WO2003048392A1 - Salts mixture for the preparation of pcr buffer solutions - Google Patents
Salts mixture for the preparation of pcr buffer solutions Download PDFInfo
- Publication number
- WO2003048392A1 WO2003048392A1 PCT/IT2001/000610 IT0100610W WO03048392A1 WO 2003048392 A1 WO2003048392 A1 WO 2003048392A1 IT 0100610 W IT0100610 W IT 0100610W WO 03048392 A1 WO03048392 A1 WO 03048392A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- pcr buffer
- buffer solutions
- pcr
- preparation
- dna
- Prior art date
Links
- 239000007853 buffer solution Substances 0.000 title claims abstract description 20
- 239000000203 mixture Substances 0.000 title claims abstract description 11
- 238000002360 preparation method Methods 0.000 title claims abstract description 7
- 150000003839 salts Chemical class 0.000 title abstract description 5
- 230000003321 amplification Effects 0.000 claims abstract description 10
- 238000006243 chemical reaction Methods 0.000 claims abstract description 10
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 10
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 11
- 239000005695 Ammonium acetate Substances 0.000 claims description 11
- 229940043376 ammonium acetate Drugs 0.000 claims description 11
- 235000019257 ammonium acetate Nutrition 0.000 claims description 11
- 235000013919 monopotassium glutamate Nutrition 0.000 claims description 8
- WDRWZVWLVBXVOI-QTNFYWBSSA-L dipotassium;(2s)-2-aminopentanedioate Chemical compound [K+].[K+].[O-]C(=O)[C@@H](N)CCC([O-])=O WDRWZVWLVBXVOI-QTNFYWBSSA-L 0.000 claims description 7
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 3
- 229910052700 potassium Inorganic materials 0.000 claims description 3
- 239000011591 potassium Substances 0.000 claims description 3
- 238000007403 mPCR Methods 0.000 claims description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 abstract description 4
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 abstract description 4
- 238000003752 polymerase chain reaction Methods 0.000 description 13
- 108020004414 DNA Proteins 0.000 description 12
- 102000004190 Enzymes Human genes 0.000 description 10
- 108090000790 Enzymes Proteins 0.000 description 10
- 238000004925 denaturation Methods 0.000 description 7
- 230000036425 denaturation Effects 0.000 description 7
- 238000009396 hybridization Methods 0.000 description 5
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 238000000137 annealing Methods 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 150000001450 anions Chemical class 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 238000002844 melting Methods 0.000 description 2
- 230000008018 melting Effects 0.000 description 2
- 239000011833 salt mixture Substances 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000009877 rendering Methods 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- -1 salts Potassium Glutamate Chemical class 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Definitions
- the present invention relates to DNA amplification techniques and more particularly to the use of Potassium glutamate and Ammonium Acetate in PCR buffer solutions in order to offer high amplification uniformity at different temperatures and higher reaction efficiency with different DNA polymerase or cocktails of two enzymes having different characteristics.
- PCR polymerase chain reaction
- the PCR reaction is based on the repetition of a single amplification cycle made of three subsequent steps :
- the double elix of DNA opens to the two complementary single DNA strains.
- the second step called annealing or hybridisation, short single strand DNA sequences of 20 bais pairs, called primers, because of bais complementarity, bound to the regions adjacent to each end of the sequence to be amplified, called target sequence.
- DNA polymerase the enzyme responsible for replication
- DNA polymerase the enzyme responsible for replication
- syntetises from the primers 3'- OH end, a single strand of DNA complementary to the desired sequence to be syntetised.
- the choice of the PCR buffer is the most critical moment for the success of experimental protocols because the proceeding of the three reactions of the basic PCR amplification cycle depends on the physical and chemical characteristics of the reaction environment, which is mostly defined by the same buffer solution.
- PCR buffer solutions capable of overcoming all the above described problems are not commercially available.
- the present invention overcomes said disadvantages providing the use of a mixture of Potassium Glutamate and Ammonium acetate for the preparation of PCR buffer solutions. Said mixture is unknown in the state of the art and among commercially available products.
- the buffer solution comprises the salt mixture of Potassium glutamate and Ammonium Acetate and, according to the present invention, shows the following advantages:
- the salt mixture is capable of rendering the local DNA elix denaturation point homogeneous even if sequences having dishomogeneous denaturation profiles are present inside the same molecule; it enables an high specific primer-template hybridisation; it permits the use of primers mixtures having various melting points; it offers high efficiency in presence of different types of polymerases, namely different enzymes, or cocktails of more than one enzyme having different characteristics.
- a PCR buffer solution comprising a mixture of Potassium Glutamate and Ammonium Acetate is provided, in order to obtain the following final concentration, to be used in the amplification reaction: POTASSIUM GLUTAMMATE from 1 to 250 mM AMMONIUM ACETATE from 5 to 200 mM
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to the use of a new salts mixture to be used for the preparation of PCR buffer solutions in order to offer high amplification uniformity at different temperatures and an higher reaction efficiency with different DNA polymerases.
Description
SALTS MIXTURE FOR THE PREPARATION OF PCR BUFFER SOLUTIONS
The present invention relates to DNA amplification techniques and more particularly to the use of Potassium glutamate and Ammonium Acetate in PCR buffer solutions in order to offer high amplification uniformity at different temperatures and higher reaction efficiency with different DNA polymerase or cocktails of two enzymes having different characteristics. As known, PCR (polymerase chain reaction) is surely the technique that changed completely molecular biology allowing inexpensive, fast and efficient DNA synthesis.
The PCR reaction is based on the repetition of a single amplification cycle made of three subsequent steps :
1. During the first reaction (denaturation) the double elix of DNA opens to the two complementary single DNA strains. 2. In the second step, called annealing or hybridisation, short single strand DNA sequences of 20 bais pairs, called primers, because of bais complementarity, bound to the regions adjacent to each end of the sequence to be amplified, called target sequence.
3. During the last step, DNA polymerisation take place and DNA polymerase (the enzyme responsible for replication) syntetises, from the primers 3'-
OH end, a single strand of DNA complementary to the desired sequence to be syntetised. In the laboratory practice, the choice of the PCR buffer is the most critical moment for the success of experimental protocols because the proceeding of the three reactions of the basic PCR amplification cycle depends on the physical and chemical characteristics of the reaction environment, which is mostly defined by the same buffer solution.
Moreover, to render the entire process economically favourable, the use of efficient buffer solutions for various amplification protocols for the analysis of different types of DNA samples with various polymerase or temperature intervals, is desiderable.
One of the most frequent problem related to the preparation of PCR buffer solutions is the achievement, in the buffer solution, of a suitable ions concentration in order to obtain homogeneous denaturation and high specificity of the hybridisation reaction between primers and templates sequence. In fact, it is well known that cations concentration affect DNA denaturation temperature and different anions act differently on DNA sequences having different structural features. Another problem is related to the use of different DNA polymerases or cocktails of two different polymerases, as in Long PCR. In this case, the buffer solution
should ensure the maximum efficiency for all the enzymes. At the present time, PCR buffer solutions capable of overcoming all the above described problems are not commercially available.
The present invention overcomes said disadvantages providing the use of a mixture of Potassium Glutamate and Ammonium acetate for the preparation of PCR buffer solutions. Said mixture is unknown in the state of the art and among commercially available products. The buffer solution comprises the salt mixture of Potassium glutamate and Ammonium Acetate and, according to the present invention, shows the following advantages:
It ensures high amplification uniformity at various temperatures because the salt mixture is capable of rendering the local DNA elix denaturation point homogeneous even if sequences having dishomogeneous denaturation profiles are present inside the same molecule; it enables an high specific primer-template hybridisation; it permits the use of primers mixtures having various melting points; it offers high efficiency in presence of different types of polymerases, namely different enzymes, or cocktails of more than one enzyme having different characteristics.
The high efficiency outcome from the fact that those anions are plentifully present in bacteria and their use permits to restore enzymes
natural working conditions, giving them the higher processivity and functional stability; it helps DNA denaturation even of very long sequences (up to 18.000 bp) because in presence of the two salt the DNA denatures easily; it avoids, during annealing, hybridisation within sequences inside the same DNA molecule
(intrachain annealing) ; it allows the treatment of amplified DNA samples, obtained from PCR, with restriction or modification enzymes without any preliminary washing step or without needing to change the buffer solution because the salts do not interfere with enzymes catalytic activity.
According to the present invention a PCR buffer solution comprising a mixture of Potassium Glutamate and Ammonium Acetate is provided, in order to obtain the following final concentration, to be used in the amplification reaction: POTASSIUM GLUTAMMATE from 1 to 250 mM AMMONIUM ACETATE from 5 to 200 mM
In a preferred embodiment the mixture object of the present invention is characterised in having the following final concentration to be used in the amplification reaction:
POTASSIUM GLUTAMMATE 20 mM
AMMONIUM ACETATE 60 mM
In conclusion, the use of the mixture of the two salts Potassium Glutamate and Ammonium Acetate allows the preparation of PCR buffer
solutions particularly suitable for the following amplification protocols:
Multiplex PCR since it enables high amplification uniformity at different temperatures, permitting the utilisation of mixtures of primers having various melting points and it ensures great hybridisation specificity between primers and templates;
Long PCR since it allows high efficiency in the presence of different types of polymerases, i.e. with various enzymes or cocktails of many enzymes with different features, it facilitates DNA denaturation even in case of very long DNA sequences (up to 18.000 bp.) .
Claims
CLAIMS 1. The use of a mixture of Potassium Glutamate and Ammonium Acetate for the preparation of PCR buffer solutions.
2. PCR buffer solution characterised in that Potassium Glutamate and Ammonium Acetate in their final concentration used in the amplification reaction are present in the following rate:
POTASSIUM GLUTAMMATE from 1 to 250 mM AMMONIUM ACETATE from 5 to 200 mM
3. PCR buffer solution as in claim 2 characterised in that Potassium Glutamate has a concentration of 20 mM and Ammonium Acetate has a concentration of a 60mM.
4. PCR buffer solutions as in claim 1 and 2 characterised by the fact of being used in multiplex PCR.
5. PCR buffer solutions as in claim 1 and 2 characterised by the fact of being used in Long PCR .
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/IT2001/000610 WO2003048392A1 (en) | 2001-12-04 | 2001-12-04 | Salts mixture for the preparation of pcr buffer solutions |
| AU2002217427A AU2002217427A1 (en) | 2001-12-04 | 2001-12-04 | Salts mixture for the preparation of pcr buffer solutions |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/IT2001/000610 WO2003048392A1 (en) | 2001-12-04 | 2001-12-04 | Salts mixture for the preparation of pcr buffer solutions |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2003048392A1 true WO2003048392A1 (en) | 2003-06-12 |
Family
ID=11133760
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/IT2001/000610 WO2003048392A1 (en) | 2001-12-04 | 2001-12-04 | Salts mixture for the preparation of pcr buffer solutions |
Country Status (2)
| Country | Link |
|---|---|
| AU (1) | AU2002217427A1 (en) |
| WO (1) | WO2003048392A1 (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0632134A2 (en) * | 1993-07-01 | 1995-01-04 | F. Hoffmann-La Roche Ag | Reagents and methods for coupled high temperature reverse transcription and polymerase chain reaction |
| US5432065A (en) * | 1993-03-30 | 1995-07-11 | United States Biochemical Corporation | Cycle sequencing with non-thermostable DNA polymerases |
| EP0669401A2 (en) * | 1994-02-25 | 1995-08-30 | F. Hoffmann-La Roche Ag | Amplification of long nucleic acid sequences by PCR |
-
2001
- 2001-12-04 WO PCT/IT2001/000610 patent/WO2003048392A1/en not_active Application Discontinuation
- 2001-12-04 AU AU2002217427A patent/AU2002217427A1/en not_active Abandoned
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5432065A (en) * | 1993-03-30 | 1995-07-11 | United States Biochemical Corporation | Cycle sequencing with non-thermostable DNA polymerases |
| EP0632134A2 (en) * | 1993-07-01 | 1995-01-04 | F. Hoffmann-La Roche Ag | Reagents and methods for coupled high temperature reverse transcription and polymerase chain reaction |
| EP0669401A2 (en) * | 1994-02-25 | 1995-08-30 | F. Hoffmann-La Roche Ag | Amplification of long nucleic acid sequences by PCR |
Also Published As
| Publication number | Publication date |
|---|---|
| AU2002217427A1 (en) | 2003-06-17 |
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