CN110527711A - A kind of rapid PCR amplification kit and its application method - Google Patents
A kind of rapid PCR amplification kit and its application method Download PDFInfo
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- CN110527711A CN110527711A CN201910504468.7A CN201910504468A CN110527711A CN 110527711 A CN110527711 A CN 110527711A CN 201910504468 A CN201910504468 A CN 201910504468A CN 110527711 A CN110527711 A CN 110527711A
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- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
It includes archaeal dna polymerase, dNTPs, reference gene primer group and buffer solution system that the present invention, which provides a kind of rapid PCR amplification kit and its application method, the kit,；The reference gene primer group includes internal reference A segment and internal reference B segment, and the internal reference A segment includes upstream primer F and downstream primer R；The internal reference B segment includes upstream primer F and downstream primer R.PCR amplification kit of the invention and its application method, Phire thermal starting II archaeal dna polymerase is used in combination, it can be achieved that more long segment target spot amplification, and error rate is lower；It is added to PCR reinforcing agent in kit, the higher structure of nucleotide sequence can be opened；And kit have the characteristics that it is convenient, saving fund, improve efficiency, one-step method PCR detection, realize minim DNA sample rapid amplifying.
Description
Technical field
The present invention relates to gene engineering technology field more particularly to a kind of rapid PCR amplification kits and its application method.
Background technique
PCR is the abbreviation of polymerase chain reaction, is a kind of for amplifying the molecular biology for expanding specific DNA fragmentation
Technology studies the influence to biology every field own profound, very close with the relationship of doctor, agricultural, bioengineering etc.,
Such as round pcr is related to the new way in agriculturally breeding, is related to medically detecting, treats genetic disease, is related to gene
Infant industry based on engineering is related to forensic identification, paternity test etc..Fast PCR includes nucleic acid Rapid extraction and PCR
Two parts are expanded, classical PCR amplification kit includes PCR buffer system, dNTPs, archaeal dna polymerase etc..Existing kit is
Different reagents can be carried out multigelation by preparation PCR reaction solution, weighed agarose every time and configured gel, and process is relatively cumbersome,
Experiment consumptive material is wasted simultaneously.
Currently, genomic DNA traditional extraction process is extracted DNA often contains more protein residue, polysaccharide
Pollution, the pollution of phenolic substances (such as DNA is brown) are the very stubborn problems being commonly encountered when extracting genomic DNA, and egg
The impurity such as white, polysaccharide and phenols have stronger inhibiting effect to downstreams experiments such as subsequent digestion, PCR reactions.However, for pole
Its minim DNA sample is difficult to capture effective DNA information since pcr amplification reaction susceptibility is low.For micro DNA sample
This, the low reaction susceptibility of conventional PCR amplification method is the main reason for causing subsequent PCR amplification effect poor.For example, in method
It cures in detection work, the identification and detection of the medical jurisprudence material evidence of scene of a crime, for timely and effectively accusing of offender, hits
Crime is played the role of vital.Fingerprint, stub, nail etc. all contain micro DNA information in scene of a crime, can be to find out
Or it accuses of suspect and scientific basis is provided.But since the DNA of scene of a crime remains little, collected trace forensic
DNA is extremely micro, and the reaction susceptibility of standard PCR amplification method is low, it is difficult to capture effective dna information to expanding effect compared with
Difference, it is low to directly result in subsequent DNA recall rate, and the failure of STR parting cannot point out crime individual.
How round pcr means are utilized, realizes that real-time, convenient, fast slowdown monitoring trace sample genetic mutation, including gene are dashed forward
The information such as change, missing, fusion have significant meaning, especially for clinical assistant diagnosis, the guidance of targeting therapeutic regimen
It is very high for technological difficulties how to realize in the amplification of ultramicron abnormal dna.
Summary of the invention
The purpose of the present invention is to solve disadvantages existing in the prior art, and a kind of realization ultramicron variation proposed
The rapid PCR amplification kit and its application method of the amplification of DNA are applicable to basic scientific research, clinical rapid PCR amplification etc. and use
On the way.
To achieve the above object, present invention employs following technical solutions:
A kind of rapid PCR amplification kit comprising archaeal dna polymerase, dNTPs, reference gene primer group and buffer
System；The reference gene primer group includes internal reference A segment and internal reference B segment, and the internal reference A segment includes upstream primer F
With downstream primer R；The internal reference B segment includes upstream primer F and downstream primer R；Wherein, the internal reference A segment it is described on
The nucleotides sequence of trip primers F is classified as SEQ ID NO:1, and the nucleotides sequence of the downstream primer R is classified as SEQ ID NO:2；It is described
The nucleotides sequence of the upstream primer F of internal reference B segment is classified as SEQ ID NO:3, and the nucleotides sequence of the downstream primer R is classified as
SEQ ID NO:4.
Preferably, the internal reference A clip size are as follows: 200-300bp, the internal reference B clip size are as follows: 1000-1100bp.
More preferably, the rapid PCR amplification kit further includes PCR reinforcing agent, and the PCR reinforcing agent includes
One or both of DMSO, glycerol, formamide.
More preferably, the working concentration of the DMSO is 1-10% (v/v), and the working concentration of the glycerol is 1-20%
(v/v), the working concentration of the formamide is 1-10% (v/v).
More preferably, the buffer solution system be 10 × buffer, including concentration be 500mmol/L Tris-HCl and
The MgCl of 20mmol/L2, the pH value of the Tris-HCl is 9.0.
More preferably, the concentration of the dNTPs is 2.5mmol/L；The DNA polymerase activity is 2.5U/ μ L.
More preferably, the concentration of the upstream primer F and the downstream primer R are 10 μm of ol/L.
More preferably, the archaeal dna polymerase is Phire thermal starting II archaeal dna polymerase.
Another object according to the present invention, the present invention also propose the application method of the rapid PCR amplification kit, answer
With the PCR reaction system of the rapid PCR amplification kit are as follows:
PCR amplification kit amplification condition are as follows: 95 DEG C of initial denaturation 3min, 95 DEG C of denaturation 10s, 60 DEG C of annealing 15s, 72 DEG C are prolonged
30s is stretched, extends 5min after 30 circulations.
Further preferably, the application method of the rapid PCR amplification kit further includes carrying out 1% agarose gel electrophoresis
Detection, burning voltage 130V, electrophoresis time 20 minutes.
Compared with prior art, the invention has the benefit that
1. Phire thermal starting II archaeal dna polymerase is used in combination in the present invention compared with prior art PCR amplification kit,
Containing enhancing archaeal dna polymerase amplification ability, the ingredient of extremely strong antidepressants ability is made it have, it can be achieved that more long segment target spot
Amplification, and error rate is lower；
2. compared with prior art, being added to PCR reinforcing agent DMSO, glycerol, one in formamide in kit of the present invention
Kind or two kinds, the higher structure of nucleotide sequence can be opened；
3. that rapid PCR amplification kit of the present invention has the characteristics that is convenient, saving fund, improves efficiency, a step
Method PCR detection, realizes minim DNA sample rapid amplifying.
Detailed description of the invention
Fig. 1 is one-step method PCR reference gene A fragment amplification testing result figure；
Fig. 2 is one-step method PCR reference gene B fragment amplification testing result figure.
Specific embodiment
To make to have further understanding to the purpose of the present invention, construction, feature and its function, hereby cooperate embodiment detailed
It is described as follows.
The object of the present invention is to provide a kind of rapid PCR amplification kit, the PCR kit include archaeal dna polymerase,
DNTPs, reference gene primer group, buffer solution system；The reference gene primer group includes (containing for expanding internal reference A segment
Upstream primer F and downstream primer R) and B segment (F containing upstream primer and downstream primer R), the sequence of the primer group see information
Table 1:
The nucleotides sequence of the upstream primer F of internal reference A segment is classified as SEQ ID NO:1, the downstream primer R's of internal reference A segment
Nucleotides sequence is classified as SEQ ID NO:2；
The nucleotides sequence of the upstream primer F of internal reference B segment is classified as SEQ ID NO:3, the downstream primer R's of internal reference B segment
Nucleotides sequence is classified as SEQ ID NO:4；
The present invention is to guarantee PCR efficient amplification, is optimized to archaeal dna polymerase, is polymerize using Phire thermal starting II DNA
Enzyme makes it have the ingredient of extremely strong antidepressants ability, it can be achieved that more long segment containing enhancing archaeal dna polymerase amplification ability
The amplification of target spot, and error rate is lower, so that rapid PCR amplification is possibly realized；
Kit of the present invention is added to one or both of PCR reinforcing agent DMSO, glycerol, formamide, it is therefore an objective to beat
Open the higher structure of nucleotide sequence.
At work, the working concentration of the DMSO is 1-10% (v/v), and the working concentration of the glycerol is 1-
20% (v/v).The working concentration of the formamide is 1-10% (v/v).
In a preferred embodiment, the size of reference gene A segment of the invention are as follows: 100-300bp；Reference gene
The size of B segment is 1000-1100bp.
It is established using the reaction system that rapid PCR amplification kit of the invention carries out PCR amplification as follows:
| 10 × buffer | 5μL |
| dNTPs(2.5mmol/L) | 4μL |
| Upstream primer F (10 μm of ol/L) | 1μL |
| Downstream primer R (10 μm of ol/L) | 1μL |
| Reinforcing agent | 2μL |
| Template DNA | 100ng |
| Archaeal dna polymerase (2.5U/ μ L) | 2μL |
| Distilled water | Complement to 50 μ L |
PCR is carried out using the pcr amplification reaction system and reacts amplification condition are as follows: 95 DEG C of initial denaturation 3min, 95 DEG C of changes
Property 10s, 60 DEG C of annealing 15s, 72 DEG C of extension 30s, 30 circulation after extend 5min.
The present invention determines that PCR annealing temperature carries out PCR expansion using formula (A+T) × 2+ (G+C) × 4- (5 DEG C~8 DEG C)
Increase；In addition, after amplification, 1% agarose gel electrophoresis detection of progress, burning voltage 130V, electrophoresis time 20 minutes.
Embodiment 1:
Using rapid PCR amplification kit of the invention and reaction system and reaction condition internal reference in human gene group DNA
Gene A segment is expanded, and takes appropriate mouse heart, liver, spleen, lung, kidney, brain, muscle, toe, rat-tail, bone, hair tissue
Carry out the experiment of nucleic acid DNA PCR amplification, the agarose gel electrophoresis testing result after amplification shown in Figure 1, wherein 1 swimming lane
For DNA marker, 2~12 swimming lanes are respectively mouse heart, liver, spleen, lung, kidney, brain, muscle, toe, rat-tail, bone, hair group
The nucleic acid DNA amplification knitted, 13 swimming lanes are human genome DNA's amplification, and 14 swimming lanes are negative control (i.e. ddH2O is
Template).
Embodiment 2:
Using rapid PCR amplification kit of the invention and reaction system and reaction condition internal reference in human gene group DNA
Gene B segment is expanded, and takes appropriate mouse heart, liver, spleen, lung, kidney, brain, muscle, toe, rat-tail, bone, hair tissue
It carries out nucleic acid DNA to extract with PCR amplification experiment, the agarose gel electrophoresis testing result after amplification shown in Figure 2, wherein
1 swimming lane be DNA marker, 2~12 swimming lanes be respectively mouse heart, liver, spleen, lung, kidney, brain, muscle, toe, rat-tail, bone,
Hair tissue nucleic acid DNA amplification, 13 swimming lanes be human genome DNA's amplification, 14 swimming lanes be negative control (i.e.
ddH2O is template).
Rapid PCR amplification kit of the invention and its application method, compared with prior art PCR amplification kit, knot
It closes and uses Phire thermal starting II archaeal dna polymerase, containing enhancing archaeal dna polymerase amplification ability, make it have extremely strong anti-inhibition
The ingredient of agent ability, it can be achieved that more long segment target spot amplification, and error rate is lower；PCR increasing is added in kit of the present invention
One or both of strong agent DMSO, glycerol, formamide, can open the higher structure of nucleotide sequence；In addition, 3. present invention
Rapid PCR amplification kit have the characteristics that it is convenient, saving fund, improve efficiency, one-step method PCR detection, realize micro
DNA sample rapid amplifying.
The present invention is described by above-mentioned related embodiment, however above-described embodiment is only to implement example of the invention.
It must be noted that the embodiment disclosed is not limiting as the scope of the present invention.On the contrary, do not depart from spirit of the invention and
It is changed and retouched made by range, belongs to scope of patent protection of the invention.
Claims (10)
1. a kind of rapid PCR amplification kit, which is characterized in that the kit includes archaeal dna polymerase, dNTPs, internal reference base
Because of primer group and buffer solution system；The reference gene primer group includes internal reference A segment and internal reference B segment, the internal reference A
Segment includes upstream primer F and downstream primer R；The internal reference B segment includes upstream primer F and downstream primer R；Wherein, described
The nucleotides sequence of the upstream primer F of internal reference A segment is classified as SEQ ID NO:1, and the nucleotides sequence of the downstream primer R is classified as
SEQ ID NO:2；The nucleotides sequence of the upstream primer F of the internal reference B segment is classified as SEQ ID NO:3, and the downstream is drawn
The nucleotides sequence of object R is classified as SEQ ID NO:4.
2. a kind of rapid PCR amplification kit as described in claim 1, which is characterized in that the interior A joins clip size are as follows:
200-300bp, the internal reference B clip size are as follows: 1000-1100bp.
3. a kind of rapid PCR amplification kit as claimed in claim 2, which is characterized in that the upstream primer F and it is described under
The concentration for swimming primer R is 10 μm of ol/L.
4. a kind of rapid PCR amplification kit as claimed in claim 1 or 2, which is characterized in that the rapid PCR amplification
Kit further includes PCR reinforcing agent, and the PCR reinforcing agent includes one or both of DMSO, glycerol, formamide.
5. a kind of rapid PCR amplification kit as claimed in claim 4, which is characterized in that the working concentration of the DMSO is
1-10% (v/v), the working concentration of the glycerol are 1-20% (v/v), and the working concentration of the formamide is 1-10% (v/
v)。
6. a kind of rapid PCR amplification kit as claimed in claim 1 or 2, which is characterized in that the buffer solution system is 10
× buffer is the MgCl of 500mmol/L Tris-HCl and 20mmol/L including concentration2, the pH value of the Tris-HCl is
9.0。
7. a kind of rapid PCR amplification kit as claimed in claim 1 or 2, which is characterized in that the concentration of the dNTPs is
2.5mmol/L；The DNA polymerase activity is 2.5U/ μ L.
8. a kind of rapid PCR amplification kit as claimed in claim 1 or 2, which is characterized in that the archaeal dna polymerase is
Phire thermal starting IIDNA polymerase.
9. a kind of application side of carry out PCR amplification of application such as the described in any item rapid PCR amplification kits of claim 1-8
Method, which is characterized in that the PCR reaction system are as follows:
PCR amplification condition are as follows: 95 DEG C of initial denaturation 3min, 95 DEG C of denaturation 10s, 60 DEG C of annealing 15s, 72 DEG C of extension 30s, 30 recycle
After extend 5min.
10. such as the application method of the carry out PCR amplification as claimed in claim 9 using rapid PCR amplification kit, feature
It is, the application method of the rapid PCR amplification kit further includes carrying out 1% agarose gel electrophoresis detection, stablizes electricity
Pressing is 130V, electrophoresis time 20 minutes.
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Citations (4)
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|---|---|---|---|---|
| US20120100545A1 (en) * | 2009-07-03 | 2012-04-26 | Tan Tock Seng Hospital | Method and/or primers for the detection of mycobacterium tuberculosis |
| CN102465120A (en) * | 2010-11-10 | 2012-05-23 | 深圳华大基因科技有限公司 | Fluorescent quantitative PCR reaction solution and fluorescent quantitative PCR method |
| CN108265113A (en) * | 2016-12-29 | 2018-07-10 | 滨江华康（北京）生物科技有限公司 | ALDH2 genetic polymorphism detection kits |
| CN109576384A (en) * | 2018-12-18 | 2019-04-05 | 北京卓诚惠生生物科技股份有限公司 | For detecting the nucleic acid reagent, kit and system of food-borne pathogens |
-
2019
- 2019-06-12 CN CN201910504468.7A patent/CN110527711A/en not_active Withdrawn
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120100545A1 (en) * | 2009-07-03 | 2012-04-26 | Tan Tock Seng Hospital | Method and/or primers for the detection of mycobacterium tuberculosis |
| CN102465120A (en) * | 2010-11-10 | 2012-05-23 | 深圳华大基因科技有限公司 | Fluorescent quantitative PCR reaction solution and fluorescent quantitative PCR method |
| CN108265113A (en) * | 2016-12-29 | 2018-07-10 | 滨江华康（北京）生物科技有限公司 | ALDH2 genetic polymorphism detection kits |
| CN109576384A (en) * | 2018-12-18 | 2019-04-05 | 北京卓诚惠生生物科技股份有限公司 | For detecting the nucleic acid reagent, kit and system of food-borne pathogens |
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