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Nucleic Acids Research, 2000, Vol. 28, No. 9 E38-e38
© 2000 Oxford University Press

Detection and mapping of mismatched base pairs in DNA molecules by atomic force microscopy

Masato Tanigawa, Masanori Gotoh1, Masayuki Machida2,*, Takao Okada and Michio Oishi3

Joint Research Center for Atom Technology, 1-1-4 Higashi, Tsukuba, Ibaraki 305-0046, Japan, 1Amersham Pharmacia Biotech K.K., 3-25-1 Hyakunin-cho, Shinjuku-ku, Tokyo 169-0097, Japan, 2National Institute of Bioscience and Human-Technology, 1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan and 3Kazusa DNA Research Institute, 1532-3 Yanauchino, Kisarazu, Chiba 292-0812, Japan

Received August 1, 1999; Revised and Accepted February 10, 2000.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Attempts were made to apply atomic force micro­scopy (AFM) imaging to the detection and mapping of the sites of base substitutions in DNA molecules. In essence, DNA fragments to be examined for possible base substitutions were mixed with an equal amount of a corresponding DNA standard and subjected to heat denaturation and subsequent annealing. The reassociated DNA was incubated with MutS protein, a protein that recognizes and binds to mismatched base pairs in duplex DNA. Bound MutS protein molecules were then detected by AFM and their positions along the DNA molecules were determined by calculating the distance from one of the DNA termini, which had been tagged with a biotin–avidin complex. Base substitutions present in DNA molecules >1 kb were effectively detected by this procedure, and the positions determined were in good agreement with the actual mutation sites. This method is quite simple, has virtually no limitations on the size of DNA fragments to be examined and requires only a very small amount of DNA sample.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Simple and efficient methods are urgently needed to detect the sites in DNA molecules that are altered by base substitutions. Such mutations account for most human genetic diseases, cancers and polymorphisms. Among a number of procedures that have been used to detect such alterations, sequencing the DNA molecules of interest is, of course, most reliable, but is quite time consuming and costly. The other methods that have been used most often for the detection of small genomic alterations include single-strand conformation polymorphism (SSCP) (1) and denaturing gradient gel electrophoresis (DGGE) (2) analyses. These methods, however, are also quite labor intensive and limited to DNA molecules no longer than several hundred base pairs. One of the approaches gaining attention these days is to make use of MutS protein, one of the components of in vivo mismatch repair systems (3). MutS protein plays a central role, recognizing and binding to mismatched base pairs. Previously reported procedures that rely on MutS protein alone for the detection of base substitutions, however, fall into two categories. In the first category, hetero­duplexes (DNA duplexes containing one or more mismatched base pairs) bind with MutS protein, and are retained upon filtration through a nitrocellulose membrane (4). In the second category, DNA is immobilized to the surface of an optical biosensor that detects the alteration in mass when MutS protein binds (5). These methods have been used optimally on relatively short DNA fragments of several hundred base pairs, similar to those for the electrophoresis-based methods (6,7).

Atomic force microscopy (AFM) is a powerful tool for analyzing DNA structures as well as DNA–protein complexes (8–10). Previously we reported that AFM imaging could be applied to determine effectively the recognition site for a transcription factor in DNA molecules as large as several kilobases (11). Major advantages of AFM imaging include ease of analysis and an extended size range of DNA molecules amenable to examination. Therefore, as a first step toward identifying all types of mutations and polymorphic sites, we explored the possibility of applying AFM to the detection and mapping of substituted bases. Employing essentially the same conditions as used for detecting the binding sites for a transcription factor, we were able to use MutS protein to detect base substitutions in DNA molecules >1 kb and furthermore to detect the locations of such changes. In this paper, we present our findings and conditions for the detection and mapping of the sites of base substitutions in DNA molecules by AFM.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
DNA fragments were prepared by PCR (Pwo DNA polymerase, Boehringer Mannheim) using Escherichia coli wild type and mutant MutS gene sequences, which were cloned as pTrc99a plasmid constructs (5). DNA fragments (1066 and 3556 bp) were prepared using primer pairs (5'-CTGGCACCCATCAACTACATGCGT-3' and 5'-GGGTGAGTGAATCCGGATCAAGAT-3') and (5'-GTTTGACAGCTTATCATC-GACTGCA-3' and 5'-CTGTTGAGATCCAGTTCGATGTA-AC-3'), respectively. pTrc99a-A, -B and -C carried mutant inserts in which GC at nucleotides 883 and 884 were substituted for CT, GC at nucleotides 640 and 641 for CG, and A at nucleotide 527 for C, respectively. For the preparation of heteroduplex DNA between the wild-type and mutant sequences, equal amounts of wild-type and mutant DNA were mixed, followed by heat denaturation and reassociation. In this way, 1066 bp fragments were prepared that contained either (G/G and A/C) or (C/C and T/G) at 883 and 884 bp (type A mismatch fragment); (G/G and C/C) or (C/C and G/G) at 640 and 641 bp (type B mismatch fragment); and either A/G or C/T at 527 bp (type C mismatch fragment). Likewise 3556 bp fragments were prepared bearing either (G/G and A/C) or (C/C and T/G) mismatch at 946 and 947 bp (type D mismatch fragment).

MutS protein was purified from E.coli overexpressing MutS protein as previously reported (6). Complex formation between MutS protein and mismatched base pairs in the hetero­duplex was performed by incubating DNA (25 ng) and MutS protein (400 ng) in a reaction mixture containing ADP (2 mM), MgCl2 (8 mM), EDTA (1 mM), DTT (1 mM) and HEPES–KOH (50 mM, pH 9.0) for 2 min at room temperature. The DNA was then fixed on mica plates, which had been treated with poly-L-lysine. For tagging one of the DNA termini with avidin, 5'-biotinylated primers were used for PCR and the DNA fragments were conjugated with avidin molecules in a buffer containing NaCl (0.15 M) and NaHCO3 (0.1 M, pH 8.5) at room temperature.

AFM analysis was carried out with Nanoscope III, Multi Mode system (Digital Instruments Inc., Santa Barbara, CA) in tapping mode at room temperature in air. Silicon cantilevers of 126 µm in length with a spring constant of 40 N/m were purchased from Nanosensors (Wetzlar-Blankenfeld, Germany). Typical resonant frequencies of these tips were ~280 kHz. The 512 x 512 pixel images were collected at a rate of two scan lines per second. The contour lengths of DNA were determined using software (NIH Image, NIH, Bethesda, MD) for digital image analysis.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Detection of base substitution sites was carried out by AFM in a series of model experiments. As a first step, each mutant DNA fragment was mixed with an equal amount of the wild-type fragment, heat-denatured and reannealed. MutS protein, which binds specifically to mismatched base pairs, was then added to form MutS protein–mismatched base pair complexes with the DNA heteroduplexes (Materials and Methods). After complex formation the DNA samples were subjected to AFM imaging. In order to determine the positions of base substitutions along the DNA molecules, the distance from the avidin-tagged DNA terminus to the bound MutS protein was calculated.

Figure 1 shows the actual AFM images of MutS–DNA complexes obtained from DNA molecules carrying single- or tandem-mismatched base pairs. The mutant fragment used for heteroduplex formation in Figure 1a carried contiguous tandem base substitutions (CT to GC). Thus heteroduplex DNA molecules formed by reannealing of wild-type to mutant strands should have a tandem base mismatch (type A). As shown in Figure 1, apparent MutS protein–mismatched base pair complexes were observed clearly after AFM imaging (indicated by arrows). The position of the MutS protein in the DNA molecules was consistent with that expected from the location of the mismatched base pairs in the molecules, although the orientation of each DNA molecule could not be determined independently from the AFM images shown here (see below). Experiments similar to those shown in Figure 1a were also performed with other DNA heteroduplexes. We show AFM images of MutS protein–mismatched base pair complexes in which MutS protein is bound to a different tandem base mismatch (type B) (Fig. 1b) or to a single base mismatch (type C) (Fig. 1c). The complexes are observed at the positions expected based upon the location of the substituted bases in the mutant segment. In the AFM imaging of even larger DNA heteroduplex molecules (3556 bp fragment) with tandem base substitutions (type D), we were also able to detect the MutS–mismatch complexes at the expected positions (Fig. 1d).



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  		Figure 1. AFM images of MutS protein–DNA complexes. Heteroduplex DNA molecules were mixed with MutS proteins and subjected to AFM imaging. (a–d) Type A, B, C and D mismatch containing fragments, respectively. Bound MutS proteins are indicated by arrows. Height is coded by color, with low regions depicted as black and higher region increasingly lighter in tone from 0 to 2 nm.

 
In order to determine the orientation of DNA molecules with MutS protein, we attempted to tag one DNA terminus. A biotinylated primer was used for PCR, and the products were subsequently bound with avidin. The avidin-bound terminus was easily recognized by AFM. In Figure 2, we show the AFM image of the DNA molecules in which one terminus was specifically tagged by avidin.



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  		Figure 2. AFM images of avidin–DNA complex. One DNA terminus in each molecule is specifically biotinylated using biotinylated PCR primers. The biotinylated PCR products were then allowed to complex with avidin. Height is coded by color as in Figure 1.

 
In Figure 3, we show AFM images of DNA molecules with base pair mismatches in which one DNA terminus was tagged with avidin before allowing the heteroduplexes to bind MutS protein. Figure 3a, b and c corresponds to the AFM images of heteroduplex DNA molecules with tandem base pair mismatches (type A), a different tandem mismatch (type B) or the single base mismatch (type C), respectively. The two associated proteins, one at the termini and the other elsewhere along the DNA molecules, were clearly observed. The distance between the tagged termini and the positions where MutS protein formed a complex was measured by a contour length analysis employing digital image analysis software. The molecular weight of bound avidin and MutS protein (each bound as a dimer) are expected to be 68 and 190 kDa, respectively. The size difference between the two molecules is apparent in the AFM image.



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  		Figure 3. AFM images of MutS–DNA complex using avidin-labeled DNA molecules and fragments are shown in (a–c). The type A, B and C mismatch-containing fragments were used in (a–c), respectively. White and yellow arrows indicate MutS proteins and avidin bound to DNA, respectively. Height is coded by color as in Figure 1. (d–f) Histograms summarizing the locations of bound MutS molecules along heteroduplexes. The y-axis shows percentages of the MutS-bound DNA over total DNA molecules. The number of the calculated DNA fragments are 112, 63 and 21 for (d–f), respectively. The arrows indicate the position of the mismatched base pair. The distances were measured by tracing the image of DNA fragments using NIH-Image (NIH).

 
These results are summarized as histograms in Figure 3d, e and f which correspond to the AFM images of the heteroduplex DNA molecules with the type A, B and C mismatches, respectively. The ratios of MutS-bound to total DNA molecules were 0.33 (112 bound over 336 total DNA molecules), 0.52 (63 over 118) and 0.51 (21 over 41), for Figure 3d, e and f, respectively. These ratios agree quite well with the proportion of hetero­duplexes expected upon re-annealing of equimolar amounts of mutant and wild type molecules. It is clear from these histograms that the distributions of internal complex sites fall within relatively narrow ranges for the tandem base mismatches at nucleotides 883–884 (d) and at nucleotides 640–641 (e). The positions of the complex along the DNA molecules as calculated from the distribution were nucleotide 868 (with a standard deviation ±30) and 603 (with a standard deviation ±134) for (d) and (e) respectively. In contrast, the histogram of complex distribution along the heteroduplexes with a single base mismatch at nucleotide 527 (f) shows a wider distribution, even though the calculated center at nucleotide 529 (standard deviation ±155) is remarkably accurate. These results suggest that at least for the DNA molecules with tandemly occurring base mismatches AFM imaging is quite useful in determining the positions of the mismatched base in the DNA molecules of interest.

In this paper, we have presented the results of our efforts to detect and map substituted base pairs in DNA molecules by AFM. We have shown that at least for certain types of the base substitutions, such as tandemly occurring ones, AFM is quite useful in detecting the sites and determining their location via MutS protein binding to DNA heteroduplexes. For single base substitutions typical of most human mutations and poly­morphisms, AFM imaging was not as reliable as for tandem base substitutions. Improved conditions for complex formation will be needed to obtain more reliable results. Nevertheless we believe that AFM has great potential in eventually analyzing most kinds of mutations and human polymorphisms. It possesses unique advantages over other biochemical procedures. These advantages include (i) easy and time-saving analytical processes, (ii) capability to detect mutations and polymorphism in large DNA molecules with essentially no size range limitation for scanning and (iii) nominal requirement for the amount of a DNA sample to be examined. Efforts are currently underway to detect a much wider spectrum of mutations including single nucleotide substitutions. To accomplish this, it will be necessary to reduce background noise, which is mostly derived from non-specific binding of MutS protein to DNA molecules. In addition, we are automating monitoring and mapping process to reduce labor time involved.


   ACKNOWLEDGEMENTS
 
Thanks are due to Dr Robert F. Whittier for reading the manuscript and making helpful suggestions. Sincere gratitude is extended to Ms Hidemi Chohnan and Ms Tomoko Ohira for their technical assistance. This work was performed under the management of a technological research association, the Angstrom Technology Partnership (ATP) in the Joint Research Center for Atom Technology (JRCAT), and was partly supported by the New Energy and Industrial Technology Development Organization (NEDO).


   FOOTNOTES
 
* To whom correspondence should be addressed. Tel: +81 298 61 6214; Fax: +81 298 61 6240; Email: machida@nibh.go.jp Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 

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