CN108949937A - One step RT-PCR premixes reagent - Google Patents
One step RT-PCR premixes reagent Download PDFInfo
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- CN108949937A CN108949937A CN201810779299.3A CN201810779299A CN108949937A CN 108949937 A CN108949937 A CN 108949937A CN 201810779299 A CN201810779299 A CN 201810779299A CN 108949937 A CN108949937 A CN 108949937A
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- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The present invention provides a kind of One step RT-PCR premix reagent characterized by comprising rna transcription is the reagent and polysaccharide of cDNA.The reagents such as enzyme, buffer needed for the present invention reacts RT-PCR are mixed in advance in a reaction tube, save the RT-PCR operating time, and reduce possible pollution of uncapping repeatedly.Further, the present invention enhances RT-PCR reaction efficiency by reagents such as addition polysaccharides.
Description
Technical field
The present invention relates to a kind of One step RT-PCRs to premix reagent, belongs to molecular biology field.
Background technique
Reverse transcription PCR (RT-PCR) is expanded by template of RNA, detection, RNA virus content in gene expression dose
Detection and Direct Cloning specific gene cDNA etc. have a wide range of applications.Reverse transcription PCR is divided into two steps, exists first
Under the action of reverse transcriptase by RNA reverse transcription be single-stranded cDNA, then single-stranded cDNA is expanded under the action of archaeal dna polymerase
For the DNA molecular of double-strand.RT-PCR process can carry out step by step, i.e., go to dezymotize after the completion of transcription, metal ion etc. it is poly- to DNA
Synthase has the component of inhibition, adds archaeal dna polymerase and corresponding buffer solution, can also be by reverse transcriptase and archaeal dna polymerase
It is mixed into a pipe and carries out single step reaction.Compared with two-step reaction, single step reaction reduces bring risk of uncapping repeatedly, thus more
It is convenient and safe to add.
Realize that one-step RT-PCR reaction is not easy to, a series of report displays work as reverse transcriptase and archaeal dna polymerase one
It can be interfered with each other when being reacted in pipe, cause the reduction of RT-PCR sensitivity.On the one hand this inhibition is from reverse transcriptase
Inconsistent with the optimal buffer of archaeal dna polymerase, on the other hand more reports point out such as avian myeloid leukemia virus
Reverse transcriptase (AMV-RT) and mouse leukemia virus reverse transcriptase (M-MLV-RT) etc. can inhibit the vigor of archaeal dna polymerase.
Summary of the invention
The purpose of the present invention is to provide a kind of new One step RT-PCRs to premix reagent, to enhance One step RT-PCR
Reaction efficiency.
Present invention employs following technical solutions:
A kind of One step RT-PCR premix reagent characterized by comprising rna transcription is the reagent and poly of cDNA
Sugar.
Further, One step RT-PCR of the invention premixes reagent: where rna transcription is that the reagent of cDNA includes: reversion
Record enzyme, archaeal dna polymerase, buffer, co-factor and surfactant.
Preferably, One step RT-PCR of the invention premixes reagent, also has a feature in that wherein, rna transcription is
The reagent of cDNA includes: reverse transcriptase: M-MLV-RT or AMV-RT, one or two kinds of archaeal dna polymerase, 5-100mM's
Tris-HCl or Tris-SO4, the MgCl of 1~10mM2, the KCl of 10~200mM, 200~400 μM of dNTP, 5~40mM's
The trehalose of DTT, 10~400mM, the sulfate of the BSA of 0.2~3mg/mL, 20~50mM, glycerol 5~35%.
Preferably, One step RT-PCR of the invention premixes reagent, further includes: SYBR dyestuff, to dye method RT-PCR
Process.
Preferably, One step RT-PCR of the invention premixes reagent, wherein sulfate is ammonium sulfate.
Preferably, One step RT-PCR of the invention premixes reagent, wherein polysaccharide are as follows: water-soluble dextran.
Preferably, One step RT-PCR of the invention premixes reagent, wherein the concentration range of water-soluble dextran is 10-
25mM。
Preferably, One step RT-PCR of the invention premixes reagent, wherein the concentration of water-soluble dextran is 20mM.
Preferably, One step RT-PCR of the invention premixes reagent, and the pH range for premixing reagent is 8.3~9.2.
The present invention also provides the One step RT-PCR of above-mentioned any one premix reagents in preparing kit for detecting nucleic acid
Application.
Advantageous effect of the invention
First, the reagents such as enzyme, buffer needed for RT-PCR is reacted are mixed in advance in a reaction tube, save RT-PCR behaviour
Make the time, and reduces possible pollution of uncapping repeatedly.
Second, by reagents such as addition polysaccharides, enhance RT-PCR reaction efficiency.
Detailed description of the invention
Fig. 1 is the RT-PCR amplification curve for adding water-soluble dextran.
Specific embodiment
Technical solution of the present invention is described in detail with reference to embodiments.
Embodiment 1:Tris-HCl and Tris-SO4Influence to RT-PCR
In order to investigate the Tris-HCl and Tris-SO of different pH4Influence to One step RT-PCR is with GAPDH gene
Example is tested, and Tris-HCl or Tris-SO is separately added into4The premix reagent of 5 gradient pH is prepared.In present embodiment
Other components and concentration are as follows: ammonium sulfate 20mM, KCl 50mM, 300 μM of glycerol, dNTP accounting for premix reagent gross mass 15%, sea
Algae sugar 300mM, DTT 20mM, BSA 0.4mg/mL.The AMV-RT and archaeal dna polymerase of normal concentration.
RT-PCR reaction condition: 42 DEG C or 50 DEG C of reverse transcriptions 5min, 95 DEG C of denaturation 10s；95 DEG C of initial denaturation 5s, 60 DEG C of expansions
Increase 20s, 40 circular responses.
As known from Table 1, Tris-HCl and Tris-SO4Optimal pH concentration be 8.8, and Tris-HCl is more advantageous to RT-
PCR process, this may be to have had the sulfate ion of suitable concentration in buffer that we prepare, add more sulphur
Acid ion is unfavorable for RT-PCR reaction instead.The concentration of reverse transcriptase M-MLV-RT and archaeal dna polymerase is adopted in each embodiment
With normal concentration.
The Tris-HCl and Tris-SO of 1. difference pH of table4Influence to RT-PCR
+, it indicates to promote RT-PCR process and the more facilitations of plus sige is more obvious；, indicate to inhibit RT-PCR process
Embodiment 2: influence of addition water-soluble dextran Ficoll 400 (Sigma-aldrich) to RT-PCR.Each reality
It applies the water-soluble dextran in mode and refers both to the Ficoll400 glucan from Sigma-aldrich.
Other components and concentration in present embodiment are as follows: ammonium sulfate 20mM, KCl 50mM, account for premix reagent gross mass 15%
Glycerol, dNTP μM, trehalose 300mM, DTT 20mM, BSA 2mg/mL.PH concentration is adjusted to 8.8.The AMV- of normal concentration
RT and archaeal dna polymerase.
RT-PCR reaction condition: 42 DEG C or 50 DEG C reversions 5min, 95 DEG C of denaturation 10s；95 DEG C of initial denaturation 5s, 60 degrees Celsius
Expand 20s, 40 circular responses.
Water-soluble dextran is added, influence of the various concentration water-soluble dextran to RT-PCR has been investigated.As known from Table 2,
As the increase RT-PCR effect presentation of glucan concentration first increases the trend reduced afterwards, when its concentration is 20mM, RT-PCR
Effect is best, and facilitation weakens when the concentration is too high.
It is tested when water-soluble dextran concentration is 20mM with different RNA template concentrations, it is same not add glucan
Component compares.From attached drawing 1 it is found that addition it is suitable water-soluble dextran it is obvious to RT-PCR facilitation effect, be obviously improved sample
Product Ct value.Curve in Fig. 1 with asterisk is the curve of non-adding water soluble glucan, and the curve without asterisk is that various concentration is added
The curve of water-soluble dextran.
Influence of the 2. various concentration water-soluble dextran of table to RT-PCR
+, it indicates to promote RT-PCR process and the more facilitations of plus sige is more obvious；, indicate to inhibit RT-PCR process
Embodiment 3:
One step RT-PCR premix reagent in present embodiment includes: reverse transcriptase: M-MLV-RT, archaeal dna polymerase,
The Tris-SO of the Tris-HCl and 5mM of 5mM4, the MgCl of 1mM2, the KCl of 10mM, the DTT of 200 μM of dNTP, 5mM, 10mM's
Trehalose, the ammonium sulfate of the BSA of 1mg/mL, 20mM account for the glycerol of gross mass 5%.The water-soluble dextran Ficoll of 10mM
400.PH value is adjusted to 8.3.
RT-PCR reaction condition: 42 DEG C or 50 DEG C reversions 5min, 95 DEG C of denaturation 10s；95 DEG C of initial denaturation 5s, 60 degrees Celsius
Expand 20s, 40 circular responses.
Embodiment 4:
Reverse transcriptase: AMV-RT, archaeal dna polymerase, the MgCl of the Tris-HCl of 100mM, 10mM2, the KCl of 200mM, 400 μ
The trehalose of the DTT of the dNTP of M, 40mM, 400mM, the ammonium sulfate of the BSA of 1.8mg/mL, 50mM account for premix reagent gross mass
15% glycerol.
The water-soluble dextran Ficoll 400 of 25mM.PH value is adjusted to 9.2.
RT-PCR reaction condition: 42 DEG C or 50 DEG C reversions 5min, 95 DEG C of denaturation 10s；95 DEG C of initial denaturation 5s, 60 degrees Celsius
Expand 20s, 40 circular responses.
Embodiment 5: influence of the different BSA to experimental result is added.
Each component and concentration in present embodiment are as follows: ammonium sulfate 20mM, KCl 50mM, account for premix reagent gross mass 15%
Glycerol, dNTP μM, trehalose 300mM, DTT 20mM, BSA0.4mg/mL.PH concentration is adjusted to 8.8.The AMV-RT of normal concentration
And archaeal dna polymerase.
RT-PCR reaction condition: 42 DEG C or 50 DEG C reversions 5min, 95 DEG C of denaturation 10s；95 DEG C of initial denaturation 5s, 60 degrees Celsius
Expand 20s, 40 circular responses.
The BSA of various concentration is added on this basis.
RT-PCR reaction condition: 42 DEG C or 50 DEG C reversions 5min, 95 DEG C of denaturation 10s；95 DEG C of initial denaturation 5s, 60 degrees Celsius
Expand 20s, 40 circular responses.
It the results are shown in Table 3.As seen from the table, adding different BSA has larger impact to RT-PCR, when BSA concentration is 2mg/mL
When, the BSA of the best 0.2~3mg/mL of RT-PCR effect, and when BSA concentration further increases, facilitation does not increase.BSA
Effect essentially consist in stabilized DNA Polymerase Activity, promote PCR amplification efficiency.
Influence of the 3. various concentration BSA of table to RT-PCR
+, it indicates to promote RT-PCR process and the more facilitations of plus sige is more obvious；, indicate not promoting RT-PCR process
Embodiment 6
Reagent is premixed using any one One step RT-PCR in embodiment 2-4, is applied in kit for detecting nucleic acid.
The RNA in various sources is expanded and detected.
Claims (10)
1. a kind of One step RT-PCR premixes reagent characterized by comprising
Rna transcription is the reagent and polysaccharide of cDNA.
2. One step RT-PCR as described in claim 1 premixes reagent, it is characterised in that:
Wherein, the rna transcription is that the reagent of cDNA includes:
Reverse transcriptase, archaeal dna polymerase, buffer, co-factor and surfactant.
3. One step RT-PCR as described in claim 1 premixes reagent, it is characterised in that:
Wherein, the rna transcription is that the reagent of cDNA includes:
Reverse transcriptase: M-MLV-RT or AMV-RT,
One or two kinds of archaeal dna polymerase,
The Tris-HCl or Tris-SO of 5-100mM4,
The MgCl of 1~10mM2,
The KCl of 10~200mM,
200~400 μM of dNTP,
The DTT of 5~40mM,
The trehalose of 10~400mM,
The BSA of 0.2~3mg/mL,
The sulfate of 20~50mM,
Glycerol 5~35%.
4. One step RT-PCR as described in claim 1 premixes reagent, which is characterized in that further include:
SYBR dyestuff.
5. One step RT-PCR as described in claim 1 premixes reagent, it is characterised in that:
Wherein, the sulfate is ammonium sulfate.
6. One step RT-PCR as described in claim 1 premixes reagent, it is characterised in that:
Wherein, the polysaccharide are as follows: water-soluble dextran.
7. One step RT-PCR as claimed in claim 6 premixes reagent, it is characterised in that:
Wherein, the concentration range of the water-soluble dextran is 10-25mM.
8. One step RT-PCR as claimed in claim 6 premixes reagent, it is characterised in that:
Wherein, the concentration of the water-soluble dextran is 20mM.
9. One step RT-PCR as described in claim 1 premixes reagent, it is characterised in that:
The pH range for premixing reagent is 8.3~9.2.
10. the One step RT-PCR premix reagent as described in any one of claim 1-9 is preparing kit for detecting nucleic acid
In application.
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| CN201810779299.3A CN108949937A (en) | 2018-07-16 | 2018-07-16 | One step RT-PCR premixes reagent |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112695019A (en) * | 2021-03-23 | 2021-04-23 | 翌圣生物科技（上海）有限公司 | Reverse transcriptase mutant and application thereof |
| CN112831551A (en) * | 2021-01-22 | 2021-05-25 | 武汉明德生物科技股份有限公司 | Fully-premixed RT-PCR reaction system and application thereof |
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| CN104024411A (en) * | 2011-08-17 | 2014-09-03 | 凯杰有限公司 | Composition and methods for RT-PCR comprising an anionic polymer |
| CN103131694A (en) * | 2011-11-25 | 2013-06-05 | 上海绿宇生物科技有限公司 | Trace and complex DNA amplification method |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112831551A (en) * | 2021-01-22 | 2021-05-25 | 武汉明德生物科技股份有限公司 | Fully-premixed RT-PCR reaction system and application thereof |
| CN112695019A (en) * | 2021-03-23 | 2021-04-23 | 翌圣生物科技（上海）有限公司 | Reverse transcriptase mutant and application thereof |
| CN112695019B (en) * | 2021-03-23 | 2021-06-25 | 翌圣生物科技（上海）有限公司 | Reverse transcriptase mutant and application thereof |
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