© 2000 Oxford University Press
Nucleic Acids Research, 2000, Vol. 28, No. 8
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One tube mutation detection using sensitive fluorescent dyeing of MutS protected DNA Pawel Sachadyn*, Anna Stanislawska and Józef Kur Technical University of Gdansk, Department of Microbiology, ul. Narutowicza 11/12, 80-952 Gdansk, Poland Resubmitted January 2, 2000; Revised February 21, 2000; Accepted March 1, 2000
ABSTRACT
MATERIALS AND METHODS
A novel, universal method for mutation detection utilising the ability of MutS protein to recognise DNA incomplementarities is proposed. The examined and reference DNA fragments are PCR amplified. The PCR products are purified, mixed, heated and cooled to form heteroduplexes. In the case of mutation the heteroduplex DNA containing mismatch is protected against exonuclease digestion by MutS, while the DNA without mismatches is degraded. The protection effect is visualised by the direct addition of a highly sensitive fluorescent dye (SYBR-Gold) selectively binding DNA. The Thermus thermophilus recombined His-tagged MutS and 3′–5′ exonuclease activity of T4 DNA polymerase were used in the assay.
The DNA fragments used in this assay were obtained by PCR amplification. The fragments of each pair were identical except for a single mutation (Table 1). The DNA sequences were chosen for the preliminary tests because they are precisely characterised and easily available. The PCR products were purified using affinity spin mini-columns (DNA clean-up kit, A&A Biotechnology, Poland). The amount of DNA was measured using DNA calculator Genequant (Pharmacia). To prepare the heteroduplexes, the DNA fragments were mixed as indicated in Table 2A and B, placed in thin wall PCR 0.2 ml tubes, 5 µl PCR buffer was added [100 mM Tris–HCl (pH 8.8 at 25°C), 500 mM KCl, 1.0% Triton X-100, 16 mM MgCl2] and completed with water to 45 µl. The closed tubes containing DNA were heated for 2 min at 90°C so as to denature DNA. In order to renature the DNA the samples were transferred to 65°C for 2 min and next to 37°C for 2 min. Then, histidinetagged Thermus thermophilus MutS was added as indicated in Table 2. The amount of added MutS was adjusted experimentally, which is why we have not given a universal DNA to MutS ratio. However, it seems that the amount of MutS protein added should be calculated in relation to the number of DNA molecules (which means that the amount of MutS per 1 µg of 500 bp DNA should be twice as large as for 1 µg of a 1000 bp DNA fragment). The mixture was incubated at 65°C for 5 min to form DNA–MutS complexes, then it was transferred to 37°C for 2 min, followed by the addition of 1 µl (5 U) of T4 DNA polymerase (Fermentas MBI, Lithuania) and incubated at 37°C for a further 10 min. Then 0.5 µl of 100× concentrated SYBR-Gold solution prepared from 10 000 concentrated original stock (Molecular Probes) was added. The following parallel control samples were prepared: background control (the sample without DNA), undigested DNA controls (DNA without MutS and T4 DNA polymerase), MutS specificity controls (homoduplex DNA without mismatches), digestion control (DNA without MutS addition, T4 polymerase treated). For details see Figure 2A and B, and Table 2A and B. The fluorescence was induced by UV transilluminator (312 nm) and the samples were photographed using a Polaroid camera. The fluorescence was measured by densitometric analysis using Scanpack 2.0 system (Biometra) (Table 2). After 10-fold dilution in water the fluorescence values of the samples were measured using a spectrofluorometer at an excitation/emission of 495/537 nm) (LS-5B, Luminescence spectrometer, Perkin Elmer).
INTRODUCTION MutS protein is an element of the DNA repair system responsible for detection of pre-mutation changes in cells by DNA binding at the sites containing mispaired bases. In vitro MutS is reported to recognise efficiently single base mismatches as well as deletions or insertions of up to three bases (1,2). Most of the methods described so far for mutation detection using MutS protein utilise either its ability to retard DNA migration (gel shift assays, gel mobility assay) (3) or the detection of labelled DNA heteroduplexes captured by MutS immobilised on filters (4). There are also reports based on the ability of MutS to protect mismatched heteroduplex DNA against DNase (footprinting) (5) or exonuclease digestion. DNA protected by MutS against 3′–5′ exonuclease digestion is visualised on sequencing gels (6), Pharmacia–EMD system. The method using protected DNA visualisation on sequencing gels allows the estimation of mutation position but requires electrophoresis and DNA labelling. The proposal to detect mutations by PCR amplification of MutS protected DNA (7) seems to be a risky approach for routine analyses as reliable results require both perfect digestion of non-protected DNA and perfect specificity of MutS binding. In the approach we propose, the effect of mismatched DNA protection against exonuclease digestion is visualised by direct dyeing of the undigested DNA using sensitive fluorescent dyes like SYBR-Gold. Thus, the mutation detection requires one tube assay, without DNA labelling and electrophoresis.
*To whom correspondence should be addressed. Tel: +48 58 347 2406; Fax: +48 58 347 1822; Email:
[email protected]
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Table 1. The description of examined DNA fragments Name of PCR product
Origin
Length (bp)
Mutation
Mutation position
Mismatch
A143(262)
β-lactamase gene, pUC19 plasmid
262
G→A (C→T) substitution
143
G-T, A-C
G143(262)
β-lactamase gene, pBR322 plasmid
262
A→G (T→C) substitution
143
G-T, A-C
InsTAA516(716)
artificial construct
716
TAA insertion
516
Trinucleotide bubble
DelTAA516(713)
artificial construct
713
TAA deletion
516
Trinucleotide bubble
Figure 1. The scheme presents SYBR-Gold-MutEx assay. The examined DNA (mut) is mixed with reference DNA (wt), the mixture is heated, cooled, then MutS is added, followed by the addition of DNA exonuclease (T4 DNA polymerase). MutS binds to DNA mismatches. The DNA complexed with MutS is partially protected against exonuclease digestion. The results are visualised by sensitive fluorescent dye (SYBR-Gold).
RESULTS AND DISCUSSION There is no doubt that MutS protein is a great tool for mutation detection because of its ability to bind to DNA mismatches. ii
Observing this effect involves nothing more than examining protein–DNA complexes. Among many possible approaches to this examination, the one we chose characterises with
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Table 2. (A) The results of SYBR-Gold-MutEx assay for T-C (A-G) mutation G-T and A-C mismatches Sample number
Sample content
Fluorescence (densitometry)
Relative fluorescence (densitometry) (%)
Fluorescence (ex/em 495/537 nm)
Relative fluorescence (ex/em 495/537 nm) (%)
22
0
8.9
0
DNA heteroduplex, mismatch, 150 ng of A143(262) + 150 ng of G143(262)
231
100
428.6
100
3
DNA homoduplex, no mismatch, 300 ng of A143(262)
231
100
406.5
100
4
DNA homoduplex, no mismatch, 300 ng of G143(262)
231
100
494.3
100
5
DNA heteroduplex, mismatch, 150 ng of A143(262) + 150 ng of G143(262), MutS (3 µg), T4 DNA polymerase
176
74
189.4
43
6
DNA homoduplex, no mismatch, 300 ng of A143(262), MutS (3 µg), T4 DNA polymerase
47
12
16.8
2
7
DNA homoduplex, no mismatch, 300 ng of G143(262), MutS (3 µg),T4 DNA polymerase
43
10
19.9
2
8
DNA heteroduplex, mismatch, 150 ng of A143(262) + 150 ng of G143(262), T4 DNA polymerase
42
10
13.0
1
9
DNA homoduplex, no mismatch, 300 ng of A143(262), T4 DNA polymerase
42
10
15.0
1.5
10
DNA homoduplex, no mismatch, 300 ng of G143(262), T4 DNA polymerase
34
6
16.0
1.5
1
Background, MutS (3 µg), T4 DNA polymerase
2
The relative fluorescence was calculated according to the following formula: (f – fb)/(fDNA – fb)/ ×100% (f, fluorescence of the sample; fb, background fluorescence; fDNA, fluorescence of the DNA non-treated by MutS and T4 phage polymerase exonuclease digestion.
appalling directness. In the procedure we propose, the mismatched heteroduplex DNA protected against 3′–5′ exonuclease digestion by MutS is visualised by the direct addition of a highly sensitive fluorescent dye like SYBR-Gold or SYBR-Green (Molecular Probes) selectively binding DNA (Fig. 1). The method utilises 3′–5′ exonuclease activity of T4 bacteriophage DNA polymerase and T.thermophilus recombined His-tagged MutS protein (cloning, purification and characterisation of the His-tagged MutS from T.thermophilus will be published elsewhere). Two DNA fragments for mutation (polymorphism) examination were PCR amplified. The 262 bp PCR products were identical except for a single nucleotide at position 143 (T-C substitution, see Table 1). The PCR products were purified, mixed, heated and cooled to form heteroduplexes. Then MutS was added, followed by the addition of exonuclease (T4 DNA polymerase). The same procedure was applied to a set of parallel control samples. The parallel controls of homoduplex DNA (without mismatches) were prepared so as to investigate the specificity of the assay (Fig. 2A, Table 2A, samples 6 and 7). Additional control samples without MutS and T4 polymerase (exonuclease) addition (samples 2–4) and without MutS addition but exonuclease-treated (samples 8–10) were prepared in order to compare the changes in fluorescence. The
samples without DNA were prepared to estimate the background fluorescence (sample 1). The fluorescence of the sample containing the mismatched heteroduplex (sample 5) was significantly higher than for both control samples without mismatches (samples 6 and 7). The difference could be easily noticed with the naked eye. The fluorescence levels for the T4 polymerase treated samples (samples 6–10) were considerably lower than for the samples without exonuclease treatment (samples 2–4) though higher than background level (sample 1). The fluorescence maintained at the similar level for at least 24 h at room temperature. A second experiment, applying the same procedure as described above, was performed using another pair of DNA fragments. The DNA fragments were >700 bp, identical except for one codon deletion in one of them (Table 1). The results of this experiment, presented in Figure 2B and Table 2B, were similar to the first one. The fluorescence of the MutS protected heteroduplex DNA (Fig. 2B and Table 2B, sample 5) was significantly higher compared with homoduplex samples (samples 6 and 7). These experiments were reproducible as similar results were obtained several times. To compare the fluorescence between the samples the relative fluorescence was calculated (Table 2A and B). The values of relative fluorescence obtained in the two described iii
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Table 2. (B) The results of SYBR-Gold-MutEx assay for trinucleotide (TAA/ATT) insertion detection Sample number
Sample content
1
Background, Muts (1 µg), T4 DNA polymerase
2
Fluorescence (densitometry)
Relative fluorescence (densitometry) (%)
Fluorescence (ex/em 495/537 nm)
Relative fluorescence (ex/em 495/537 nm) (%)
2
0
1.9
0
DNA heteroduplex, mismatch, 180 ng of InsTAA516(716) + 180 ng of DelTAA516(716)
219
100
105.7
100
3
DNA homoduplex, no mismatch, 360 ng of InsTAA516(716),
219
100
107.4
100
4
DNA homoduplex, no mismatch, 360 ng of DelTAA516(716)
217
100
101.9
100
5
DNA heteroduplex, mismatch, 180 ng of InsTAA516(716) + 180 ng of DelTAA516(716), Muts (1 µg), T4 DNA polymerase
120
54
32.4
29
6
DNA homoduplex, no mismatch, 360 ng of InsTAA516(716), Muts (1 µg), T4 DNA polymerase
35
15
8.0
6
7
DNA homoduplex, no mismatch, 360 ng of DelTAA516(716), Muts (1 µg), T4 DNA polymerase
59
26
11.8
10
8
DNA heteroduplex, mismatch, 180 ng of InsTAA516(716) + 180 ng of DelTAA516(716), T4 DNA polymerase
25
11
5.0
3
9
DNA homoduplex, no mismatch, 360 ng of InsTAA516(716), T4 DNA polymerase
18
7
3.9
2
10
DNA homoduplex, no mismatch,360 ng of DelTAA516(716), T4 DNA polymerase
37
16
7.4
5
The relative fluorescence was calculated as in Table 2A.
experiments were not identical for the respective samples, which should be explained by different sizes and polymorphisms of the examined DNA fragments. The differences between the values of relative fluorescence derived from the results obtained by densitometry and measured using spectrofluorometer are not surprising, as in the first method a range of emission wavelengths was measured, while in the latter only a single emission peak at 537 nm was used. The system, which turned out to be suitable for 700 bp DNA fragments, could possibly be applied in the case of bigger fragments. However, it seems to be essential that the amount of T4 DNA polymerase is adjusted to the mass of DNA, whereas the amount of MutS should be adjusted to the number of the examined DNA molecules. This is because the processivity of exonuclease is limited by the mass of DNA, while MutS–DNA binding is described by a molar ratio. The undigested excess of DNA could give a strong background, while the excess of MutS may result in non-specific protection. As the signals from the unprotected samples were always above the background level, the parallel control without MutS is necessary to avoid false positive results. It is also recommended, but not absolutely required, to prepare controls without DNA (to estimate background) and containing only DNA (at the same iv
concentration as in the examined samples) for comparison with the exonuclease treated samples. We assume that other fluorescent dyes could also be used in this approach (SYBR-Green, PicoGreen, results not shown), but among those we tested SYBRGold was the most useful. Ethidium bromide was not sensitive enough (results not shown). The results may be measured using spectrofluorometers, Elisa readers or densitometric equipment but also estimated with the naked eye. The advantage of this method is the elimination of electrophoresis and DNA labelling, combined with the fact that only tiny amounts of DNA are necessary for the assay. It seems that MutS from T.thermophilus, which is reported to detect efficiently all eight possible types of mismatches and insertion/deletions up to three bases (1), is the best choice for mutation detection among the MutS proteins that have been characterised so far. The recombined MutS protein possessing a histidine tag at the N-terminus that was used in the study was obtained in the one step purification procedure and was proven to be functional. The approach does not limit the spectrum of point mutations possible for detection, but it does not define the detected mutation. It seems that for these reasons it will be most appropriate for rapid, preliminary tests and scanning for unknown polymorphisms. The rapid visualisation of protein protected
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DNA using fluorescent dyes may be also applied to the study of other DNA binding proteins. As MutS is able to recognise some chemical modifications of DNA (8,9) the test could possibly be useful in examination of DNA adducts, crosslinking etc. The method, which we have named the SYBR-Gold-MutEx assay, is cheap, simple and rapid. It does not require any instrumentation other than basic PCR equipment. It may be applied to routine tests for mutation scanning and could be easily automated. We conclude that the proposed method for mutation detection could be useful for routine laboratory and clinical tests. ACKNOWLEDGEMENT We thank A&A Biotechnology for the supply of minicolumns. This work was supported by the Technical University of Gdansk and DNA-Gdansk II s.c. REFERENCES
Figure 2. The results of SYBR-Gold-MutEx assay for a single substitution (A) and a trinucleotide deletion (B). (A) Sample 1 contained no DNA (background control); samples 2–4 contained undigested DNA; sample 5 contained heteroduplex DNA protected against exonuclease digestion by MutS; samples 6 and 7 contained homoduplex DNA (no mismatches) digested by exonuclease, although MutS was present; and samples 8–10 contained DNA without MutS addition digested by exonuclease. The detailed contents of the samples and results are presented in Table 2A. (B) Sample 1 contained no DNA (background control); samples 2–4 contained undigested DNA; sample 5 contained heteroduplex DNA protected against exonuclease digestion by MutS; samples 6 and 7 contained homoduplex DNA (no mismatches) digested by exonuclease, although MutS was present; samples 8–10 contained DNA without MutS addition digested by exonuclease. The detailed contents of the samples and results are presented in Table 2B.
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