DE19818422A1 - Method for determining absolute mutagenicity and error rate of polymerases in amplification reactions - Google Patents

Abstract

A differential method for determining the absolute mutagenicity of a substance, is new.

Description

introduction

The invention relates to a molecular biological method of genetic engineering.

With mutagenicity and polymerase inaccuracies u. a. (Kunkel 1984), (Kunkel 1985)), (Kunkel & Alexander 1986), (Boosalis et al. 1987), (Tindall & Kunkel 1988), (Krawczak et al. 1989), (Keohavong & Thilly 1989), (Reiss et al. 1990), (Eckert & Kunkel 1990), (Lundberg et al. 1991), (Kohler et al. 1991), (U.S. Patent No. 5,347,075), (Barnes 1992), (Kok et al. 1997).

The problem arises from the long-known discrepancy between the Accuracy of in vitro polymerization reactions and the accuracy of in vivo Polymerization reactions.

In order to optimize polymerization reactions with regard to accuracy, it is necessary to establish a simple, straightforward assay that can be used and standardized for all types of mutations. The error rate or the absolute mutagenicity is uniform with f in

specified.

With sequencing alone you can determine which differences in a SE quantity of occurrences. So if you compare the error rate in multiple sequence sets before and after polymerization, as well as the degree of polymerization, i.e. the amplifica determined, the error rate of the reaction can be calculated with formula (3). For this invention is sought protection.

One can determine error rates of polymerases with this invention, but also the absolute mutagenicity of reaction additives such. B. of food or Measure medication and absolute cancer-generated cancer rates z. B. from Detect TVs or computer monitors.

All types of mutations can be considered (substitutions, inserts tions and deletions) and together or individually. Influences  of repair mechanisms or selection mechanisms that exist in vivo Not. These are pure in vitro (S1) experiments, animal experiments are unnecessary.  

Description of the pictures

Fig. 1: Product composition after n cycles

Fig. 2: Error distribution after n cycles

Fig. 3: Filtration profile of column-purified EN primed NF1 cDNA; Caught in two drop fractions and examined with C / E-PCR (30 cycles)

Fig. 4: Amplification of NF1 from rat brain cDNA; Temperature profile: 94 ° C, 30 sec; 68 ° C, 9 min. (last step 68 ° C 10 min); 22 then amplified for 20 cycles; Lane 1: λ-Hind III standard; 2: -; 3: -; 4: FAN / AD without SK; 5: FAN / AD amplification with SK; 6: standard;

Fig. 5: Determination of the error rates of NF1 pieces

Fig. 6: Product composition after 22 cycles

Fig. 7: Product composition after 42 cycles

Fig. 8: Table of values from Fig. 6 and Fig. 7; Coefficients developed according to Fig. 2; % based on 2 ²² and 2 ⁴² ; y in cm: 20% ≈ 8 cm

Fig. 9: Sketch of NF1 with the position of the primers

Derivation

During a polymerization reaction, a polymerase makes mistakes when incorporating L nucleotides into the resulting double strand F. So the response has an error rate of

When copying a single-stranded molecule of length 1, a polymerase in the 1st approximation makes mistakes. Probabilities are additive. When synthesizing the copy of the copy of the molecule of length l, the enzyme makes lf mistakes again in a first approximation. After two synthesis cycles, one molecule contains 1.2 f more errors. After n cycles, a molecule of length l has five errors more than before the synthesis. The original has the error or. Without a copy, there are l.or errors in the source material. After n copies, a molecule contains F (n, f) errors:

F (n, f) = l. (Or + n.f) (1)

It is now important, however, how many molecules with how many defects are in the end product. The end product contains both the original and its copies. Fig. 1 describes the product composition after n cycles, whereby each molecule is only characterized by how often it has been copied.

This distribution corresponds to the triangular distribution published by B. Pascal (1665) (Chevalier 1954). Fig. 2 is the more mathematical spelling of Fig. 1. B. after 6 reaction cycles 20 molecules that were copied 3 times, but only one molecule that was copied 6 times. As can be clearly seen ( Fig. 2, Fig. 6, Fig. 7), the majority of the molecules have after amplification

Error.

Since the frequency of the molecules that were copied n times is symmetrically distributed with respect to n ( Fig. 6, Fig. 7), and the error rate is proportional to the number of these synthesis cycles, the error rate is also distributed symmetrically. The mean error rate is:

When determining error rates before and after amplification by determining the number of errors F in a set of L sequenced nucleotides:

the error rate of the reaction can be determined from the difference:

So

The distribution model can be tested by checking whether the product has the predicted error distribution for a given number of cycles ( Fig. 6, Fig. 7).

Because DNA is double-stranded and one strand is always a copy of the other Is strand, the number of errors of the copied strand is always l.f larger. The Sequencing templates are double-stranded DNA fragments. With a probability l.f one finds two nucleotides in a sequencing of both strands for  a position. This error was generated by the polymerase in the last cycle the. Such an error is also sequenced to the set L = 2.l of the total Nucleotides related.  

Generation of an amplification template

Adult rat brain is homogenized in filtered solution (approx. 2 ml per 100 mg tissue):

• 5 M guadinium thiocyanate,
• - 100 mM Tris-HCl (pH 7.6),
• - 10 mM EDTA,
• - 0.5% lauryl sarcosine and
• - 0.1 M 2-mercaptoethanol

Become a clear solution

• - 2/10 volumes of chloroform
• - 1 volume of acidic phenol
• - 1/10 volume sodium acetate (pH 4)

given. The mixture is then extracted and centrifuged, the aqueous phase is removed and precipitated with 1 volume of isopropanol (-20 ° C., 1 hour) (Chomczynski & Sacchi 1987). The RTA thus obtained is used in

• - 100 mM Tris-HCl (pH 7.6)
• - 10 mM EDTA
• - 0.5% lauryl sarcosine

dissolved and mixed with 1 g / ml CsCl. In a cellulose nitrate centrifuge tube, a CsCl cushion is overlaid with RNA solution in a ratio of 1/3. RNA precipitated by centrifugation (30,000-50,000 rpm, 16 ° C, 16 hours) (Meselson et al. 1967) is used in:

• - 100 mM Tris-HCl (pH 7.6)
• - 10 mM EDTA
• - 0.5% lauryl sarcosine

solved. CsCl is separated by reprecipitation twice (100 mM NaCl, 2.5 volumes of ethanol, -20 ° C., 1 hour) (Glisin et al. 1974). The RNA obtained is dissolved (65 ° C., 5 min):

• - 74 mM Tris-HCl (pH 7.6)
• - 10 mM EDTA
• - 0.1% SDS

and brought to 500 mM NaCl. Solid phase-coupled oligo dT is added and hybridized (37 ° C., 10 min) (Gilham 1964). It is washed three times in:

• - 74 mM Tris-HCl (pH 7.6)
• - 10 mM EDTA
• - 0.1% SDS
• - 0.5 M NaCl

and three times with:

• - 74 mM Tris-HCl (pH 7.6)
• - 10 mM EDTA
• - 0.1% SDS
• - 0.1 M NaCl

Elute at 60 ° C for 5 min with:

• - 74 mM Tris-HCl (pH 7.6)
• - 10 mM EDTA
• - 0.1% SDS

For NF1 cDNA synthesis, 20 µg mRNA are dissolved in water with primer EN (AGC TTC ACA CGA TCT TCT TAA TGC) and 1 U RNAsin and denatured (94 ° C, 1 min). At the synthesis temperature (42 ° C) reverse transcriptase conditions are set:

• - 50 mM Tris-HCl (pH 8.3),
• - 50 mM KCl,
• 10-3 mM MgCl ₂ ,
• 1-5 mM DTT,
• - 1 mM dNTP's and
• - 5 U RNAsin.
• - 200-20 nM primer

200 U MMLV are added and an incubation (42 ° C., 1/2 hour) follows. 1-5 U AMV and 100 U MMLV are added and incubation is continued (60 ° C, 1/2 hour). Then the sample is mixed with 1 µl RNAse A (stock solution: 1 mg / ml in 5 mM Tris-HCl (pH 8); cooked: 10 min. 100 ° C) denatured at 1 min 94 ° C and store the sample at 4 ° C.

The whole batch is gel filtered with a Sepharose 4-bcl column. Solvent is:

• - 10 mM Tris-HCl (pH 8)
• - 1mM EDTA  
• - 100 mM NaCl

The eluate is collected in two drops of fractions and examined. 2 µl of the fractions are amplified with PCR (Primer C: CAG AAT TCC CCC CTC AAC TTC GAA GT and Primer E: AAG TGT TGC CAT CTT ATC AAA AGG TC):

• - 20 mM Tris-HCL (pH 8.8)
• - 10 mM KCl
• - 2 mM MgSO ₄
• - 0.1% Triton X-100
• - 100 µg / ml BSA
• - 400 µM dNTPs
• - 0.25 U heat-stable DNA polymerase
• - 400 nM primer
• - temperature profile:
  • - 94 ° C, 1 min,
  • - 60 ° C, 1 min,
  • - 73 ° C, 1 min

The samples are mixed with 5 × sample buffer:

• - 0.025% bromophenol blue,
• - 0.025% xylene cyanol,
• - 20% Ficoll and
• - 100mM EDTA

added and in a 2% agarose gel stained in an ethidium bromide:

• 89 mM Tris-borate (pH 8),
• - 89 mM boric acid,
• - 2 mM EDTA,
• - 0.4 µg / ml ethidium bromide and
• - 2% agarose

examined (

Fig.

3).

Fractions containing NF1 are pooled and precipitated. The cDNA is in

• - 10 mM Tris-HCl (pH 8)
• - 1mM EDTA

solved.

Amplification

8 µl of the cDNA approach are diluted with 12 µl 25 mM Mg (OAc) ₂ and 8 µl water. This mixture is denatured at 94 ° C for 1 min. 7 µl become a preheated reaction mix:

• - 15.15 µl 3.3 × polymerase buffer
• - 2 µl 5 µM primer CAT TGT ACC TAG TTA ATT AAT CTC CAC CAT GGT CGC ACA CAG GCC GGT GGA (FAN)
• - 1 µl SK primer (Harjes 1998)
• - 2 µl 5 µM primer AAT GGA TTG GTT GCG GCC GCC TAT CAC ACG ATC TTC TTA ATG CTA TTA CGT TTG AAA CTG CCA GCA CTC CTT TGC (AD)  
• - 1 U Tth _XL polymerase (Myers & Gelfand nd)
• - 1 µl 10 mM dNTPs
• - 20.85 µl water

given (94 ° C). 22 cycles with the following temperature profile are amplified:

• - 68 ° C, 9 min
• - 94 ° C, 30 sec
• - last step: 68 ° C, 10 min

With 12 µl 25 mM Mg (OAc) ₂ and 12 µl water, 4 µl of this amplification is diluted, heated to 94 ° C, the reaction mix is added and amplified again (20 cycles). The products of the 42 cycle amplification are shown in Fig. 4.

Cloning

In order to be able to form the differences of formula (3), one must generate sequencing templates for the product and template under the same conditions as possible. For this purpose, part of the amplification from Fig. 4. Lane 5 is reamplified after 22 and 42 cycles with partially homologous primers. (Primer ES-5: CCG GAATTC _EcoRI GCC GCC ATG GGG AGG CCT TTT AAT GAT TTT GTG; and Primer ES-3: GC T CTA GA _XbaI C TAC TTG TGC TCC GGA GGA CCC with XbaI):

• - 20 mM Tris-HCL (pH 8.8)
• - 10 mM KCl
• - 2 mM MgSO ₄
• - 0.1% Triton X-100
• - 100 µg / ml BSA  
• - 400 µM dNTPs
• - 0.25 U heat-stable DNA polymerase
• - 400 nM primer
  • - temperature profile:
  • - 94 ° C, 1 min
  • - 50 ° C, 1 min
  • - 73 ° C, 1 min

In order to enable the primers to hybridize to the sequence to be cloned, the first annealing step is carried out at approximately 40 ° C. (21/19 3'-bases of the primers hybridize to the NF1 sequence). The 22 cycle approach is reamplified 20 cycles, the 42 cycle approach is reamplified 15 cycles. The PCR products are ethanol precipitated and EcoRI / XbaI cut:

• - 50 mM Tris-HCl (pH 7.5)
• - 10 mM MgCl ₂
• - 100 mM NaCl
• - 1mM DTE
• - 30 U EcoRI
• - 150 U XbaI
• - in 100 µl water

The restriction endonucleases are heat inactivated (70 ° C, 10 min) and the DNA is precipitated (2.5 volumes of CH ₃ CH ₂ OH, -20 ° C, 1 hour). The fragments are dissolved in H ₂ O and alloyed in precut (EcoRI / XbaI) dephosphorylated cloning vector (15 ° C., 2 hours).

• - 100ng cloning vector
• - 100 ng EcoRI / XbaI fragment ≈ 3 × molar excess of fragment)
• - 1 × ligation buffer
• - 1 U ligase
• - 1 mM ATP

The ligation approaches are transfected into competent bacteria. These are spread on agar plates and multiplied for 12 hours. 5 ml cultures are inoculated from individual colonies and multiplied. Plasmid is purified and examined. Plasmid containing fragment is sequenced according to the methods of (Sanger et al. 1977). Clones 42 ₁ and 42 _{2 were} sequenced from the amplification after 42 cycles, clones 22 ₁ to clone 22 _{6 were} sequenced from the amplification after 22 cycles ( FIG. 5).

evaluation

The following was measured ( Fig. 5):

For f:

If one assumes a sequencing accuracy of 1 nucleotide in 1398 sequenced nucleotides during the sequencing, there is a fluctuation of Δf ≈

For exact measurements, it is necessary to determine the amplification quantitatively. Here Δn ≈ 10 ^-3 ... 30 is generously estimated.

With these error bounds, the range is:

so

Summary

For the first time, the accuracy f of the heat-stable DNA polymerase Tth _XL in 1 × Tth _XL buffer with NF1 cDNA was measured differentially:

This method can be used to calculate absolute mutagenicity and polymerase standardize errors.

With differential in vitro measurements one can mutagenicities of in vivo Si absolutely characterize tuations.  

literature

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Boosalis, MS, Petruska, J. & Goodman, MF (1987), Dna polymerase insertion fidelity, The Journal of Biological Chemistry 262 (30), 14689-14696.
Chevalier, J. (1954), uvres completes, Édition Gallimard. P. 91.
Chomczynski, P. & Sacchi, N. (1987), "Single-step method of rna isolation by acid guadinium thiocyanate-phenol-chloroform extraction", Analyti cal Biochemistry 162, 156-159.
Eckert, KA & Kunkel, TA (1990), "High fidelity dna synthesis by the ther mus aquaticus dna polymerase", Nucleic Acids Research 18 (13), 3739-3744.
Gilham, PT (1964), "The synthesis of polynucleotide-celluloses and their use in the fractionation of polynucleotides", Journal of the American Chemical Society 86, 4982-4985.
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Harjes, S. (1998), Neurofibromin, PhD thesis, Ruhr-Universität-Bochum, in preparation.
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Kunkel, TA (1984) "Mutational specifity of depurination", Proc. Natl. Acad. Sci. USA 81, 1494-1498.
Kunkel, TA (1985), "The mutational specificity of dna polymerase-β during in vitro dna synthesis", The Journal of Biological Chemistry 9, 5787-5796.
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Claims (6)

1. Method for determining the absolute mutagenicity, characterized in that it is determined differentially. 2. Method for determining the absolute mutagenicity, characterized in that the error rate
is determined with sequencing. 3. Method for determining the absolute mutagenicity, thereby ge indicates that the amplification is determined quantitatively. 4. Method for determining the absolute mutagenicity, thereby ge indicates that this is calculated according to Formula 3. 5. Method for determining the absolute mutagenicity, thereby ge indicates that the average error rate of the DNA to be determined is proportional to half the amplification. 6. Method for determining the absolute mutagenicity, thereby ge indicates that the error rate is symmetrically distributed.