DE4411588C1 - Buffer, contg. betaine, for RNA- and DNA-polymerase reactions - Google Patents

Buffer, contg. betaine, for RNA- and DNA-polymerase reactions

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DE4411588C1
DE4411588C1 DE4411588A DE4411588A DE4411588C1 DE 4411588 C1 DE4411588 C1 DE 4411588C1 DE 4411588 A DE4411588 A DE 4411588A DE 4411588 A DE4411588 A DE 4411588A DE 4411588 C1 DE4411588 C1 DE 4411588C1
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betaine
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Thomas Weissensteiner
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Abstract

Buffer for RNA and DNA-polymerase reactions, which contains betaine, is new.

Description

Die Erfindung betrifft einen Puffer für DNA- und RNA- Polymerase-Reaktionen, der Betain enthält, und die Ver­ wendung von Betain in DNA- und RNA-Polymerase-Reaktio­ nen, insbesondere in der spezifischen Polymerase-Ket­ ten-Reaktion und in der reversen Transkription von RNA in cDNA (RT).The invention relates to a buffer for DNA and RNA Polymerase reactions containing betaine and Ver Use of betaine in DNA and RNA polymerase reactions NEN, especially in the specific polymerase chain ten reaction and in the reverse transcription of RNA in cDNA (RT).

Es ist bekannt, daß die in der Polymerase-Ketten-Reak­ tion verwendeten Puffer Tris-HCL, Tricin, Kaliumchlorid und Magnesiumchlorid beinhalten. Auch wurde der Zusatz von DMSO (Winship, P. R. et al., 1989, Nucl. Acids Res. 17, 1266) oder nicht-ionischen Detergenzien (Bachmann, B. et. al., 1990, Nucl. Acids Res. 18, 1309) vorge­ schlagen, um die DNA-Sequenzierungsreaktion zu verbes­ sern und Basenmißpaarungen zu vermeiden. It is known that the polymerase chain react tion used buffer Tris-HCL, tricin, potassium chloride and include magnesium chloride. Also the addition by DMSO (Winship, P.R. et al., 1989, Nucl. Acids Res. 17, 1266) or non-ionic detergents (Bachmann, B. et. al., 1990, Nucl. Acids Res. 18, 1309) to improve the DNA sequencing reaction and base mismatches to avoid.  

Hung et. al. beschreiben in Nucl. Acids Res. Vol. 18, No. 16, 4953 (1990) den Zusatz von Tetrameythylammoni­ umchlorid zu den üblichen PCR-Puffern, um die Spezifi­ tät der PCR zu erhöhen.Hung et. al. describe in Nucl. Acids Res. Vol. 18, No. 16, 4953 (1990) the addition of tetrameythylammoni umchloride to the usual PCR buffers to the specifi the PCR.

Um DNA-Moleküle mit mehr als 6 kbp zu amplifizieren, wird von M. R. Ponce et. al. in Nucl. Acids Res. Vol. 20, No. 3, 623 (1992) der Zusatz von Beta-Mercaptoetha­ nol, Gelatine und Thesit beschrieben.To amplify DNA molecules with more than 6 kbp, is by M. R. Ponce et. al. in nucl. Acids Res. Vol. 20, No. 3, 623 (1992) the addition of beta-mercaptoetha nol, gelatin and thesite are described.

Alle genannten PCR-Puffer weisen jedoch den Nachteil auf, daß durch die vorhandene Salzkonzentration die Am­ plifikation bestimmter DNA-Moleküle negativ beeinflußt werden kann. Daneben sind Dimethylsulfoxid und Tetramethylammoniumchlorid toxisch.However, all the PCR buffers mentioned have the disadvantage on that the Am plication of certain DNA molecules negatively affected can be. In addition are dimethyl sulfoxide and Tetramethylammonium chloride toxic.

Es wurde nun gefunden, daß Puffer, die N, N, N-Trime­ thylglycin (Betain) und keine Kalium-, Ammonium- oder Tetraalkylammoniumionen enthalten, sehr gut für spezifische Polymerase-Ketten-Reaktionen geeignet sind.It has now been found that buffers, the N, N, N trime thylglycine (betaine) and no potassium, ammonium or Contain tetraalkylammonium ions, very good for specific polymerase chain reactions are suitable.

Die Konzentration von Betain im erfindungsgemäßen Puf­ fer kann 200 bis 1000 mM betragen. So hat sich zum Bei­ spiel bei der spezifischen PCR von HLA-Antigenen ge­ zeigt, daß eine Zusammensetzung des Puffers aus 500 bis 800 mM Betain und 1 bis 6 mM Magnesiumchlorid optimal ist. Insbesondere enthält der Puffer zur spezifischen Amplifikation von HLA-B-Allelen 500 bis 800 mM Betain, 1 bis 6 mM Magnesiumchlorid, 8 bis 12 mM N-Tris(hydro­ xymethyl)methylglycin mit pH 8,3 und 93 bis 106 µg/ml BSA V. Dabei wird die PCR eines Fragmentes des HLA-B-Gens enthaltend Sequenzen von Exon 2 bis Exon 3 wie zum Beispiel von B 27, B27-Subtypen, B 12, B 44, B 45, B 7, BW 22, B 13, B 14, B 18, B 73, B 41, B*4001 oder B 42 mit 2 Allel-spezifischen Oligo­ desoxynukleotid-Primern durchgeführt, die am 3′-Ende in den drei letzten Nukleotiden mit der gesuchten Allelsequenz übereinstimmen und in der übrigen Sequenz zu mindestens 80% mit der Allelsequenz homolog sind.The concentration of betaine in the buffer according to the invention can be 200 to 1000 mM. For example, it has been shown in the specific PCR of HLA antigens that a composition of the buffer of 500 to 800 mM betaine and 1 to 6 mM magnesium chloride is optimal. In particular, the buffer for the specific amplification of HLA-B alleles contains 500 to 800 mM betaine, 1 to 6 mM magnesium chloride, 8 to 12 mM N-tris (hydroxymethyl) methylglycine with pH 8.3 and 93 to 106 µg / ml BSA V. The PCR of a fragment of the HLA-B gene containing sequences from exon 2 to exon 3, such as, for example, of B 27, B27 subtypes, B 12, B 44, B 45, B 7, BW 22, B 13 , B 14, B 18, B 73, B 41, B * 4001 or B 42 carried out with 2 allele-specific oligo deoxynucleotide primers which match at the 3'-end in the last three nucleotides with the allele sequence sought and in the rest Sequence are at least 80% homologous to the allele sequence.

Überraschend wurde auch gefunden, daß Betain-enthal­ tende Puffer für die reverse Transkription von RNA in cDNA sehr gut geeignet sind. Ein positiver Einfluß auf die reverse Transkription von sekundärstrukturreicher RNA wurde festgestellt.Surprisingly, it was also found that betaine-containing buffers for the reverse transcription of RNA in cDNA are very suitable. A positive influence on reverse transcription of secondary structures RNA was detected.

Insbesondere können die erfindungsgemäßen Puffer für die reverse Transkription von RNA in cDNA unter Verwen­ dung eines Oligo-dT-Primers und von M-MLV reverser Transkriptase bis zu 3 M Betain und ggf. auch Kaliumchlorid enthalten. Die Aktivität der reversen Transkriptase wird, wie auch die Aktivität der Taq- Polymerase, durch Betain nicht merklich beeinträchtigt.In particular, the buffers according to the invention for reverse transcription of RNA into cDNA using of an oligo-dT primer and M-MLV reverser Transcriptase up to 3 M betaine and possibly also Contain potassium chloride. The activity of the reverse Transcriptase, like the activity of Taq- Polymerase, not noticeably affected by betaine.

Vorzugsweise enthält ein erfindungsgemäßer Puffer zur reversen Transkription von RNA in cDNA zum Beispiel un­ ter Verwendung von Oligodesoxythymidin ((dT)12-18) und M-MLV reverser Transkriptase 1 bis 3 M Betain, 60 bis 75 mM Kaliumchlorid und 1 bis 6 mM Magnesiumchlorid.A buffer according to the invention for reverse transcription of RNA into cDNA preferably contains 1 to 3 M betaine, 60 to 75 mM potassium chloride and 1 to 6 mM magnesium chloride, for example using oligodeoxythymidine ((dT) 12-18 ) and M-MLV reverse transcriptase .

In einer anderen bevorzugten Ausführungsform kann der Puffer zur reversen Transkription von RNA in cDNA auch 1 bis 3 M Betain, 20 bis 75 mM Kalium- oder Ammoniumionen und 1 bis 10 mM Magnesiumionen enthalten.In another preferred embodiment, the Buffer for reverse transcription of RNA in cDNA too  1 to 3 M betaine, 20 to 75 mM potassium or Contain ammonium ions and 1 to 10 mM magnesium ions.

Zusammenfassend ist festzustellen, daß Betain eine Reihe von Vorteilen herkömmlicher, bisher in der PCR- und reversen Transkription verwendeten Kosolventien kombiniert:
Es ist chemisch inert und nicht-ionisch, d. h. es kann mit einer Anzahl anderer Kosolvenzien und Salzen kombiniert werden, um optimale Reaktionsbedingungen für die jeweiligen Primer und Template zu schaffen.In summary, betaine combines a number of advantages of conventional cosolvents previously used in PCR and reverse transcription:
It is chemically inert and non-ionic, meaning that it can be combined with a number of other cosolvents and salts to create optimal reaction conditions for the respective primers and templates.

Daneben erleichtert es die Transkription GC-reicher Domänen. Da nur GC-Basenpaare destabilisiert werden, können Primer so gewählt werden, daß ihre Hybridi­ sierung mit der Zielsequenz möglichst wenig beeinträchtigt wird.It also makes transcription easier for GC-rich people Domains. Since only GC base pairs are destabilized, primers can be chosen so that their hybridi sation with the target sequence as little as possible is affected.

Allgemeine Destabilisierung von dsDNA, z. B. durch Glycerin, Dimethylsulfoxid oder Formamid setzt auch die Hybridisierungstemperatur der Primer herab und hat damit sowohl positive wie negative Auswirkungen auf die Ausbeute von DNA-Polymerase-Reaktionen. Betain ist unter den nicht-ionischen Kosolvenzien einzigartig in seiner Eigenschaft, AT-Basenpaare zu binden und dadurch zu stabilisieren. Da nicht-ionische Kosolvenzien allgemein dsDNA destabilisieren, erfolgt in Anwesenheit von Betain insgesamt eine spezifische Destabilisierung GC-reicher Domänen in DNA und RNA. Betain ist jedoch erheblich billiger als Deoxyguanidin-Analoge wie sie zu diesem Zweck in Sequenzierung und PCR erfolgreich ein­ gesetzt wurden und kann außerdem auch in der RT verwendet werden. AT-reiche Primer wie z. B. (dT)12-18 in der RT werden jedoch weniger beeinflußt als GC- reiche Domänen im Templat.General destabilization of dsDNA, e.g. B. by glycerol, dimethyl sulfoxide or formamide also lowers the hybridization temperature of the primers and thus has both positive and negative effects on the yield of DNA polymerase reactions. Betain is unique among non-ionic cosolvents in its ability to bind and thereby stabilize AT base pairs. Since non-ionic cosolvents generally destabilize dsDNA, a specific destabilization of GC-rich domains in DNA and RNA takes place in the presence of betaine. However, betaine is considerably cheaper than deoxyguanidine analogues, such as those used successfully in sequencing and PCR, and can also be used in RT. AT-rich primers such as B. (dT) 12-18 in RT, however, are less affected than GC-rich domains in the template.

Magnesiumchlorid ist ein essentieller Kofaktor für DNA- Polymerasen, jedoch hängt die optimale Konzentration in der PCR stark von den jeweiligen Primern oder Templaten ab. In Gegenwart von Betain erweitert sich dieser optimale Bereich, wodurch sich die Optimierung, insbesondere von Koamplifikationen ("Multiplex-PCR"), vereinfacht.Magnesium chloride is an essential cofactor for DNA Polymerases, however, the optimal concentration depends on the PCR strongly from the respective primers or templates from. In the presence of betaine, it expands optimal range, which optimizes especially of co-amplifications ("multiplex-PCR"), simplified.

Desweiteren ist bekannt, daß sowohl NaCl als auch Heparin als Verunreinigungen in DNA-Proben auftreten können. So hat Heparin in Konzentrationen zwischen 2,5 · 10-4 - 10-3 U/µl ähnliche Effekte wie NaCl. Im Stand der Technik wurde deshalb Heparinase-Verdau oder Vorbehandlung Heparin-enthaltender DNA mit Chelex 100 vorgeschlagen (Francesca Poli, Rosa Catteano, Loretta Crespiatico, Angelea Nocco und Girolamo Sirchia, PCR Methods and Applications, 1993, 2, 356-358).Furthermore, it is known that both NaCl and heparin can occur as contaminants in DNA samples. In concentrations between 2.5 · 10 -4 - 10 -3 U / µl, heparin has similar effects to NaCl. Heparinase digestion or pretreatment of heparin-containing DNA with Chelex 100 has therefore been proposed in the prior art (Francesca Poli, Rosa Catteano, Loretta Crespiatico, Angelea Nocco and Girolamo Sirchia, PCR Methods and Applications, 1993, 2, 356-358).

Es wurde nun gefunden, daß die PCR-Ausbeuten, insbesondere von HLA-B, bei Heparin-enthaltenden DNA- Proben in Gegenwart von Betain deutlich verbessert werden können. Vorzugsweise zeigen sich diese Befunde bei Betainkonzentrationen von 500-800 mM. In Gegenwart von 0,8 M Betain werden bis zu 10fach höhere Heparin­ konzentrationen toleriert.It has now been found that the PCR yields especially of HLA-B, in heparin-containing DNA Samples in the presence of betaine improved significantly can be. These findings are preferred at betaine concentrations of 500-800 mM. In present of 0.8 M betaine is up to 10 times higher heparin concentrations tolerated.

Betain macht also die Aufreinigung von Salz- oder Heparin-enthaltenden DNA-Proben weitgehend überflüssig, was vor allem in der routinemäßigen Untersuchung klinischer und forensischer Proben von Vorteil ist.So betaine does the purification of salt or DNA samples containing heparin largely unnecessary,  what especially in the routine examination clinical and forensic samples is beneficial.

Daneben ist Betain ist im Gegensatz zu den Kosolvenzien DMSO, Formamid und Methyl-Quecksilberhydroxid ungiftig und ein natürlicher Schutz, den Zellen im Laufe der Evolution gegen thermische und ionische Denaturierung ihrer Proteine entwickelt haben.Next to it is betain is in contrast to the cosolvencies DMSO, formamide and methyl mercury hydroxide are non-toxic and a natural protection that cells receive over the course of Evolution against thermal and ionic denaturation have developed their proteins.

Die Aktivität der Taq-Polymerase und der reversen Transkriptase wird durch Betain nicht merklich beeinträchtigt.The activity of Taq polymerase and the reverse Transcriptase is not noticeable through betaine impaired.

Die Erfindung soll nachstehend durch Ausführungsbei­ spiele näher erläutert werden:The invention will hereinafter be described by way of example games are explained in more detail:

AusführungsbeispieleEmbodiments 1. Polymerase-Ketten-Reaktion von HLA-B-Allelen1. Polymerase chain reaction of HLA-B alleles a) Reaktionsansatza) Reaction approach

Die Amplifikationen werden in DNA-Thermocyclern (z. B. dem DNA-Thermocycler 480 der Firma Perkin-Elmer-Cetus) in 500 µl"Eppendorf"-Reaktionsgefäßen durchgeführt. DNA wird durch Verdauung von zu untersuchenden Proben mit Proteinase K und nachfolgende Phenol/Chloroform- Extraktion gewonnen. Zur Reaktion werden Mengen von 50 bis 1000 ng in einem 20 µl Reaktionsvolumen eingesetzt. The amplifications are carried out in DNA thermocyclers (e.g. the DNA thermal cycler 480 from Perkin-Elmer-Cetus) performed in 500 µl "Eppendorf" reaction vessels. DNA is generated by digestion of samples to be examined with proteinase K and subsequent phenol / chloroform Extraction won. Amounts of 50 to 1000 ng in a 20 µl reaction volume used.  

Der Ansatz enthält außerdem:The approach also includes:

10 mM N-Tris(hydroxymethyl)methylglycin, pH 8,3 bei 20°C (Tricin)
100 µg/ml Rinder-Serumalbumin (BSA V)
0,2 mM je Nukleotidtriphosphat (dATP, dCTP, dGTP, dTTP)
3 ng/µl je Primer
0,025 U/µl Taq-Polymerase10 mM N-tris (hydroxymethyl) methylglycine, pH 8.3 at 20 ° C (tricin)
100 µg / ml bovine serum albumin (BSA V)
0.2 mM per nucleotide triphosphate (dATP, dCTP, dGTP, dTTP)
3 ng / µl per primer
0.025 U / µl Taq polymerase

mit TNFβ als Kontrolle:with TNFβ as control:

600 mM N,N,N-Trimethylglycin (Betain)
  3 mM Magnesiumchlorid600 mM N, N, N-trimethylglycine (betaine)
3 mM magnesium chloride

mit Genen XA/XB als Kontrolle:with genes XA / XB as control:

800 mM N,N,N-Trimethylglycin (Betain)
  4 mM Magnesiumchlorid 800 mM N, N, N-trimethylglycine (betaine)
4 mM magnesium chloride

b) Temperaturschritteb) temperature steps

Die PCR-Programme werden in folgenden Temperaturschritten durchgeführt:The PCR programs are described in the following Temperature steps carried out:

Erfindungsgemäß erweisen sich in Gegenwart von Betain Hybridisierungstemperaturen, die ca. 10°C niedriger liegen als die theoretische Denaturierungstemperatur der Primer, und MgCl₂-Konzentrationen zwischen 3 und 4 mM als vorteilhaft. According to the invention, they are found in the presence of betaine Hybridization temperatures that are about 10 ° C lower than the theoretical denaturation temperature the primer, and MgCl₂ concentrations between 3 and 4 mM as beneficial.  

c) Detektionc) detection

Erfolgreiche Amplifikation der internen Kontrolle und gegebenenfalls eines allelspezifischen HLA-B-Fragments wird durch Elektrophorese in einem 1,5%-igen Agarosegel, 0,5x TBE-Puffer geprüft. DNA-Banden werden durch Anfärbung mit Ethidiumbromid und Fluoreszenz unter einer UV-Lampe sichtbar gemacht. Die Länge der erhaltenen PCR-Produkte ist eine Kontrolle für die Spezifität der PCR für HLA-B.Successful amplification of internal control and optionally an allele-specific HLA-B fragment is by electrophoresis in a 1.5% Agarose gel, 0.5x TBE buffer tested. DNA bands by staining with ethidium bromide and fluorescence made visible under a UV lamp. The length of the PCR products obtained is a control for that Specificity of the PCR for HLA-B.

d) Ergebnissed) results

HLA-B27, B 7, B 8, B 41, B 42, B 60, B 61 und B 73 wurden unter obigen Bedingungen erfolgreich amplifiziert. Die Spezifität der HLA-B PCR wurde mit Hilfe der folgenden Standard-Zellinien getestet:
Positive Kontrollen warenHLA-B27, B 7, B 8, B 41, B 42, B 60, B 61 and B 73 were successfully amplified under the above conditions. The specificity of the HLA-B PCR was tested using the following standard cell lines:
There were positive controls

  • a) für B27 und seine Subtypen: HOM2, JESTHOM, WT24, LS40 sowie die nicht-standartisierten Linien LH, NW, R69 und Wewak,a) for B27 and its subtypes: HOM2, JESTHOM, WT24, LS40 and the non-standardized lines LH, NW, R69 and Wewak,
  • b) für B60 (B*40012): SLE, MADURA, MT14B, PE117, BRU, RLO, LS40 (B27/B60)b) for B60 (B * 40012): SLE, MADURA, MT14B, PE117, BRU, RLO, LS40 (B27 / B60)
  • c) für B7: HHKB, SAVC, LD2B, R69 (B7/B27)c) for B7: HHKB, SAVC, LD2B, R69 (B7 / B27)
  • d) für B8: VAVY, LH (B8/B27)d) for B8: VAVY, LH (B8 / B27)
  • e) für B42: RSHe) for B42: RSH
  • f) für B41: RLO (B38/B41)f) for B41: RLO (B38 / B41)
  • g) für B61 (B*4002) : SWEIGg) for B61 (B * 4002): SWEIG

Als Negativkontrolle dienten außerdem zahlreiche weitere, für die häufigsten HLA-B-Allele repräsentative, Standardlinien oder Linien, deren Allelsequenz zu einem der beiden verwendeten Primern vollständig komplementär war (z. B. B14 und B16 mit B7CREG1 in der B27-PCR).Numerous also served as a negative control further, for the most common HLA-B alleles representative, standard lines or lines whose Allele sequence to one of the two primers used was completely complementary (e.g. B14 and B16 with B7CREG1 in the B27-PCR).

Von 50 sowohl serologisch als auch mit Hilfe der PCR auf HLA-B27 untersuchten Spendern wurde in 43 Fällen positive Übereinstimmung zwischen beiden Tests und in 6 Fällen negative Übereinstimmung gefunden. Eine Probe eines Patienten mit Bechterev war serologisch B27- negativ und positiv anhand des PCR-Ergebnisses. Einmal wurde kein PCR-Produkt von einem serologisch positiven gesunden Spender erhalten. Die Spezifität der PCR- Produkte wurde weiterhin durch Gelelektrophorese (Groesse) und in den Amplifikationsprodukten von 22 verschiedenen vollständig HLA-B typisierten B27- heterozygoten Proben durch Verdau mit Restriktionsenzymen bestätigt.Of 50, both serologically and with the help of PCR HLA-B27 donors were examined in 43 cases positive agreement between both tests and in 6 Cases found negative match. A sample of a patient with Bechterev was serologically B27- negative and positive based on the PCR result. once was not a PCR product from a serologically positive one receive healthy donors. The specificity of the PCR Products were continued through gel electrophoresis (Size) and in the amplification products of 22 various fully HLA-B typed B27 heterozygous samples by digestion with Restriction enzymes confirmed.

Unspezifische (Co-)Amplifikationen wurden unter den obigen Bedingungen nicht beobachtet.Nonspecific (co) amplifications were found among the not observed above conditions.

Um den Einfluß von Tetramethylammoniumchlorid, Betain und NaCl auf die Amplifikation von B7, B8 und B27 zu untersuchen wurden vier Puffer enthaltend 50 mM KCl und 1,5 mM MgCl₂ getestet: zwei handelsübliche (Boehringer Mannheim, Promega) mit Tris sowie zwei eigene mit Tris oder Tricine. To the influence of tetramethylammonium chloride, betaine and NaCl towards the amplification of B7, B8 and B27 four buffers containing 50 mM KCl were examined and 1.5 mM MgCl₂ tested: two commercially available (Boehringer Mannheim, Promega) with Tris and two own with Tris or Tricine.  

Es zeigte sich, daß 50 mM KCl sowie geringe Verunreinigungen mit 5-10 mM NaCl (final) die PCR der HLA-B-Allele der internen Kontrollen erst bei 70 mM NaCl-Konzentrationen behindern. Verbesserte Ampli­ fikation wird jedoch in Gegenwart von 50 mM TMACl erzielt und optimale PCR von HLA-B7, B8 und B27 bei 0,6-1,0 M Betain- und 2,5-4 mM MgCl₂-Konzentrationen. Oberhalb dieser Grenzen wirkt Betain inhibierend auf eine PCR mit den oben genannten Primern. Während die Amplifikation der internen Kontrollen in Abwesenheit isostabilisierender Substanzen robuster als die HLA-B PCR ist, kehrt sich dieses Verhältnis in Gegenwart von Betain und TMACl um.It was found that 50 mM KCl as well as low Impurities with 5-10 mM NaCl (final) the PCR of the HLA-B alleles of the internal controls only at 70 mM Obstruct NaCl concentrations. Improved ampli However, fication is in the presence of 50 mM TMACl achieved and optimal PCR of HLA-B7, B8 and B27 0.6-1.0 M betaine and 2.5-4 mM MgCl₂ concentrations. Above these limits, betaine has an inhibitory effect a PCR with the above primers. While the Absence of internal controls isostabilizing substances more robust than the HLA-B Is PCR, this ratio reverses in the presence of Betain and TMACl around.

2. Reverse Transkription mit M-MLV reverser Transkriptase2. Reverse transcription with M-MLV reverser Transcriptase a) Reaktionsansatza) Reaction approach

3,0 M N,N,N-Trimethylglycin (Betain)
50 mM Tris(Hydroxymethyl)Aminomethan (Tris) pH 8,3 bei 20°C
75 mM KCl
3 mM MgCl₂
10 mM 1,4 Dithio-Threitol (DTT)
400 mM je Nukleotidtriphosphat (dATP, dCTP, dGTP, dTTP)
100 mM Oligodesoxythymidin ((dT)12-18)
10 U/41 Murine Moloney-Leukaemie-Virus reverse Transkriptase (M-MLV) (Hersteller: GIBCO BRL)3.0 MN, N, N-trimethylglycine (betaine)
50 mM Tris (hydroxymethyl) aminomethane (Tris) pH 8.3 at 20 ° C
75 mM KCl
3 mM MgCl₂
10 mM 1.4 dithio-threitol (DTT)
400 mM per nucleotide triphosphate (dATP, dCTP, dGTP, dTTP)
100 mM oligodeoxythymidine ((dT) 12-18 )
10 U / 41 Murine Moloney-Leukaemie virus reverse transcriptase (M-MLV) (manufacturer: GIBCO BRL)

RNA von ca. 2 × 10⁵ Blutzellen wird durch Guanidin-Iso­ thiocyanat/Phenolextraktion gewonnen und in destillier­ tem Wasser gelöst.RNA from approx. 2 × 10⁵ blood cells is isolated by guanidine iso thiocyanate / phenol extraction obtained and distilled dissolved water.

Vordenaturierung geschieht in Gegenwart von Oligo­ desoxythymidin für 10 Minuten bei 65°C. Dann werden die übrigen Reagenzien zugefügt und für 60 Minuten bei 37°C, 43°C oder 51°C inkubiert.Pre-denaturation occurs in the presence of oligo deoxythymidine for 10 minutes at 65 ° C. Then be the remaining reagents added and added for 60 minutes Incubated 37 ° C, 43 ° C or 51 ° C.

Die erhaltene cDNA wird durch semiquantitative PCR der T-Zellrezeptorgene V-beta8, V-beta20 und C-alpha geschätzt.The cDNA obtained is obtained by semiquantitative PCR T cell receptor genes V-beta8, V-beta20 and C-alpha estimated.

Es zeigte sich, daß Betain in Konzentrationen bis zu 3 M keinen sichtbaren Nachteil auf die cDNA-Ausbeute bei den üblichen Reaktionstemperaturen hat, jedoch den Vorteil, daß die erhöhte Temperatur von 51°C von M- MLV-reverser Transkriptase erheblich besser als in her­ kömmlichen RT-Puffern ohne Betain toleriert wird.It was found that betaine was found in concentrations up to 3 M no visible disadvantage on the cDNA yield at the usual reaction temperatures, however Advantage that the elevated temperature of 51 ° C from M- MLV reverse transcriptase significantly better than in ago conventional RT buffers without betaine is tolerated.

Claims (15)

1. Puffer für DNA- und RNA-Polymerase-Reaktionen, ent­ haltend Betain.1. Buffer for DNA and RNA polymerase reactions, ent holding betaine. 2. Puffer nach Anspruch 1 für die spezifische Polyme­ rase-Ketten-Reaktion (PCR), vorzugsweise für die PCR von Histokompatibilitäts-Antigenen (HLA).2. Buffer according to claim 1 for the specific polymer rase chain reaction (PCR), preferably for the PCR of histocompatibility antigens (HLA). 3. Puffer nach Anspruch 1 oder 2 für die spezifische PCR von HLA-B-Allelen unter Verwendung von Taq- Polymerase.3. Buffer according to claim 1 or 2 for the specific PCR of HLA-B alleles using Taq Polymerase. 4. Puffer nach einem der Ansprüche 2 oder 3, enthaltend 200-1000 mM Betain.4. Buffer according to one of claims 2 or 3, containing 200-1000 mM betaine. 5. Puffer nach einem der Ansprüche 2 bis 4, enthaltend 200 bis 1000 mM Betain und 1 bis 6 mM Magnesium­ chlorid.5. Buffer according to one of claims 2 to 4, containing 200 to 1000 mM betaine and 1 to 6 mM magnesium chloride. 6. Puffer nach einem der Ansprüche 2 bis 5, enthaltend 500 bis 800 mM Betain, 1 bis 6 mM Magnesiumchlorid, 8 bis 12 mM N-Tris(hydroxymethyl)methylglycin mit pH 8,3 und 93 bis 106 µg/ml BSA V.6. Buffer according to one of claims 2 to 5, containing 500 to 800 mM betaine, 1 to 6 mM magnesium chloride, 8 to 12 mM N-tris (hydroxymethyl) methylglycine with pH 8.3 and 93 to 106 µg / ml BSA V. 7. Puffer nach Anspruch 1 für die reverse Transkription von RNA in cDNA, vorzugsweise unter Verwendung eines Oligo-dT-Primers und M-MLV reverser Transkriptase. 7. Buffer according to claim 1 for the reverse Transcription of RNA into cDNA, preferably under Use of an oligo-dT primer and M-MLV reverse transcriptase.   8. Puffer nach Anspruch 1 oder 7 für die reverse Transkription von RNA in cDNA unter Verwendung von Oligodesoxythymidin ((dT)12-18).8. Buffer according to claim 1 or 7 for the reverse transcription of RNA in cDNA using oligodeoxythymidine ((dT) 12-18 ). 9. Puffer nach Anspruch 7 oder 8, enthaltend bis zu 3 M Betain.9. Buffer according to claim 7 or 8, containing up to 3 M betaine. 10. Puffer nach einem der Ansprüche 7 bis 9, enthaltend 1 bis 3 M Betain, 20 bis 75 mM Kalium- oder Ammoniumionen und 1 bis 10 mM Magnesiumionen.10. Buffer according to one of claims 7 to 9, containing 1 to 3 M betaine, 20 to 75 mM potassium or Ammonium ions and 1 to 10 mM magnesium ions. 11. Puffer nach Anspruch 10, enthaltend 1 bis 3 M Betain, 60 bis 75 mM Kaliumchlorid und 1 bis 6 mM Magnesiumchlorid.11. Buffer according to claim 10, containing 1 to 3 M betaine, 60 to 75 mM potassium chloride and 1 to 6 mM magnesium chloride. 12. Verwendung von Betain für DNA- und RNA-Polymerase- Reaktionen.12. Use of betaine for DNA and RNA polymerase Reactions. 13. Verwendung gemäß Anspruch 12 für die spezifische PCR, vorzugsweise für die PCR von Histokompatibili­ täts-Antigenen (HLA).13. Use according to claim 12 for the specific PCR, preferably for the PCR of histocompatibility activity antigens (HLA). 14. Verwendung gemäß Anspruch 12 oder 13 zur Amplifikation eines Fragmentes des HLA-B-Gens enthaltend Sequenzen von Exon 2 bis Exon 3.14. Use according to claim 12 or 13 for Amplification of a fragment of the HLA-B gene containing sequences from exon 2 to exon 3. 15. Verwendung gemäß Anspruch 12 für die reverse Trans­ kription von RNA in cDNA, vorzugsweise unter Ver­ wendung eines Oligo-dT-Primers und M-MLV reverser Transkriptase.15. Use according to claim 12 for the reverse trans description of RNA in cDNA, preferably with Ver using an oligo-dT primer and M-MLV reverser Transcriptase.
DE4411588A 1994-03-30 1994-03-30 Buffer, contg. betaine, for RNA- and DNA-polymerase reactions Expired - Lifetime DE4411588C1 (en)

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