CN108220411A - A kind of PCR primer and detection method for detecting human gene CEBPA - Google Patents
A kind of PCR primer and detection method for detecting human gene CEBPA Download PDFInfo
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Abstract
The present invention provides two pairs of PCR primers, 60 DEG C of the PCR primer annealing temperatures；Forward and reverse primer Tm difference is no more than 2 DEG C；Target fragment length is 867bp and 632bp；19 21bp of primer length；G/C content is 40 60%, and base is in random distribution situation；Its specificity is good simultaneously, and PCR product does not form secondary structure, is particularly suitable for the PCR amplification for CEBPA genes.On the basis of this, the present invention develops detection method for CEBPA genes, while realize the verification to primer performance around primer feature based on first generation gene sequencing technology.This method has carried out brand-new design to reaction system ingredient, PCR reaction conditions, products therefrom is verified by agarose gel electrophoresis finds that target gene has clear band, it is found after gel extraction through purifying, sequencing analysis, product is definitely CEBPA genes, it was demonstrated that experimental result is good.
Description
Technical field
The present invention relates to technical field of molecular biology, further to genetic test and PCR primer designing technique, specifically
It is related to a kind of PCR primer and detection method for detecting human gene CEBPA.
Background technology
It is the important transcription factor for maintaining the differentiation of hemopoietic system grain system to enhance bindin alpha, is adjusting cell Proliferation with dividing
Crucial effect is played in the balance of change.This transcription factor contains 358 amino acid, is encoded by gene C EBPA.Gene C EBPA
It is positioned at 19q 13l, overall length 3318bp, intronless, cDNA overall lengths 2385bp includes multiple translation initiation sites.Its structure packet
It includes：The abundant dimerization functional areas of the leucine in the transcriptional activity area at N- ends, DNA combined areas and C- ends.Under normal circumstances with complete
Based on long 42KD albumen, a small amount of 30KD albumen, the change that the change of ratio will lead to its function therebetween are existed simultaneously.First
It is one of main method for detecting gene C EBPA for gene sequencing (Sanger methods).
Sanger method gene sequencing is to extend the primer being incorporated on sequence template undetermined using a kind of archaeal dna polymerase,
Until mixing a kind of terminating nucleotide with fluorescent marker, analyzed by capturing fluorescence, finally obtain a system
The peak figure of row is analyzed it can be seen that its sequence.
Since CEBPA gene G/C contents are higher, PCR amplification target fragment easily generates nonspecific products, causes to be sequenced
Of poor quality, experimentation is complicated；And gene order is longer, the logical the primer number of survey completely is more, increases experimental implementation number；
Required reagent cost is higher.
Invention content
The present invention is directed to be directed to the technological deficiency of the prior art, provide a kind of PCR primer for detecting human gene CEBPA and
Detection method, to solve to utilize required primer a fairly large number of skill during the progress CEBPA genetic tests of Sanger methods in the prior art
Art problem.
Another technical problem to be solved by the present invention is that lead to CEBPA genes since primer performance is bad in the prior art
Detection method is cumbersome.
For realization more than technical purpose, the present invention uses following technical scheme：
A kind of PCR primer for detecting human gene CEBPA, as forward primer and sequence of the sequence as shown in SEQ ID NO.1
Arrange the reverse primer as shown in SEQ ID NO.2.
Preferably, the PCR primer annealing temperature is 60 DEG C；Forward and reverse primer Tm difference is no more than 2 DEG C；Target fragment
Length is 867bp and 632bp；Primer length 19-21bp；G/C content is 40-60%.
The PCR primer of another kind detection human gene CEBPA, as forward primer of the sequence as shown in SEQ ID NO.3 and
Reverse primer composition of the sequence as shown in SEQ ID NO.4.
Preferably, the PCR primer annealing temperature is 60 DEG C；Forward and reverse primer Tm difference is no more than 2 DEG C；Target fragment
Length is 867bp and 632bp；Primer length 19-21bp；G/C content is 40-60%.
A kind of method using above-mentioned PCR primer detection human gene CEBPA, this method belong to Sanger PCR sequencing PCRs,
Middle PCR reactions include the following steps into program：95 DEG C of pre-degeneration 3min；95 DEG C of denaturation 30s, 52~70 DEG C of annealing 30s, 72 DEG C are prolonged
Stretch 70s, 32 cycles；In 72 DEG C of last extension 5min.
Preferably, it is 60 DEG C that PCR, which is reacted into annealing temperature in program,.
Preferably, the every 20.1 μ L of target fragment amplification reaction system contain following component in this method：GCracker
10 μ L of PCR Buffer, 9 μ L, GCracker PCR Enzyme of distilled water 0.1 μ L, 10 μM of each 0.5 μ L of forward and reverse primer,
The 0.5 μ L of DNA profiling of concentration 40~60ng/ μ L.
Preferably, wherein PCR reactions are into after program finishes execution, to PCR reaction products into row agarose gel electrophoresis
Experiment, the reaction condition of the agarose gel electrophoresis experiment are：2.5% Ago-Gel adds in the DNA of 2000bp
Marker, constant pressure 140V, electrophoresis 30min.
The present invention provides two pairs of PCR primers, the design of the primer is found by human genome browser UCSC
CEBPA gene orders design two pairs of primers using it as according to first with IDT PrimerQuest Tool, then it are carried out
Blast is verified, primer synthesis is carried out after verification, and universal primer M13F/M13R is then recycled to be obtained after modifying its 5 ' end
It arrives.60 DEG C of the PCR primer annealing temperature；Forward and reverse primer Tm difference is no more than 2 DEG C；Target fragment length for 867bp and
632bp；Primer length 19-21bp；G/C content is 40-60%, and base is in random distribution situation；Its specificity is good simultaneously, PCR
Product does not form secondary structure, is particularly suitable for the PCR amplification for CEBPA genes.
On this basis, the present invention is developed based on first generation gene sequencing technology for CEBPA bases around primer feature
The detection method of cause, while realize the verification to primer performance.This method carries out reaction system ingredient, PCR reaction conditions
Brand-new design, products therefrom is verified by agarose gel electrophoresis finds that target gene has clear band, is passed through after gel extraction
Purifying, sequencing analysis find that product is definitely CEBPA genes, it was demonstrated that experimental result is good.
In above scheme, " human genome browser UCSC finds CEBPA gene orders " refers in title
For the CEBPA genes in database in the website of " UCSC ", searched from the genome of entitled Feb.2009 (GRCh37/hg19)
Sequence, network address http://genome-asia.ucsc.edu/cgi-bin/hgGatewayRedirect=manual＆
Source=genome.uc sc.edu.
In above scheme, " the IDT PrimerQuest Tool " refer on the website of entitled " IDT "
Line design of primers program, network address http://sg.idtdna.com/Primerquest/Home/Index.
In above scheme, the CEBPA gene orders refer to whole base sequences of human genome CEBPA.
In above scheme, primer synthesis, is conventional Primed synthesis method, and specific implementation can be
Foundation this field routine techniques is implemented on the basis of specifying its sequence, common biotechnology company can also be entrusted to be closed certainly
Into.
In above technical scheme, described pair is modified 5 ' ends, is referred at 5 ' ends of designed forward and reverse primer
Universal primer sequence M13F/M13R is introduced respectively.
The sequence of the universal primer M13F is：TGTAAAACGACGGCCAGT；The sequence of the M13R is：
CAGGAAACAGCTATGACC。
Description of the drawings
Fig. 1 is the electrophoretogram of pcr amplification product in embodiment 1；
Fig. 2 is the electrophoretogram of pcr amplification product in embodiment 2.
Specific embodiment
The specific embodiment of the present invention will be described in detail below.In order to avoid excessive unnecessary details,
It will not be described in detail in following embodiment to belonging to well known structure or function.In addition to being defined, institute in following embodiment
Technical and scientific term has the identical meanings being commonly understood by with those skilled in the art of the invention.
Embodiment 1
A kind of PCR primer for detecting human gene CEBPA, as forward primer and sequence of the sequence as shown in SEQ ID NO.1
Arrange the reverse primer composition as shown in SEQ ID NO.2.In the primer pair, forward and reverse primer Tm difference is no more than 2 DEG C；
Target fragment length is 867bp and 632bp；Primer length 19-21bp；G/C content is 40-60%.
Utilization more than PCR primer verifies its PCR reaction effect by the following method：
The every 20.1 μ L of target fragment amplification reaction system contain following component：GCracker PCR Buffer 10 μ L, it is double
Steam 9 μ L, GCracker PCR Enzyme of water, 0.1 μ L, the DNA moulds of 10 μM of forward and reverse primer each 0.5 μ L, concentration 60ng/ μ L
0.5 μ L of plate.
PCR reactions include the following steps into program：95 DEG C of pre-degeneration 3min；95 DEG C denaturation 30s, 60 DEG C annealing 30s, 72 DEG C
Extend 70s, 32 cycles；In 72 DEG C of last extension 5min.
PCR is reacted into after program finishes execution, PCR reaction products is tested into row agarose gel electrophoresis, the agar
The reaction condition of sugared gel electrophoresis experiment is：2.5% Ago-Gel adds in the DNA Marker of 2000bp, constant pressure 140V, electricity
Swim 30min.As shown in Figure 1, condensation electrophoretogram in target gene CEBPA have clear band, it was demonstrated that using the primer by with
Upper method detection CEBPA genes work well.
Embodiment 2
A kind of PCR primer for detecting human gene CEBPA, as forward primer and sequence of the sequence as shown in SEQ ID NO.3
Arrange the reverse primer composition as shown in SEQ ID NO.4.In the primer pair, forward and reverse primer Tm difference is no more than 2 DEG C；
Target fragment length is 867bp and 632bp；Primer length 19-21bp；G/C content is 40-60%.
Utilization more than PCR primer verifies its PCR reaction effect by the following method：
The every 20.1 μ L of target fragment amplification reaction system contain following component：GCracker PCR Buffer 10 μ L, it is double
Steam 9 μ L, GCracker PCR Enzyme of water, 0.1 μ L, the DNA moulds of 10 μM of forward and reverse primer each 0.5 μ L, concentration 40ng/ μ L
0.5 μ L of plate.
PCR reactions include the following steps into program：95 DEG C of pre-degeneration 3min；95 DEG C denaturation 30s, 60 DEG C annealing 30s, 72 DEG C
Extend 70s, 32 cycles；In 72 DEG C of last extension 5min.
PCR is reacted into after program finishes execution, and PCR reaction products are tested into row agarose gel electrophoresis.Such as Fig. 2 institutes
Show, condensing target gene CEBPA in electrophoretogram has clear band, it was demonstrated that detects CEBPA by above method using the primer
Gene works well.
Embodiment 3
A kind of PCR primer for detecting human gene CEBPA, as forward primer and sequence of the sequence as shown in SEQ ID NO.1
Arrange the reverse primer composition as shown in SEQ ID NO.2.
Utilization more than PCR primer is included the following steps by PCR reaction detection CEBPA genes, wherein PCR response procedures：
95 DEG C of pre-degeneration 3min；95 DEG C of denaturation 30s, 70 DEG C of annealing 30s, 72 DEG C of extension 70s, 32 recycle；In 72 DEG C of last extensions
5min。
The embodiment of the present invention is described in detail above, but the content is only presently preferred embodiments of the present invention,
It is not intended to limit the invention.All all any modification, equivalent and improvement done in the application range of the present invention etc., should all
It is included within protection scope of the present invention.
Claims (8)
1. a kind of PCR primer for detecting human gene CEBPA, as forward primer and sequence of the sequence as shown in SEQ ID NO.1
Reverse primer composition as shown in SEQ ID NO.2.
2. PCR primer according to claim 1, it is characterised in that the PCR primer annealing temperature is 60 DEG C；Forward and reverse primer
Tm values difference is no more than 2 DEG C；Target fragment length is 867bp and 632bp；Primer length 19-21bp；G/C content is 40-60%.
3. a kind of PCR primer for detecting human gene CEBPA, as forward primer and sequence of the sequence as shown in SEQ ID NO.3
Reverse primer composition as shown in SEQ ID NO.4.
4. PCR primer according to claim 3, it is characterised in that the PCR primer annealing temperature is 60 DEG C；Forward and reverse primer
Tm values difference is no more than 2 DEG C；Target fragment length is 867bp and 632bp；Primer length 19-21bp；G/C content is 40-60%.
A kind of 5. method using the PCR primer detection human gene CEBPA of claim 1 or 3, it is characterised in that this method
Belong to Sanger PCR sequencing PCRs, wherein PCR reactions include the following steps into program：95 DEG C of pre-degeneration 3min；95 DEG C denaturation 30s, 52
~70 DEG C of annealing 30s, 72 DEG C of extension 70s, 32 recycle；In 72 DEG C of last extension 5min.
6. the method for detection human gene CEBPA according to claim 5, it is characterised in that PCR is moved back in reacting into program
Fiery temperature is 60 DEG C.
7. the method for detection human gene CEBPA according to claim 5, it is characterised in that target fragment expands in this method
Increase the every 20.1 μ L of reaction system and contain following component：10 μ L of GCracker PCR Buffer, distilled water 9 μ L, GCracker
PCR Enzyme 0.1 μ L, the 0.5 μ L of DNA profiling of 10 μM of forward and reverse primer each 0.5 μ L, concentration 40~60ng/ μ L.
8. the method for detection human gene CEBPA according to claim 5, it is characterised in that wherein PCR is reacted into program
After being finished, PCR reaction products are tested into row agarose gel electrophoresis, the reaction item of the agarose gel electrophoresis experiment
Part is：2.5% Ago-Gel adds in the DNA Marker of 2000bp, constant pressure 140V, electrophoresis 30min.
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Cited By (1)
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| CN112575071A (en) * | 2020-12-25 | 2021-03-30 | 北京思尔成生物技术有限公司 | Method for directly performing Sanger sequencing on PCR amplification product without depending on purification |
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| CN112575071A (en) * | 2020-12-25 | 2021-03-30 | 北京思尔成生物技术有限公司 | Method for directly performing Sanger sequencing on PCR amplification product without depending on purification |
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Application publication date: 20180629 |