Proc. Natl. Acad. Sci. USAVol. 83, pp. 586-590, February 1986Biochemistry

Detection of single base-pair mismatches in DNA by chemicalmodification followed by electrophoresis in 15%polyacrylamide gel

(carbodiimide/heteroduplex)

DAVID F. NOVACK, NANCY J. CASNA, STUART G. FISCHER, AND JOHN P. FORDLifecodes Corporation, 4 Westchester Plaza, Elmsford, NY 10523

Communicated by James E. Darnell, Jr., September 13, 198S

ABSTRACT We have developed a method for distinguish-ing fragments of DNA that contain single-base mismatchesfrom their perfectly paired homologues. Single-stranded re-gions within a duplex fragment are accessible to 1-cyclohexyl-3-{2-[4-(4-methyl)morpholinyl]ethyl}carbodiimide, which re-acts with unpaired guanidylate and thymidylate residues inDNA. Intact linear duplex DNA molecules do not react withcarbodiimide, whereas DNA molecules containing single-basemismatches react quantitatively. After carbodiimide reaction,the DNA molecules are electrophoresed in high-percentagepolyacrylamide gels so that modified and unmodified frag-ments can be resolved. Application of this technique shouldmake it possible to locate and purify DNA fragments thatexhibit sequence differences from those that do not; these mightbe used to signal phenotypic variation as well as to diagnoseinherited disease.

A method for rapid comparison of the sequences of short,homologous DNA molecules to reveal differences amongthem as slight as a single base-pair substitution would providean important step in elucidating the genetic basis for pheno-typic variation and heritable disease.

Several approaches have been tried in the past. (i) S1 andmung bean nucleases cleave at unpaired bases in heterodu-plex DNA molecules (1, 2); however, these enzymes cleavesingle-base mismatches with relatively low efficiency. (ii)Changes in electrophoretic mobility in denaturing gradientpolyacrylamide gels have been reported, but so far thistechnique is able to detect only a fraction of the differencesin DNA base sequence (3). (iii) Differential hybridizationwith oligonucleotide probes that differ by a single base couldbe carried out (4); this procedure is useful only if priorsequence information is available.Our strategy is to apply a "tag" after denaturation and

reannealing of a restriction endonuclease digest of a mixtureof the samples to be compared. The tag is specific forunpaired regions of DNA. Reassociation of single strandsfrom identical fragments produces perfectly pairedhomoduplexes, whereas, reassociation of single strands fromfragments that differ at a single base generates heteroduplex-es containing a mismatched pair. The tag, when bound, altersthe electrophoretic mobility of only the heteroduplex in apolyacrylamide gel and thus has the potential for facilitatingspecific extraction of the fragment of interest.The chemical probe we chose is 1-cyclohexyl-3-{2-[4-(4-

methyl)morpholinyl]ethyl}carbodiimide (hereafter referredto as carbodiimide), since it modifies only unpaired guanidyl-ate and thymidylate residues in supercoiled DNA withoutaffecting intact Watson-Crick base pairs (5, 6). At least oneheteroduplex formed from the reassociation of any two

molecules that differ by a single base pair will contain amismatched guanidylate or thymidylate; thus all substitu-tions should be accessible.The ends of a blunt-ended DNA duplex are less stable than

interior portions and their availability to carbodiimide mod-ification could obscure differences resulting from internalmismatches. However, Kelly and Maden (7) have shown thatalthough rG-rC pairs at the terminus of a double-strandedregion of rRNA are subject to modification by the base-specific reagent bisulfite, they are unreactive to carbodiim-ide, suggesting an advantage in our choice of carbodiimide.Finally, carbodiimide modification is reversible upon incu-bation under mild, slightly alkaline conditions (pH 10.5, 210C)which do not affect duplex DNA (8).We describe here an assay involving electrophoresis in

high-percentage polyacrylamide gels of heteroduplexedDNA molecules containing sequence alterations as minor asa single-base mismatch. Carbodiimide quantitatively modi-fies the guanines and thymines in such mismatches, resultingin slower migration through the polyacrylamide gel.

MATERIALS AND METHODSRestriction enzymes were purchased from Bethesda Re-search Laboratories or New England Biolabs and used asdirected by the supplier. Radiochemicals were purchasedfrom Amersham International. Acrylamide and N,N'-methylenebisacrylamide were purchased from Bio-Rad. M13pentadecamer primer and Klenow fragment of DNA poly-merase I were purchased from Bethesda Research Labora-tories. 1-Cyclohexyl-3-[2-(4-morpholinyl)ethyl]carbodiimidemetho-p-toluenesulfonate was purchased from Aldrich. andprepared as a 0.5 M aqueous solution. pT24, a human Ha-rasoncogene-containing plasmid (9) and pTPT, the wild-type-homologue-containing plasmid (10) were obtained from ColdSpring Harbor Laboratories.

Construction of M13 and pSP64 Derivatives. A Pst Ifragment including the 5' noncoding region and the first exonfrom pT24 and pTPT were sequenced by interrupted synthe-sis (11). A 371-base-pair (bp) Pst I fragment of Ha-rasoncogene DNA and a 365-bp Pst I fragment from homologouswild-type DNA were isolated from pT24 and pTPT, respec-tively. These Pst I fragments are of different lengths due tosix additional base pairs in pT24 (Fig. 1). Pst I fragmentscontaining the 5' noncoding region were ligated (16 hr at 14'Cin 50 mM Tris Cl, pH 7.6/10 mM MgCl2/10 mM dithiothre-itol/1 mM ATP) into the Pst I site of M13 mp8. Ligationreactions were carried out with 40 ng of DNA/pi, using a4-fold excess of insert to vector, and 8 units of T4 DNAligase/,g of DNA. Escherichia coli strain JM103 was trans-formed with these ligated DNAs (12) and plated on YT (13)plates containing 5-bromo-4-chloro-3-indolyl ,B-D-galactopy-

Abbreviations: bp, base pair(s).

586

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Proc. Natl. Acad. Sci. USA 83 (1986) 587

5' G C 3

T6 G ... GCCCTGTGGGGCCT mGGGC GGGCCTGCGCCTG * * * G C *.* CA **CTGCAACGTC ... CGGGACACCCCGGA [CCCG CCCGGACCCGGAC ...C A G... GT A.. G1 76 220 266 371

5 G C 3

ACGT ... TG...G C . TCAGGCCCAGGCCC AGCCCC AGGCCCCACAGGGC... CTGCAACGTC ..T AC C T G -GTCCGGGTCCGGG TCGGGG TCCGGGGTGTCCCG ..G

371 266 220 76 1

FIG. 1. Nucleotide sequence of 371-bp Pst I fragment of the pT24 oncogene. T6 refers to the Pst I fragment inserted into the Pst I site ofM13 mp8 in the direction of transcription. T9 refers to the fragment inserted in the opposite orientation. The boxed sequences are differencesbetween the oncogene and its nontransforming homologue pTPT. Thus, the boxed sequence at positions 90-95 is not present in pTPT. Single-basesubstitutions in the oncogene are shown at position 220 and position 266. Horizontal arrows are above 6-bp direct repeats.

ranoside. DNA was prepared by a miniprep procedure (14)from insert-containing colonies. Recombinants contained thePst I fragment inserted in both orientations (Fig. 2). These PstI fragments were similarly inserted into the Pst I site of theplasmid pSP64 (15).

Heteroduplex Formation. Labeled DNA. Recombinantswere labeled by primer extension (16), and the double-stranded DNA was digested with Pst I or Sma I. A reactionmixture composed of the labeled restriction enzyme digest (5x 107 cpm/pg) and a 50-fold molar excess of homologousunlabeled M13 replicative-form DNA that had been cleavedwith the same enzyme was heat-denatured (100TC, 5 min) ina final volume of 150 Al. (Excess unlabeled DNA was addedto favor incorporation of labeled DNA into heteroduplex.)The fragments were hybridized at 420C for 60 min at a final

Smo I

T6

PstI Smo I

Smi I 15 tI

T9

Smo

Pst I

I~ 213SmoaI Pst I

P6 P9

FIG. 2. M13 mp8/Ha-ras recombinants. The heavy line repre-sents the 371-bp Pst I fragment from pT24 or the 365-bp Pst Ifragment from pTPT that was inserted into the M13 mp8 polylinker.The small arrow indicates the site of hybridization of the M13pentadecameric primer that was used for primer extension. The longwavy arrow shows the 5'-.3' direction of primer extension. (SeeMaterials and Methods for details.) Oncogene recombinants aredesignated T6 and T9. In oncogene recombinant T6, the internal SmaI site of the 371-bp Pst I fragment is closest to the Sma I site of theM13 polylinker. In oncogene recombinant T9, the Pst I fragment isinserted in the opposite orientation. Homologous wild-type recom-binants are designated P6 and P9. In wild-type recombinant P6, the365-bp Pst I fragment is inserted in the same orientation as the 371-bpPst I fragment in T6. In wild-type recombinant P9, the 365-bp Pst Ifragment is in the same orientation as the 371-bp Pst I fragment in T9.

concentration of0.08 gg//l and then were ethanol-precipitat-ed. Homoduplex fragments from primer-extendedDNA werenot denatured and renatured.

UnlabeledDNA. pSP64 derivatives containing the 365- and371-bp inserts were digested with EcoRI. Wild-type andoncogenic DNAs were mixed in an equimolar ratio (asverified by gel electrophoresis). The mixture (50-100 ;kgtotal) was brought to 100 mM with 5 M NaOH and incubatedat room temperature for 5 min. The reaction was neutralizedby the addition of 2.5 volumes (relative to 5 M NaOH) of 3M Mops. The fragments were hybridized at 420C for 2 hr ata final concentration of 0.1 ;kg/;LI and then were ethanol-precipitated.Fragment Purification. Polyacrylamide was prepared as a

30% stock solution (30:0.8, acrylamide/bisacrylamide). La-beled DNA fragments were electrophoresed in a 5% poly-acrylamide gel containing 0.1 M Tris borate (pH 8.0) and 1mM EDTA and were visualized by autoradiography. Thebands were cut from the gel and fragments were electroelutedfrom the gel strip into dialysis tubing in 0.05 M Tris boratebuffer (pH 8.0) overnight at 2 V/cm. Then the current wasreversed for 5 min at 150 V. The buffer containing the DNAwas removed from the dialysis tubing and extracted threetimes with phenol and once with chloroform. DNA wasprecipitated in 0.35 M ammonium acetate and washed with70%o (vol/vol) ethanol.

Carbodiimide Reaction. DNA fragments were incubated in0.1 M carbodiimide/0.1 M sodium borate, pH 8.5, for 4 hr ina volume of 50-100 ,ud at various temperatures. Samples wereadjusted to 0.25M with 2.5M ammonium acetate and broughtto 400 ,ul with 0.25 M ammonium acetate. Carrier tRNA (50,ug) then was added to each reaction mixture. The DNA wasethanol-precipitated three times and then washed once with70%6 ethanol. Pellets were dried and resuspended in 10 mMTris Cl, pH 7.5/0.1 mM EDTA. In some cases, the DNA wascleaved with restriction enzymes after carbodiimide reaction(see Results). It is important that the pH of the restrictionenzyme buffer be s8.0 to avoid alkaline hydrolysis of thecardodiimide adduct.

Gel Electrophoresis. Separation of fragments after reactionwith carbodiimide was achieved using 12-15% polyacrylam-ide gels. Gels were run in a recirculating buffer (40 mM Trisacetate, pH 8.0/20 mM NaCl/1 mM EDTA) at 30°C for 7-10hr at 150 V (Figs. 4-6A) or 18 hr at 200 V (Fig. 6B). In orderto visualize nonradioactive modified mismatched DNA,30-60 ng of the fragment was electrophoresed at least 10 cminto a 16-cm 15% polyacrylamide gel, which was then stainedwith ethidium bromide.

RESULTSWe used a short region including the initial protein-codingportion of the Ha-ras oncogene as a model system to test the

Biochemistry: Novack et al.

588 Biochemistry: Novack et al.

carbodiimide reaction with mismatched DNA. When thisfragment is hybridized to nontransforming homologousDNA, the heteroduplex molecule contains a 6-base unpairedregion and two single-base mismatches (Fig. 3). Thesemismatched regions within the DNA segments are potentialsites for modification by carbodiimide.

Reaction of Carbodihmide at Fragment Ends. We tested theeffect of carbodiimide modification upon the migration ofDNA segments in high-percentage polyacrylamide gels. Re-striction enzyme-digested DNA that either was perfectlydouble-stranded (Sma I digest) or had protruding single-stranded ends (Pst I digest) was treated with carbodiimidebefore electrophoresis.

Digestion with Pst I generates 3' "overhangs" with thesequence 5' TGCA 3'. Thymidylate and guanidylate residuesare potential sites of carbodiimide modification. Sma Iproduces blunt-ended fragments with three GC pairs at thefragment end. Such fragments should be resistant tocarbodiimide modification.A 365-bp Pst I fragment was isolated from primer-extended

P6 (Fig. 2). The purified fragment was treated with carbodi-imide at 30TC, 37TC, and 450C and analyzed by autoradiog-raphy following polyacrylamide gel electrophoresis (Fig. 4A).After carbodiimide modification at 30TC, the Pst I fragmentmigrated as a single band with decreased mobility comparedto the unmodified fragment (Fig. 4A, lanes 4 and 1). Higherreaction temperatures (37TC and 450C) resulted in a progres-sive diffusion of the banding pattern (Fig. 4A, lanes 5 and 6).After reaction with carbodiimide at 30TC and 37TC, a 228-bpblunt-ended Sma I fragment isolated from primer-extendedT9 (Fig. 2) did not exhibit a mobility shift (Fig. 4B, lanes 5 and6). Higher temperatures (45TC and 550C) caused changes inbanding patterns similar to those described above (Fig. 4B,lanes 7 and 8).

T632p P4I

Sma I

15

Pst I

;GGGCT

152

T

91 - 3A 12P2

Sma I Sau3A PstI

B TTCGG32p-T9<TI r \Y

P9SnI 122 91 1iP521SMOI Pst I G Sou ASma I Pst I

CCCCGA32P-T6 -

/A\_/A\

Sma I Pst I Sma I Sou3A Pst I

AGCCCCT9 I N_

32p_ Pa|1 1 122 9 t 152

Smo I PstI Sou 3A Sma I PstI

FIG. 3. Heteroduplex pairs formed by mismatch hybridization.Heteroduplex pair A was formed from the labeled strand of primer-extended P6 and its complementary strand from unlabeled T6replicative form. Heteroduplex pair B was formed from the labeledstrand of primer-extended T9 and its complementary strand from P9replicative form. Heteroduplex pair C was formed from the labeledstrand of T6 and its complementary strand from P6 replicative form.Heteroduplex pair D was formed from the labeled strand of P9 andits complementary strand from T9 replicative form. The 6-baseunpaired region and single-base mismatches are indicated for eachheteroduplex. Restriction enzyme sites used in the study are shownas well as restriction fragment lengths (in bp).

Abp 2 3 4 5 6

365- *MI a b -

Bbp 1 2 3 4 5 6 7 8

228-

FIG. 4. Reaction ofcarbodiimide at restriction fragment ends. (A)Isolated 365-bp Pst I fragment from primer-extended P6 was treatedas described below and then electrophoresed in a 12% polyacrylam-ide gel. Lanes 1-3: incubated at 300C, 370C, and 450C withoutcarbodiimide. Lanes 4-6: incubated with carbodiimide at 30TC, 37C,and 450C. (B) Isolated 228-bp Sma I fragment from primer-extendedP9 was treated as described below and electrophoresed in a 12%polyacrylamide gel. Lanes 1-4: incubated at 30TC, 370C, 450C, and550C without carbodiimide. Lanes 5-8: incubated with carbodiimideat 30TC, 370C, 450C, and 55C.

Reactivity of Carbodilmide with a 6-Base Internal UnpairedRegion. To test the reactivity of carbodiimide with internalDNA mismatches, heteroduplexes were formed between afragment of the Ha-ras oncogene and its homologue (Fig. 3).The 6-base unpaired region that occurs in the heteroduplexDNA molecule causes a large decrease in migration of thefragment through a polyacrylamide gel. This lower mobilityallowed us to quantitatively separate heteroduplex fragmentsfrom any reannealed, perfectly paired fragments. For a167-bp Sma I fragment isolated from heteroduplex A (Fig. 3),the 6-base unpaired region causes a decrease in mobility suchthat the fragment migrates in a 12% gel with an apparentlength of 650 bp (Fig. SA, lane 3).DNA fragments were electroeluted from a gel slice and

purified (see Materials and Methods) prior to carbodiimidemodification. The 6-base unpaired region in the 167-bp SmaI fragment from heteroduplex A has five potential sites ofcarbodiimide modification within the sequence GGGGCT. Achange in electrophoretic mobility after reaction withcarbodiimide would correlate with the predicted modificationof these unpaired bases. After reaction with carbodiimide at30'C, the heteroduplex migrated through the 12% polyacryl-amide gel more rapidly, as evidenced by the appearance ofseveral diffuse new bands (Fig. SA, lane 4). Under the sameconditions of carbodiimide modification, the 167-bphomoduplex showed no change in electrophoretic mobility(Fig. 6, lane 2).The Sma I fragment isolated from heteroduplex C (Fig. 3)

contains a 6-base unpaired region with the complementarysequence (CCCCGA). This unmodified heteroduplex alsomigrates with fragments 650 bp long but contains only onepotential site of modification by carbodiimide. After reactionwith carbodiimide at 30TC, the fragment showed a single,distinct band of greater mobility, with an apparent length of580 bp (Fig. SB, lane 4).

Reaction of Carbodlimide with Single-Base Mismatches. Theobservation of mobility shifts upon modification of small

A

C

Proc. Natl. Acad. Sci. USA 83 (1986)

Proc. Natl. Acad. Sci. USA 83 (1986) 589

B1 2 3 4A 1 2 3 4

A

bpbp

- 650

1 2 3 4 5 6 7 8

137-

91-

B

bp

-167

613-

564-

FIG. 5. Carbodiimide modification of6-base unpaired region withthe sequences GOGGCT (A) and CCCCGA (B) analyzed on a 12%polyacrylamide gel. Lane 1: 167-bp Sma I homoduplex fragment.Lane 2: 167-bp Sma I homoduplex fragment after reaction withcarbodlimide at 30TC. Lane 3: 167-bp Sma I heteroduplex fiagment.Lane 4: 167-bp Sma I heteroduplex fragment after reaction withcarbodiimide at 30TC.

unpaired regions of DNA encouraged us to apply the tech-nique to a DNA fragment containing a single-base mismatch.A 228-bp Sma I fragment was isolated from heteroduplex B(Fig. 3). In this fragment, there are two single-base mismatch-es, separated by 45 bp. The fragment was incubated in thepresence or absence ofcarbodiimide at various temperatures.After reaction with carbodiimide, cleavage at a unique Sau-3A1 site between the mismatches generated a 91-bp fragmentcontaining one T-C mismatch and a 137-bp fragment con-taining one T-G mismatch. The DNA fragments were elec-trophoresed in a 15% polyacrylamide gel (Fig. 6A). Frag-ments containing a single-base mismatch exhibited the samemobility as the corresponding homoduplex fragment; there-fore, a single-base mismatch per se, is not sufficient forDNAseparation under these conditions.After carbodiimide reaction at 30'C, the 91-bp

heteroduplex containing a T-C mismatch yielded a band ofdecreased mobility compared to the 91-bp homoduplex (Fig.6A, lanes 4 and 3). The 137-bp heteroduplex containing a T-Gmismatch and treated with carbodiimide at 30°C showed nomobility change in comparison to the untreated molecule.However, after carbodiimide reaction at 37°C, the 137-bpheteroduplex also yielded a band of decreased mobilitycompared to the identically treated homoduplex (Fig. 6A,lanes 6 and 5). Under these conditions, the 91-bpheteroduplex containing the T-C mismatch migrated to thesame position as that seen following reaction at 30°C.Reaction of DNA with carbodiimide at 45°C resulted indiffusion of the banding pattern for both homoduplex andheteroduplex molecules (Fig. 6A, lanes 7 and 8).To investigate the extent of mobility shift due to

carbodiimide modification of single-base mismatches withinlonger fragments of DNA, pSP64 subclones of T6 and P6were made linear by digestion with EcoRI. The unlabeled,digested DNAs were mixed in equimolar ratios, denatured,and reannealed. The fragments were treated with carbodi-imide at 30°C and 37°C and then cleaved with Sma I and SphI. Digestion with these enzymes generates fragments of 2568bp, 613 bp, 179 bp, and 15 bp. The 613-bp fragment containstwo single-base mismatches. The unlabeled DNA containsboth possible heteroduplex DNA molecules, one with A-Gand A-C mismatches and one with T-C and T-G mismatches.The fragments were separated in a 15% polyacrylamide gel

FiG. 6. Mobility shifts due to carbodiimide modification. (A) The228-bp Sma I fragment was treated as indicated below and then wascleaved with Sau3Al; the fragments were separated in a 15%polyacrylamide gel. Lane 1: unmodified homoduplex (primer-extend-ed P9). Lane 2: unmodified heteroduplex B (see Fig. 3). Lanes 3, 5,and 7: homoduplex treated with carbodiimide at 30°C, 37°C, and45°C, respectively. Lanes 4, 6, and 8: heteroduplex B treated withcarbodiimide at 30°C, 37°C, and 45°C, respectively. (B) Aheteroduplex mixture ofpSP634 subclones of T6 and P6 (Fig. 2) wasincubated in the presence or absence of carbodiimide at 30°C and37°C, cleaved with Sma I and Sph I, and electrophoresed in a 15%polyacrylamide gel. Lane 1: 30°C, minus carbodiimide. Lane 2: 30°C,plus carbodiimide Lane 3: 37°C, minus carbodiimide. Lane 4: 37°C,plus carbodiimide. Arrows indicate the positions of the modifiedbands.

(Fig. 6B). At least one-half of the mixture shown in each laneof Fig. 6B is homoduplex and is not expected to react. Aftercarbodiimide reaction at 30°C, a new band is observed withmobility lower than that of the homoduplex (Fig. 6B, lanes 2and 1). This mobility shift is due to modification of the T ofthe T-C mismatch in one heteroduplex and possibly tomodification of the G of the A-G mismatch in the otherheteroduplex. Reaction with carbodiimide at 37°C (Fig. 6B,lanes 3 and 4) gives a faint, second modified band of slowermobility, indicating a shift due to carbodiimide modificationof the T-G mismatch. The faintness of the band is due todiffusion of the banding pattern that is observed at higherreaction temperatures and with longer DNA fragments.

DISCUSSIONWe have used reaction of carbodiimide with single-strandedregions of duplex DNA (5, 6) as the basis for development ofa system capable of distinguishig DNA fragments with minorsequence differences. Mismatches and unpaired regions inotherwise perfectly paired DNA fragments were generatedby formation of heteroduplexes between the denaturedstrands of homologous fragments containing base substitu-tions and additions or deletions. Altered mobility on ahigh-percentage polyacrylamide gel was used to detect thereactivity of these heteroduplex molecules with carbodiim-ide. Single-base mismatches and unpaired regions were foundto react with carbodiimide at 30°C and 37°C, whereas per-fectly paired DNA remained unreactive. DNA duplexescontaining mismatches are less stable than perfectly pairedduplexes, although the stability depends both on which basesare mismatched and on the surrounding DNA sequence (17,18). In the present case, the more thermodynamically stableT-G mismatch must be treated at 37°C to be modified,whereas the less stable T-C mismatch can be modified at

Biochemistry: Novack et al.

590 Biochemistry: Novack et al.

30'C. Even higher temperatures may result in "breathing" oropening within the molecule and at the fragment ends (19),allowing carbodiimide access to internal bases. Breathing atthe fragment ends could be prevented by the addition of aG+C-rich "clamp" (20, 21).

Reduction in electrophoretic mobility was observed aftercarbodiimide modification of the T and/or G residues of thePst I staggered ends, of the T of a T-C mismatch in anotherwise duplex fragment, and of the T and/or G of a T-Gmismatch. These results are consistent with the followingexplanation. In gels of 12-15% acrylamide, the size of theaverage "pore" is a small multiple of the cross-sectional areaofDNA (22). With the DNA threading in a primarily end-onmotion ("reptation," see ref. 23) through the matrix, one orseveral carbodiimide molecules bound to the single-strandedPst I ends add only a small increment to the cross-sectionalarea and result in a small reduction in gel mobility.Carbodiimide bound to the T of a T-C mismatch would beexpected to lie outside the helix, adding significantly to thecross-sectional area and resulting in a greater reduction in gelmobility. Reaction ofcarbodiimide with aG-T mismatch mayresult in an adduct with two molecules of carbodiimidebound, both lying outside the helix; further reducing gelmobility because ofthe still greater local cross-sectional area.

Mobility shifts due to carbodiimide modification of asingle-base mismatch can be seen with fragments of differentsizes. The 91-, 137-, and 613-bp fragments described aboveshow clear mobility shifts. We have seen mobility shifts infragments up to 1500 bp long (data not shown), althoughlonger fragments show diffusion of the banding pattern(especially at higher temperatures), which may result fromrandom reaction of carbodiimide along the length of themolecule. Changes in reaction and washing conditions mayreduce or eliminate this problem. We have seen similarmobility shifts after carbodiimide treatment at 30'C ofheteroduplex DNA from point mutants within a fragment ofthe cII gene of bacteriophage X. In addition to a T-Cmismatch, we have seen modification and mobility shifts ina DNA fragment containing a G-G mismatch and a fragmentcontaining a T-T mismatch with this system (unpublishedresults). Thus, at least one of the two mismatches generatedby any single base change can be tagged by carbodiimide. Theelectrophoretic mobility of the modified fragment cannot asyet be predicted because it may be influenced by the positionof the mismatch within the fragment, the length of thefragment, and the sequences surrounding the mismatch.The 167-bp Sma I fragment isolated from heteroduplex A

(Fig. 3) shows a completely different pattern of change in gelmobility following carbodiimide modification. The singlestrands from which the heteroduplex was formed differ byonly six contiguous base pairs, yet these molecules migrateconsiderably slower than either perfectly paired parenthomoduplex (Fig. 5). It is unlikely that such a large reductionin electrophoretic mobility is caused by just six unpairedbases in the duplex. The six unpaired bases are flanked by a6-bp direct repeat (see Fig. 1) that may induce the formationof a cruciform secondary structure, distorting the linearmolecule. Modification of the altered structure withcarbodiimide may partially relieve the distortion, resulting ina faster-migrating molecule.

Radiolabeled or unlabeled DNA can be used as a substratefor carbodiimide modification. By radioactively labeling onestrand of a fragment used to generate mismatches,heteroduplex formation can be driven by the addition ofexcess nonradioactive DNA. In addition, the mobility shiftsfor each individual mismatch can be determined without thepresence of the overlapping mobility shifts caused by thecomplementary mismatch. Alternatively, modification of anunlabeled heteroduplex will establish the presence of DNA

segments containing carbodiimide-modified mismatches aswell as their perfectly paired, reannealed homologues.

Reactivity ofrestriction enzyme-generated single-strandedends must be taken into consideration. Solutions to thisproblem include (i) generating the substrate to be modifiedwith a restriction enzyme that leaves blunt ends and (ii)removing the reacted ends with a restriction enzyme(s) aftermodification of large heteroduplex molecules (see Fig. 6B).Carbodiimide modification of single-base mismatches

should be useful in elucidating the genetic basis of bothphenotypic variation and heritable human disease. Manysingle-base changes of clinical significance do not occur at aknown restriction enzyme site (24). Current oligonucleotideprobe technology, although it allows detection of any DNAbase change, is technically diffcult and requires prior se-quence information. Carbodiimide modification may providea useful alternative to current methods of mutant detection.

Finally, the search for sequence differences in complexmixtures, such as restriction enzyme digests of humangenomic DNA, requires a purification step to sort variant andinvariant sequences. By tagging mismatches with carbodi-imide, molecules that include critical sequence differencescan, in principle, be purified.We thank Stanley Nawoschik for technical assistance, Drs.

Sebastiano Gattoni-Celli and Jacob Grimberg for helpful discussionsduring the course of the work, Drs. Nadrian Seeman and NevilleKallenbach for reviewing the manuscript, and Ms. Denise Reinalterand Ms. Donna Cucchiarella for preparation of the manuscript.

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Proc. Natl. Acad Sci. USA 83 (1986)