention then analyzes theoretically and what time following proof is by experiment:

One, the over-all properties with high preciseness ultra-long primer of the present invention is better than having the existing general primer of very high preciseness.

Table four ultra-long primer PCR of the present invention and current standards PCR principal character synopsis

Project	Current standards PCR	Ultra-long primer PCR of the present invention
			Common primer length	15-30 alkali base	31-50 alkali base
Primer melting temperature(Tm) (Tm) under the common PCR condition	55-82℃	82-92℃
			Product Tm and primer Tm's is poor under the common PCR condition	10-35℃	-4-10℃
Common PCR circulation step	3	2
			Common PCR denaturation temperature	90-95℃	95-98℃
Common PCR annealing temperature	45-65℃	72-82℃
			Common PCR elongating temperature	68-72℃	72-82℃

Two, the chain melting temperatur (Tm) of ultra-long primer of the present invention improves

Melting temperature(Tm) of oligonucleotide (Tm) and chain length, alkali base are formed, and arrange relevant with solution properties etc.Long during for 31-50 when primer alkali base, the Tm of primer under PCR reaction conditions usually is between 82 ° to 92 ℃, and than the high 15-30 of Tm temperature range ℃ of general primer, the annealing temperature between can 72 ℃ to 82 ℃ also extends.Annealing is extended in this temperature range, has both overcome the formation of non-single-minded product and primer dipolymer, makes the Taq enzyme remain stable again and shows higher enzyme activity.

Three, the single-minded bonding strength height of ultra-long primer of the present invention and target, mispairing bonded intensity is constant, and the clean difference of the two is big

Putting in order of a Nucleotide that contains 20 alkali bases above 1,000,000,000,000 kinds, this numerical value is the hundred times of human genome total length, so the possibility that runs into the identical nonstandard target DNA template of order for 20 alkali base long primers of a setting has only more than one percent.Two primer orders of PCR reaction needed are all mated, and matching order must compare near just finishing.From this angle analysis, non-single-minded PCR product may form hardly.Its reason is that the PCR reaction will take place when the mispairing of primer and target and non-target reaches certain intensity.Obviously, the single-minded bonding strength of primer and target is high more good more, and the mispairing bonding strength of primer and target and non-target is low more good more, and the single-minded combination of primer is the bigger the better with the clean difference of mispairing bonded.These characteristics of ultra-long primer and general primer have been compared in the present invention's test, and the statistical results show ultra-long primer is significantly superior than homology general primer.The present invention by random fashion in glycero-3-phosphate dehydrogenase enzyme gene, choose G+C% be respectively 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70% and 75% contain 20 alkali base oligonucleotide each 5 as general primer; Extend 20 alkali bases at these oligonucleotide 5 ' end then and obtain containing the oligonucleotide of 40 alkali bases as ultra-long primer.Combine and the test of mispairing bonding strength is calculated statistics and listed in table five from 50 primers of glycero-3-phosphate dehydrogenase enzyme gene picked at random and target are single-minded.

Table five general primer and the ultra-long primer and the target bonded characteristic of extending at 5 ' end

Item compared	Primer length	Minimum value	Maximum value	Mean value	Standard error
						Primer combines with target is single-minded	20	328	569	426	77
40	462	734	574	79
					Primer combines with the target mispairing		20	34	246	132	52
40	63	259	138	48
						With target single-minded combine and mispairing in conjunction with poor	20	149	445	294	78
40	261	594	435	87
					The single-minded bonding strength of primer poor		40 primers-20 primer	124	191	147	18
Primer mispairing bonding strength poor	40 primers-20 primer	-60	52	6		21

Data show that 50 ultra-long primers and target bonded average intensity are 574 points, considerably beyond standard 360 points to very high preciseness primer defined, also than high about 150 points of the mean value of homologous general primer.Though the single-minded bonded intensity of ultra-long primer is far above general primer, the mispairing of ultra-long primer in conjunction with mean value than only high 6 points of general primer.The single-minded combination of ultra-long primer combines difference average out to 435 points with mispairing, considerably beyond desired 200 points of very high preciseness primer, also than high 140 multiple spots of homology general primer mean value.More valuable is that the mispairing intensity of many ultra-long primers does not only increase on the contrary and reduces to some extent, have in addition than low 50 multiple spots of homology general primer, indivedual single-minded combinations of ultra-long primer are clean poor up to 600 points with the mispairing bonding strength.

Four, the mispairing bonding strength of ultra-long primer of the present invention and non-target and general primer is close

In the PCR reactive system content of nonstandard target DNA often template DNA several ten thousand to several thousands of times.Owing to the restriction of primer-design software, do not understand the mispairing intensity of primer and nonstandard target DNA during the design primer.Though application BLAST etc. can know the homology situation of primer order, the mispairing bonding strength is not to be determined by the homology degree simply, but depends on homology sequence length, and Nucleotide is formed and arranged, particularly 3 ' end situation in proper order.The mispairing bonding strength of ultra-long primer and general primer and non-target has been compared in the present invention's test.Choose 11 oligonucleotide that contain 20 alkali bases as general primer in mode at random from glycero-3-phosphate dehydrogenase enzyme gene.Equally, obtain 11 homologous at 20 alkali bases of its 5 ' end prolongation and contain the oligonucleotide of 40 alkali bases as ultra-long primer.Getting the long nucleic acid of one section 20,000 alkali base from one the 4th chromosomal clone tests as non-target.The statistics and the test result shown in the table five of listing in table six are in full accord, and promptly ultra-long primer is more much higher than general primer with the single-minded bonding strength of target, and with about the mispairing bonded mean value of target and non-target is compared slightly with general primer.

Table six general primer with 5 ' end extends ultra-long primer and non-target mispairing bonding strength

The primer of 11 picked at random is to the test statistics result of the long non-target of 18,000 alkali bases

Item compared	Primer length	Minimum value	Maximum value	Mean value	Standard error
						Primer combines with target is single-minded	20	326	537	425	57
40	540	694	597	61
					Primer combines with the target mispairing		20	56	191	132	62
40	59	284	130	60
						Primer combines with the maximum mispairing of non-target	20	95	273	165	60
40	95	277	152	57
					Primer combines with the second largest mispairing of non-target		20	84	190	116	33
40	50	128	99	23
						Primer combines with the third-largest mispairing of non-target	20	72	123	97	16
40	47	120	91	20

Five, the Tm of ultra-long primer of the present invention is still higher when target contains sudden change

Might there be sudden change in the target DNA that is amplified in guiding region, ultra-long primer still has higher Tm in this case, thereby more superior than general primer.When there was a mispairing alkali base in one 20 alkali base oligonucleotide, the chain melting temperatur reduced about six degree, and 40 alkali base oligonucleotide then only reduce about three degree.

The present invention from the glycero-3-phosphate dehydrogenase acid gene seven G+C% of picked at random at the 20 alkali base oligonucleotide of 40-55% as general primer, extend 20 alkali bases at 5 ' end then and obtain containing 40 alkali base homology ultra-long primers.Calculate according to the most contiguous alkali base method, the average T m of general primer is 62 degree, has one to suddenly change then that Tm is reduced to 56 degree as target.Otherwise the average T m of ultra-long primer is 92 degree, its Tm of sudden change is arranged still up to 89 degree as target.So ultra-long primer still can be at higher annealing temperature when sudden change appears in target.

Six, it is less that the single-minded bonding strength of ultra-long primer of the present invention is subjected to the influence of target sudden change

The present invention has the long primer of very high preciseness 20 alkali bases one of picked at random and tests from some, and the single-minded bonding strength of this primer and target is 393 points.Extend 20 alkali bases at 5 ' end and obtain containing 40 alkali base homology ultra-long primers, this ultra-long primer and target bonding strength are 540 points.Sudden change is relevant closely with the position of sudden change to the influence of bonding strength, and is also relevant with the kind of sudden change alkali base.The single-minded bonding strength of primer and sudden change target when the present invention's test occurs in different positions when suddenling change.Three kinds of sudden changes of each position measurement are also averaged.The result shows, on the one hand, occurs in which position no matter suddenly change, the bonding strength of ultra-long primer and target all surpasses 300 points, when sudden change occur in apart from 3 ' the three, the four of end or during greater than any position of the 7th alkali base, the bonding strength of primer and target is all between 400 and 500; And under the kindred circumstances general primer 300 and 400 between.So, ultra-long primer to the sudden change tolerance than general primer height.In other words, if target contains a unknown mutation, ultra-long primer can be finished amplification under the condition than the low about 5-10 degree (promptly more than 65 ℃) of its highest annealing temperature (75-80 ℃), and keeps the PCR product more single-minded.Under the kindred circumstances, general primer must be annealed with suitable low temperature, and non-single-minded product disturbs and just becomes serious problems.On the other hand, distinguish allelotrope if desired, then ultra-long primer is also superior than common primer.When allelic mutation occurred in first or second of primer 3 ' end, the bonding strength difference of ultra-long primer and gene reached 200 multiple spots, can it be made a distinction with PCR in bigger annealing region.Ultra-long primer has more probability to run into sudden change because of it is long than its homology general primer, when sudden change occur in 3 of ultra-long primer ' end half, general primer also can run into.As occur in 5 of ultra-long primer ' end half, general primer has been avoided this sudden change, though ultra-long primer can not avoid, this sudden change to ultra-long primer single-minded in conjunction with the influence very little.

Seven, ultra-long primer of the present invention can relax the area requirement of G+C%

Usually require the C+G% of primer between 45 to 55 in the general primer design.When C+G% was lower than 45%, not only the Tm of primer was on the low side, and the single-minded bonding strength of primer and target does not often reach the required standard of very high preciseness primer.For example contain in the primer of 20 alkali bases, have only single-minded 360 points that reached very high preciseness primer defined that combine of a primer and target between 30% to 40% 15 of the G+C% of picked at random.Increase G+C% no doubt can increase Tm and reach and the single-minded bonded intensity of target, but has often also strengthened the secondary structure of primer.Ultra-long primer can under the relatively low situation of G+C%, still have sufficiently high Tm and with the single-minded bonded intensity of target.So the ultra-long primer design can be relaxed the requirement of G+C%.Also might design the primer that 3 ' end is rich in A and T, make the mispairing intensity of primer and target and non-target reduce the minimizing of favourable non-single-minded PCR product and elimination.

Eight, the over-all properties of ultra-long primer of the present invention obviously is better than general primer

The present invention has very high preciseness 20 alkali base long primers with the primer-design software search, the long 150-800 alkali of regulation PCR product base obtains 31 pairs of primers altogether and meets the requirements, according to the position and the product size of primer, 31 pairs of primers can be divided into 7 groups, get a pair of the representative for every group.With search 44 pairs of high preciseness 40 alkali base long primers with quadrat method, 44 pairs of primers can be divided into 4 groups, also get a pair of the representative for every group.Table eight has compared 7 pairs comprehensively and has had non-preciseness 20 alkali base long primers and 4 pairs of characteristics with high preciseness 40 alkali base long primers of growing tall.

Table seven has very high preciseness general primer and of the present inventionly has a high preciseness ultra-long primer performance comparison sheet

Item compared	General primer (20 alkali base)								Ultra-long primer (40 alkali base)
															1	2	3	4	5	6	7	On average	1	2	3	4	On average
Positive primer location	17	17	366	366	601	652	711		4	4	343	601
														The anti-primer position	654	507	505	721	970	972	975		602	342	1021	1011
PCR product length	657	507	159	375	389	340	275		638	378	706	450
														The positive primer 3 ' complementary alkali radix of end	2	2	2	2	2	2	0		0	0	0	2
The complementary alkali radix of anti-primer 3 ' end	2	2	2	2	0	3	2	3	2	0	2
												The complementary alkali radix of 3 ' end between primer	2	2	2	2	2	2	2	0	0		2	2
The complementary alkali radix of 5 ' end between primer	2	2	2	2	0	2	2	2	0	2	2
												Positive primer hair clip handle alkali radix	3	3	0	0	3	0	0		4		4	3	3
Anti-primer hair clip handle alkali radix	0	0	0	3	3	3	3	3	3	3	3
												Positive primer hairpin loop alkali radix	3	3	0	0	4	0	0			3	3	3	5
Anti-primer hairpin loop alkali radix	0	0	0	3	7	7	7	5	3	7	7
												Positive primer combines with target is single-minded	480	480	435	435	451	499	469	453		610	610	633	581	589
Anti-primer combines with target is single-minded	477	443	439	454	417	428	428	584	640	526	525
												The maximum mispairing of positive primer and normal chain	95	95	123	123	106	89	130		98	94	94	74	93		99
The maximum mispairing of positive primer and minus strand	107	107	66	66	78	91	151	57	57	102	88
												The maximum mispairing of anti-primer and normal chain	83	64	81	142	100	89	89	104		132	109	109
The maximum mispairing of anti-primer and minus strand	126	30	45	99	88	151	151	166	90	87	130
												Positive primer strand melting temperatur	68	68	69.4	69.4	70.4	76.1	74.1	70.4		87.1	87.1	92.9	91.7	89.7
Anti-primer chain melting temperatur	74.5	68.7	68.2	69.8	67.6	70.4	70.4	91.2	93.5	86.9	87.5
												PCR product chain melting temperatur	88.1	87.3	86.1	89	90.1	89.9	89.2		88.5	87.8	86.9	89.4	89.4		88.4
Maximum chain melting temperatur difference	20.1	19.3	16.7	19.6	19.7	19.5	18.8	18.1	0.7	-0.2	2.5									1.9						-1.3

As shown in Table 73 of 4 pairs of ultra-long primers ' and the complementary alkali radix of end, the alkali radix of hair clip handle and ring is about having mutually with the master data such as the mispairing intensity of target and the 7 pairs of general primers are identical or very approaching.And at primer Tm, primer Tm and the clean difference of PCR product Tm and with aspect such as the single-minded bonding strength of target, ultra-long primer obviously is better than general primer.

Description of drawings

Fig. 1 is a general primer PCR product electrophoretic band photo (annealing temperature 52-76 ℃)

Fig. 2 is a ultra-long primer PCR product electrophoretic band photo (annealing temperature 52-76 ℃)

Fig. 3 is a ultra-long primer PCR product electrophoretic band photo (annealing temperature 74-80 ℃)

Fig. 4 is ultra-long primer PCR product electrophoretic band photo (5,10,20, the 30 seconds extension time of annealing)

Fig. 5 is a ultra-long primer PCR product electrophoretic band photo (Taq enzyme dosage 100%, 50%, 40%, 33%, 25%).

Fig. 6 is ultra-long primer PCR product electrophoretic band photo (68 ℃, 70.3 ℃ of an annealing temperature), (glycerol concentration 0%, 10%, 15%, 30%).

Fig. 7 is ultra-long primer PCR product electrophoretic band photo (72.9 ℃, 74.8 ℃ of an annealing temperature), (glycerol concentration 0%, 10%, 15%, 20%).

Fig. 8 is ultra-long primer PCR product electrophoretic band photo (72 ℃-80 ℃ of an annealing temperature).

Embodiment

Embodiments of the present invention and existing PCR are basic identical, and its necessary composition still is hot resistant DNA polymerase, four kinds of deoxyribonucleotides, magnesium ion, damping fluid, target DNA and a pair of positive and negative primers.Special feature is that a pair of positive and negative primer is the ultra-long primer that length is 31-50 alkali base.But preferred length 35-45 alkali base, and its PCR circulation step is high-temperature denatured and annealing extended for two steps, annealing temperature and elongating temperature are 72-82 ℃.Following table is the composition of embodiment PCR reaction solution.

The composition of table eight 25 microlitre PCR reaction tubess is formed

Reagent	Dosage	Ultimate density
			The 10X reaction buffer	2.5 microlitre	40mM Tricine-KOH pH8.7，15mM KOAc
Four kinds of deoxynucleoside acid mixtures of 10mM	0.5 microlitre	0.2mM
			35mM magnesium	2.5 microlitre	3.5mM
The 50XTaqDNA polysaccharase	0.5 microlitre	The IXTaq archaeal dna polymerase
			The positive primer of 10uM	2.5 microlitre	IuM
The 10uM anti-primer	2.5 microlitre	IuM

The used DNA of embodiment of the invention PCR is cDNA, is to synthesize with commodity reverse transcription test kit from commodity people total tissue RNA.Unless point out separately, per 20 microlitre PCR reaction solutions add the cDNA that is equivalent to by the total RNA generation of 10ng.The used PCR reagent of the embodiment of the invention is the commodity PCR test kit that contains the Taq enzyme antibody.PCR sex change first be 95.5 ℃ 1 minute, circulation sex change condition except that pointing out separately, be 95.5 ℃ 30 seconds.PCR and electrophoresis equipment are respectively the commodity PCR instrument and the vertical plate electrophoresis of the commodity polyacrylamide system of band gradient function, and running gel dyes with ethidium bromide or other commodity nucleic acid staining agent.PCR product band record analysis commodity image analyzer.Embodiment of the invention institute drawings attached photo sees that Fig. 1-Fig. 8 leftmost side is standard DNA, and center strip is 500 alkali bases, and other bands are decremented to 100 alkali bases or are incremented to 1000 alkali bases by 100 alkali bases.The ultra-long primer length of three embodiment PCR of the present invention is respectively embodiment 1, forward 39, reverse 37; Embodiment 2, forward 31, reverse 31; Embodiment 3, forward 49, reverse 46.

The length of the ultra-long primer of table nine embodiment 1,2,3 and order

	Templet gene	Primer length	Primer location	Primer order (5 ' to 3 ')
					Embodiment 1	The glycero-3-phosphate dehydrogenase enzyme	Forward 39	553-591	CTG GCC AAC CTC ATC CAT GAC AAC TTT GGT ATC GTG GAA
Reverse 37	904-940	CGC TGT TGA AGT CAG AGG AGA CCA CCT GGT GCT CAG T
			Embodiment 2	The Beta-actomyosin			Forward 31	199-228	GGC GTG ATG GTG GGC ATG GGT CAG AAG GT
Reverse 31	666-695	TCC CGC TCG GCC GTG GTG GTG AAG CTG TAG
					Embodiment 3	Adenylate cyclase	Forward 49	1078-1126	TCC TGC TAG TAT TCT GCA TCT GCT TCC TGG TGG CCT GTG TCC TGT ACC T
Reverse 46	1787-1832	CCC ATG TTG TTG CCG TCC AGC TCG ATG TAG AAG TCA TTC AAG TTG

Every data of other of primer and characteristic are listed in table ten.

The relevant data and the characteristic of the ultra-long primer of table ten embodiment 1,2,3 PCR

Project	General primer		The ultra-long primer of embodiment 1		The ultra-long primer of embodiment 2		The ultra-long primer of embodiment 3
									Forward	Oppositely	Forward	Oppositely	Forward	Oppositely	Forward	Oppositely
	Primer location	573-591	904-920	553-591	904-940	199-228	666-695	1078-1126									1787-1832
Primer length									19	17	39	37	31	31	49	46
	PCR product length	348		348		497		754
Primer G+C%										42	59	48.7	56.8	60	66.7	53.1	18.9
	Primer Tm under the PCR condition	63.0	63.2	89.1	88.9	88.7	90.7	92	91.3
PCR product Tm										90.2		90.2		91.4		91.1
	Primer and PCR product Tm are poor	27.2	27.0	1.1	1.3	2.7	0.7	-0.9	-0.2
3 ' end pairing alkali radix										2	2	2	2	2	2	2	2
	The most stable pairing alkali of complete sequence radix	3	4	6	4	4	6	4	4
3 ' end pairing alkali radix between primer										no		3	2	3	n0	n0	2	2
	The most stable pairing alkali radix between primer	4		4		3		4
The most stable hair clip handle alkali radix										＜2	3	3	3	3	3	3	3
	With the target bonding strength	383	353	569	525	547	591	596	576
With the strongest mispairing bonding strength of normal chain										134	81	66	73	89	70	110	61
	With the strongest mispairing bonding strength of minus strand	105	117	122	76	102	82	122	77

1) the highest annealing temperature and elongating temperature

Embodiment has tested in different annealing temperature (52-76) formation of PCR product down.Fig. 1 and Fig. 2 are respectively the test-results that embodiment 1 uses common and ultra-long primer.Annealing temperature is respectively 52,55.8,58.6,62,66,69.4,72.1,74.1 and 76 ℃ from left to right, annealing time 1 minute, extend to 76 ℃ 1 minute.Fig. 1 illustrates that using general primer makes PCR, and the product that just responds when annealing temperature is 52 to 62 ℃ forms; When annealing temperature does not just have the product band to be detected greater than 69.4 ℃.Fig. 2 illustrates that using ultra-long primer makes PCR, and annealing temperature all has the PCR product to form for from 52 to 76 ℃.Advance a test ultra-long primer in higher annealing temperature (74-80 ℃) formation of PCR product down, among Fig. 3 from left to right annealing temperature be respectively 74,74.5,75,75.7,76.7,77.7,78.5,79.1 and 80 ℃, annealing time is 1 minute, extend to 80 ℃ 20 seconds.The result shows when annealing temperature still has the PCR product during near 78 ℃ and forms.Figure eight is the test-results that embodiment 3 uses ultra-long primer.Shown in figure eight, 78.8 ℃ have obvious product band, and 79.5 ℃ have faint product band, and showing can be 79 ℃ of annealing.

2) non-single-minded PCR product

As shown in Figure 1, use general primer and make PCR, have non-single-minded product band to form if annealing temperature is low, and if the higher target P CR product of annealing temperature just disappears.So the suitable annealing degree of moving back that can obtain single-minded PCR product is about 66 ℃, scope is quite narrow.When as shown in Figure 2, using extraordinary primer when annealing temperature during greater than 66 ℃ just single-minded nothing but product band occur.In very wide annealing region, can obtain pure PCR product.

3) formation of primer dipolymer

Being formed in the PCR reaction of primer dipolymer is quite general.Some experiments of the embodiment of the invention show that the primer dipolymer that all has when using general primer in various degree forms.Fig. 2 and Fig. 3 demonstration are used ultra-long primer and are made PCR, also have the primer dipolymer to form under common annealing temperature, just weaken greatly or elimination fully but annealing temperature is improved 70 ℃ of left and right sides primer dipolymers formation.

4) PCR stability and repeated

Ultra-long primer PCR all adopts two step method, simplifies than traditional three-step approach.And the difference of sex change and annealing elongating temperature only 15 to 20 the degree, far below the 30-50 degree of conventional P CR.Annealing and extension under high temperature have have not only reduced or eliminated non-single-minded band and primer dipolymer and have formed, and also make the stability and the repeatability raising of PCR reaction, have reduced the error between the tube and tube.The embodiment of the invention has been made 36 replications altogether.Test divides to be carried out for 4 times, and each test contains 9 and repeats pipe, and the PCR cycle number is respectively 25,28,29 and 32.General primer PCR was 62 ℃ of annealing 30 seconds, and 72 ℃ were extended 1 minute; Ultra-long primer PCR extended 1 minute 76 ℃ of annealing.The relative scanning mean value of each test is 11.11, and the standard error of 4 tests of general primer is respectively 0.73,0.75,0.91 and 1.28; Be respectively 0.43,0.38,0.48 and 0.52 with the standard error of parallel 4 the ultra-long primer PCR tests of general primer PCR.The stability of these presentation of results ultra-long primers PCR is better than general primer.Ultra-long primer PCR can fix and use two step method, and the design of PCR instrument also can be simpler.

5) the PCR reaction times

The optimum temperuture of Taq enzyme is between 75-80 ℃, and the extension speed of this moment is 70 ℃ more than one times.The elongating temperature of conventional P CR is 68-72 ℃, may cause unwinding as extending under at higher temperature.Ultra-long primer PCR then can extend between 75-80 ℃, and the maximum vigor of performance Taq enzyme shortens the PCR reaction times.The embodiment of the invention has been tested ultra-long primer under 74 ℃ and 76 ℃ of annealing and elongating temperature, finishes the PCR required time.Extension time of annealing from left to right among Fig. 4 was respectively 5,10,20 and 30 seconds.Test shows to anneal extends and can finish in 20 seconds.The test the sex change condition be 98 ℃ 10 seconds.Each round-robin calculated value only is 30 seconds.In fact in 32 minutes, finished the whole PCR of 30 round-robin reaction, be Standard PC R required time 1/4th to half.

6) Taq enzyme dosage

From the practical application angle, the Taq enzyme has no difference in stability between 75-80 ℃ and the stability between 40-72 ℃.Between 68-72 ℃, extend the enzyme activity of having brought into play Taq in ratio of elongation between 75-80 ℃ biglyyer.So, if keep the common extension time that enzyme dosage is reduced.The embodiment of the invention has been tested and has been finished PCR and react needed enzyme concentration.Fig. 6 is from the right side on a left side, and the Taq enzyme dosage is that test kit is recommended consumption, also is 100%, 50%, 40%, 33% and 25% of conventional amount used of the present invention.The annealing extension time is 1 minute.In the scope of test, all show close PCR product band.

The Tm of primer is given in the multiplication of primer length, with the difference of PCR product Tm, and with the single-minded bonded intensity of target, the variation that very big amplitude has been brought in aspects such as annealing and elongating temperature.Except above test statistics analysis and the illustrated content of embodiment, ultra-long primer PCR is inevitable in the PCR detection sensitivity, magnesium ion concentration scope, the influence of total ionic strength adjustment buffer degree, the effect of Taq enzyme inhibitors, aspects such as template DNA purity requirement produce the variation that deep favourable PCR improves and uses.

Claims (5)

1, uses ultra-long primer instead to improve the polymerase chain reaction method of gene amplification efficacy, it is characterized in that the primer in the system of said polymerase chain reaction uses the positive and negative primer that a pair of length is the 31-50 base instead, the chain melting temperatur of positive and negative primer is 82-92 ℃, its PCR circulation is sex change and two steps of annealing extension, and the annealing elongating temperature is 72-82 ℃.

2, polymerase chain reaction method according to claim 1, it is characterized in that said a pair of positive and negative primer and the complete complementation of target order maybe can have indivedual base mismatch, the difference that the chain melting temperatur of PCR product subtracts positive and negative primer strand melting temperatur is-4-10 ℃.

3, polymerase chain reaction method according to claim 1 is characterized in that said a pair of positive and negative primer length is the 35-45 base.

4, polymerase chain reaction method according to claim 1 is characterized in that the chain melting temperatur of said a pair of positive and negative primer is 85-90 ℃.

5, polymerase chain reaction method according to claim 1 is characterized in that said annealing elongating temperature is 75-80 ℃.

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