Printer Friendly

Chemical mismatch cleavage combined with capillary electrophoresis: detection of mutations in exon 8 of the cystathionine [beta]-synthase gene.

The method based on chemical mismatch cleavage (CMC) [1] for mutation analysis was developed by Cotton et al. (1) and has been used successfully for the detection and identification of mutations in several genes (2-5). When compared with denaturing gradient gel electrophoresis (6) and single-stranded conformation polymorphism (7), CMC has a higher diagnostic sensitivity and can analyze larger DNA fragment lengths (8).

The principle of the CMC method is the formation of heteroduplex double-stranded DNA (dsDNA) by annealing single-stranded DNA (ssDNA) from the wild-type and mutant alleles. The two alleles can either be derived from a heterozygous DNA sample or by combining two samples, one with wild-type DNA and one containing the mutant allele. The base at the mismatch reacts either with hydroxylamine or osmium tetroxide (Os[O.sub.4]), which modify unpaired cytosine or thymine residues, respectively. DNA is then cleaved at the modified base by piperidine, and the products are separated by denaturing gel electrophoresis and detected by autoradiography (1, 3) or by fluorescence (5, 9,10).

Capillary electrophoresis (CE) in entangled polymers has become an attractive alternative to gel electrophoresis techniques for the analysis of DNA fragments. CE can be automated and is characterized by short analysis times, small sample requirements, high resolution and separation efficiency, and when coupled to a laser-induced fluorescence (LIF) detector, unsurpassed sensitivity (11-15). Low viscosity sieving media like short-chain linear polyacrylamide (SLPA) have the additional advantage of efficiently filling small diameter (<75 [micro]m) capillaries with sieving medium. Small diameters ensure efficient heat dissipation and thereby fast analysis without loss of resolution (16-18).

There is extensive documentation of efficient analysis of dsDNA by CE. Data on the separation of ssDNA are sparse (15), however, and analysis of CMC cleavage products by CE has been suggested but not investigated (8).

We have previously described single-stranded conformational polymorphism analysis by CE, demonstrating high resolution of ssDNA by CE using SLPA as the sieving matrix (18). Notably, a recent study compared CE analysis of ssDNA and dsDNA and showed the enhanced resolution and selectivity of ssDNA migrating in 50-[micro]m capillaries filled with linear polyacrylamide (19). In the present study, we transferred CMC mutation analysis to the capillary format by analyzing the fluorescein-tagged cleavage product by CE-LIF, using SLPA as the sieving matrix. The expected fragment lengths for T833C and G919A mutations in exon 8 of the cystathionine [beta]-synthase (CBS) gene (20-23) were obtained, demonstrating the detection of both known and unknown mutations by CMC combined with CE-LIF.

Materials and Methods


The acrylamide, N,N,N',N'-tetramethylenediamine, and ammonium peroxydisulfate were purchased from BioRad Laboratories. The 3-methacryloxypropyl-trimethoxysilan and fluorescein-labeled dsDNA markers (fragment sizes of 50-500 bp) were from Pharmacia LKB Biotechnology AB. The hydroxylamine, diethylamine, 40 g/L Os[O.sub.4] solution, HEPES, and tRNA were from Sigma Chemical Co. The hydroxylamine stock solution (6 mol/L) was aliquoted into 1-mL Eppendorf tubes and stored at -70 [degrees]C; it was stable for at least 3 months under these conditions. The Os[O.sub.4] solution was stored at 4 [degrees]C and was stable for at least 3 weeks. The pyridine was from Chemical Limited Walkerburn. The piperidine was a product of Merck. The reaction tubes (thin-walled, Gene Amp) for PCR reactions were from Perkin-Elmer. The fused capillaries (50 [micro]m i.d., 192 [micro]m o.d.) were products of Polymicro Technologies Inc. The QIAquick PCR Purification Kit and QIAamp Blood Kit were products of QIAGEN Co. The 5'-fluorescein-labeled primers were synthesized by Eurogentec. Water, doubly distilled and purified on a Mi11iQ Plus Water Purification System (Millipore), was used for preparation of all aqueous solutions. The short-chain linear polyacrylamide (SLPA) was synthesized according to a slight modification (18) of the procedure described by Grossman (16).

Os[O.sub.4] and piperidine are toxic chemicals, and skin and eye contact must be avoided. The handling and chemical cleavage reactions were performed under a fume hood. The supernatants of the ethanol precipitation after Os04 modification were collected for safe disposal (9).


Blood from four different subjects was used. One subject had a presumably wild-type CBS genotype, one was heterozygous for the T833C mutation, one heterozygous for the G919A mutation, and one was homozygous for the G919A mutation. The CBS mutations in these samples have been determined by DNA sequencing (24).

Template DNA used in PCR reaction was extracted from whole blood, using the QIAamp Blood Kit according to the instructions from the manufacturer. The PCR reaction mixture contained 10 mmol/L Tris-HCI, pH 9.0, 50 mmol/L KCI, 1.5 mmol/L Mg[Cl.sub.2], 0.1 g/L gelatin, 1 mL/L Triton X-100, 125 [micro]mol/L each dNTP, 0.2 [micro]mol/L each primer, 0.2 U of Taq polymerase (Super Taq, HT Biotechnology Ltd.), and ~100 ng of template DNA in a final volume of 100 [micro]L. The PCR reaction was performed on a Perkin-Elmer 480 thermocycler, using a three-step thermocycling profile with 35 cycles of 94 [degrees]C for 30 s, 58 [degrees]C for 40 s, and 72 [degrees]C for 20 s, preceded by 94 [degrees]C for 3 min and followed by 72 [degrees]C for 5 min.

The primers used were 5'-fluorescein-ACT000CTT-GAGCCCTGAA-3' (F1, forward) derived from intron 7 and 5'-fluorescein-AGGCCGGGCTCTGGACTC3'(F2, reverse) from intron 8 of the CBS gene (21-23). The primers define a 186-bp PCR product that includes both the T833C and G919A mutations, as depicted in Fig. 1.


The generation and cleavage of heteroduplex DNA have been described before (1, 2, 5). Briefly, heteroduplex DNA was formed by mixing 100 [micro]L of PCR product (containing both the wild-type and mutated alleles) with 60 [micro]L of 3 mmol/L Tris-HCl, pH 7.7, containing 1.2 mol/L NaCl and 3.5 mmol/L Mg[Cl.sub.2], followed by heating at 96 [degrees]C for 6 min and annealing at 42 [degrees]C for 1.5 h. The DNA was then precipitated with ethanol and purified with the QIAquick PCR Purification Kit, according to the instructions from the manufacturer. This purification step was included to remove fluorescent material and fluorescent primers, which interfered with the analysis of the CMC cleavage products by CE-LIF. The final volume of the purified DNA sample was 50 [micro]L.


Specific modification of unpaired C and T residues was obtained with hydroxylamine and Os04, respectively. Five microliters of DNA was mixed with a solution of 20 [micro]L of hydroxylamine adjusted by diethylamine to pH 6.0 (final concentration, 2.0-4.0 mol/L) or Os[O.sub.4] containing 20 mL/L pyridine, 0.5 mmol/L [Na.sub.2]EDTA, and 5 mmol/L HEPES, pH 8.0 (final concentration, 0.8-5.6 g/L), and then incubated for 5-105 min at 20-45 [degrees]C. The reaction conditions are given in the figure legends. The reaction was stopped by transferring the solution to ice and adding 200 [micro]L of 0.3 mol/L sodium acetate buffer with 0.1 mmol/L [Na.sub.2]EDTA and 25 mg/L tRNA, pH 5.2. The DNA was precipitated with ethanol, and the DNA pellet was washed once with 700 mL/L ethanol. The DNA was then cleaved at the modified base by dissolving the DNA pellet in 50 [micro]L of 1 mol/L piperidine, followed by incubation at 90 [degrees]C for 30 min. To minimize the loss of DNA, the sample was finally lyophilized. Before electrophoresis, the lyophilized DNA was dissolved in 8 [micro]L of 800 mL/L formamide, incubated at 95 [degrees]C for 5 min, and then cooled in ice water. Fluorescein-labeled dsDNA markers and PCR products, dissolved in 800 mL/L formamide, were converted into ssDNA using the same procedure.


CE was performed on a commercial CE instrument (Prince Technologies). The LIF detector was built in-house with a sheath-flow cuvette constructed essentially as described by Dovichi et al. (25). An argon ion laser (Uniphase Ltd.) with 488 nm emission (20 mW) was focused on the sheath-flow cuvette 30 [micro]m below the capillary outlet. A fluorescence emission signal was collected at 90[degrees] with a microscope objective and amplified by a photomultiplier (Hamamatsu); the signal was then transferred to a computer. We used Prince software (Ver. 1.14) and Caesar software (Ver. 4.0), both from Prince Technologies, for instrument control and data collection, respectively.


The capillary (length, 40 cm), coated with linear polyacrylamide (18), was rinsed with Tris-borate-EDTA buffer (89 mmol/L Tris-borate buffer containing 1 mmol/L EDTA, pH 8.3) for 5 min and then filled with sieving medium consisting of 60 g/L SLPA in Tris-borate-EDTA buffer containing 7 mol/L urea (pH 8.3). Tris-borate-EDTA was used as the electrophoresis buffer. Samples were introduced by electrokinetic injection at -30 kV for 18 s. Electrophoresis was performed at reverse polarity mode under the conditions specified in the figure legends. The sieving medium in the capillary was replaced between each electrophoresic run.


After heteroduplex DNA formation, a heterozygous sample contains four dsDNAs, i.e., two homoduplexes and two heteroduplexes. For each mutation, only one out of the two heteroduplexes is a target for the site-directed probe, i.e., either hydroxylamine or Os[O.sub.4]. Notably, only one of the two strands is cleaved, which implies that the maximal efficiency of the cleavage is one out of eight fluorescein-labeled single strands. The cleavage yield ([gamma]) in percentage of the theoretical maximum is given by the equation:

[gamma](%) = C x 8 x 100/[summation]C (1)

where C refers to the concentration of the cleaved product, and [summation]C is the sum of the cleaved and uncleaved products.

In CE, the mass (M) injected is the product of the injection volume (V) and the analyte concentration (C), which in turn are proportional to the corrected peak area (A):

M = V x C = k x A ?? C = k x A/V (2)

where k is constant for all analytes labeled with the same fluorescent reagent.

When samples are injected electrokinetically, the injection volume V of a given analyte depends on its injection velocity [[upsilon].sub.1]:

V = [pi] x [r.sup.2] x l = [pi] x [r.sup.2] x [v.sub.i] x [t.sub.i] (3)

where r denotes the radius of the capillary, [t.sub.i] is the injection time, and l is the length of the injected sample plug.

The injection velocity, [[upsilon].sub.i], in turn is a function of the ionic mobility of the analyte, [micro] which is proportional to the electric field strength [E.sub.i] applied during the injection (26, 27):

[v.sub.i] = [micro] x [E.sub.i] (4)

The ionic mobility [micro] of the analyte can be determined using the following equations:

[v.sub.s] = [micro] x [E.sub.s] = L/[t.sub.s] ?? [micro] = L/([E.sub.s] x [t.sub.s]) (5)

where [[upsilon].sub.s] is the migration velocity, [E.sub.s] refers to the electric field strength during the separation, L is the length of the capillary from the inlet to the detection window, and is [t.sub.s] the migration time.

By combining Eqs. 3, 4, and 5, we can rearrange Eq. 2:

C = k x [E.sub.s] x [t.sub.s] x A/([pi] x [r.sub.2] x [t.sub.i] x [E.sub.i] x L) (6)

Combining Eqs. 1 and 6 gives an expression of the percentage of yield:

[gamma](%) = 8 x A x [t.sub.s] x 100/([summation]A x [t.sub.s]) (7)

Results and Discussion

Genetic defects in CBS are the most common cause of the inborn error homocystinuria (28). However, the number of pathogenic mutations in this gene now number ~30 (29), which makes mutation detection in these patients a demanding task. CMC is a useful technique for screening under conditions of genetic heterogeneity because it not only detects new mutations but also indicates their location (8). In the present work, we demonstrate the use of CMC coupled to CE-LIF for the rapid detection of the T833C and G919A substitutions that are the most prevalent pathogenic mutations in the CBS gene.


The principles of the CMC method for detection of the T833C and G919A mutations in the CBS gene are shown in Fig. 1. Hydroxylamine selectively modifies the mismatched cytosine residues, probably by binding to its 5-6 double bond, whereas Os04 catalyzes the dihydroxylation of thymine residues. After such chemical modification, the polynucleotide chain becomes susceptible to cleavage by piperidine (30). In our present study of the CBS gene, the amplified DNA strand is labeled only at the 5' end. Hence, the chemical cleavage yields a fluorescent fragment at the 5' end, whereas the other cleavage product (3' end) is not detectable by CE-LIF.

A DNA sample that is heterozygous for the G919A or the T833C mutation will contain one heteroduplex with an A:C mismatch, which can be modified by hydroxylamine, whereas the other heteroduplex has a T:G mismatch, which can be modified with Os[O.sub.4]. The uncleaved amplified DNA strand is 186 bp. In samples with the T833C mutation, the fluorescent cleavage product is from the coding strand. The length from the mutation site to the 5' end of this strand is 40 nucleotides (nt). The cleavage in the G919A mutation occurs in the non-coding strand, and produces a 61-nt fluorescent fragment (Fig. 1).

The electropherograms from chemically modified samples with wild-type CBS, the T833C mutation, or the G919A mutation are shown in Figs. 2 and 3. In samples with wild-type CBS, heteroduplexes are not formed, and as expected, there was no cleavage product. In samples containing two different alleles, hydroxylamine treatment produced distinct peaks of the size expected for both mutations. In contrast, cleavage products after Os04 treatment were only observed for the T833C mutation. This is in accordance with a previous observation (31) that the extent of cleavage is influenced by the base context of the mutation site. We also observed that the G919A exposed to hydroxylamine and the T833C mutation exposed to Os[O.sub.4] produced double peaks in the electropherogram. This phenomenon can probably be explained by adjacent mismatch cleavage (5, 9).


We used the CMC technique for the detection of the G919A mutation in a homozygous sample. Heteroduplex formation was obtained by mixing the patient's DNA sample with a wild-type DNA sample. The highest yield of the cleavage product (61 nt) was observed when these samples were mixed in a 1:1 ratio. The electropherogram was identical to that obtained with the heterozygous G919A sample (Fig. 2D)


The cleavage efficiency with both hydroxylamine and Os[O.sub.4] was much higher for the T833C mutation than for the G919A mutation, although both create the same mismatch (Fig. 1). This may be related to differences in the base context of these two mutations. The T883C site is located between two T:A pairs, whereas the G919A mutation is located between two C:G pairs, and a mutation in the latter context is more resistant to chemical cleavage (31). In addition, there have been consistent reports that the T:G mismatch is only weakly reactive towards Os[O.sub.4] (9,10, 31), which may explain why the G919A mutation created no cleavage product with Os[O.sub.4] (data not shown).




Hydroxylamine and Os[O.sub.4] are known to modify their respective base targets (C and T) in a concentration-, time-, and temperature-dependent manner (1). This was confirmed for both probes in our study (Figs. 4 and 5). However, the relative fluorescence intensity declined when optimal cleavage conditions were obtained. This reduction of relative fluorescence intensity during high probe concentration, high temperature, or long incubation time (Figs. 4 and 5) may be related to loss of selectivity for mismatched residues, degradation of dsDNA, and degradation or oxidation of the fluorescent group (1). Hence, optimal cleavage conditions should be balanced against an acceptable signal-to-noise ratio, including small degradation peaks (Fig. 5) possibly derived from the cleaved fragment.

For hydroxylamine, the optimal relative fluorescence intensity relative to yield was obtained at a concentration of 3.0-3.8 mol/L, a temperature of 35-37 [degrees]C, and with a reaction time of 45-75 min (Fig. 4). The corresponding parameters for Os[O.sub.4] were 2.0-4.0 g/L Os[O.sub.4], 25-30 [degrees]C, and 15-25 min (Fig. 5).


We used the denatured fluorescenn-labeled DNA calibrator (50-500 nt) as the size calibrator. The calibrator was mixed and analyzed together with the intact PCR product (186 nt), which eluted as a double peak, presumably because of the different mobilities of the two strands (Fig. 6). Sufficiently high resolution of ssDNA was obtained to resolve a 1-nt difference in size (Fig. 2).


For the subsequent analyses of the cleavage products, we used the first component of this doublet as an internal standard. The sizes of the products were then estimated on the basis of their relative migration times, using the equation for the calibration curve (size M vs relative migration time [t.sub.r]) for the size makers (Fig. 6, inset). Table 1 compares the estimated and real sizes, and small but important differences were observed. Furthermore, the estimates were characterized by high precision (relative standard deviation <0.12%, Table 1).

The small difference between estimated and real size of the cleavage products (Table 1) as well as the separation of the two opposing strands of the intact PCR product (Fig. 6) may be attributed to sequence-specific migration, as has previously been demonstrated by Gunman et al. (32) for oligonucleotides separated by CE under denaturing conditions. Likewise, separation of opposing strands has also been observed during slab gel electrophoresis of CMC products (5, 9). This anomalous migration impedes the exact localization of the mutation site by CMC, which should be performed by subsequent DNA sequencing.

Summary and Conclusion

The present work demonstrates the precise estimation of molecular size and yield of CMC cleavage products by CE-LIF and exploits the efficient separation of ssDNA, demonstrating the fast analysis, the quantitative detection, and the small sample requirements of this technology. Both primers were labeled with the same fluorophore, and the products were detected by a single-channel detector. This precludes differentiation between cleavage of the coding or non-coding strand, which requires labeling with different fluorophores and dual-channel detection. An alternative and less expensive approach is repeated analysis using only one labeled primer. Thus, this technology is suitable for screening of known mutations giving expected CMC products. The main advantage compared with mutation assays based on restriction cleavage or primer extension is that unknown mutations are also detected and distinguished from known mutations, making this technology suitable for newborn screening for homocystinuria. However, because sequence-specific electrophoretic migration may prevent accurate assessment of fragment size, the exact localization of new mutations would require subsequent DNA sequencing.

The main disadvantage of the CMC technique is the use of toxic reagents, which could be avoided by using enzyme mismatch cleavage. However, not all mutations are detected by the latter technique, more than one enzyme is required, and unspecific cleavage of homoduplexes has been observed (33, 34). When these problems are solved, the combination of enzyme mismatch cleavage and CE-IF detection should be feasible.


This work was funded by European Union Commission Demonstration Project Contract No. BMH4-CT 95-0505. We thank Anne Berit Guttormsen for supplying the blood samples from homocystinuria patients and Norman J. Dovichi (University of Alberta, Canada) for providing a sheath-flow cell for our laser-induced fluorescence detector built in-house.

Received April 27, 1998; revision accepted July 29, 1998.


(1.) Cotton RGH, Rodrigues NR, Campbell RD. Reactivity of cytosine and thymine in single-base pair mismatches with hydroxylamine and osmium tetroxide and its application to the study of mutations. Proc Natl Acad Sci U S A 1988;85:4397-401.

(2.) Montandon AJ, Green PM, Giannelli F, Bentley DR. Direct detection of point mutations by mismatch analysis: application to haemophilia B. Nucleic Acids Res 1989;17:3347-58.

(3.) Rodrigues RN, Rowan A, Smith MEF, Kerr IB, Bodmer WF, Gannon JV, Lane DP. p53 mutation in colorectal cancer. Proc Natl Acad Sci U S A 1990;87:7555-9.

(4.) Forrest SM, Dahl HH, Howells DW, Dianzani I, Cotton RGH. Mutation detection in phenylketonuria by using chemical cleavage of mismatch: importance of using probes from both normal and patient samples. Am J Hum Genet 1991;49:175-83.

(5.) Verpy E, Biasotto M, Brai M, Misiano G, Meo T, Tosi M. Exhaustive mutation scanning by fluorescence-assisted mismatch analysis discloses new genotype-phenotype corrections in angioedema. Am J Hum Genet 1996;59:308-19.

(6.) Khrapko K, Hanekamp JS, Thilly WG, Belenku A, Foret F, Karger BL. Constant denaturant capillary electrophoresis (CDCE): a high resolution approach to mutational analysis. Nucleic Acids Res 1994;22:364-9.

(7.) Orita M, Iwahana H, Kanazawa H, Hayashi K, Sekiya T. Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc Natl Acad Sci U S A 1989;86:2766-70.

(8.) Nollau P, Wagener C. Methods for detection of point mutations: performance and quality assessment. Clin Chem 1997;43: 1114-28.

(9.) Verpy E, Biasotto M, Meo T, Tosi M. Efficient detection of point mutation on color-coded strands of target DNA. Proc Natl Acad Sci U S A 1994;91:1873-7.

(10.) Haris II, Green PM, Bentley DR, Giannelli F. Mutation detection by fluorescent chemical cleavage: application to hemophilia B. PCR Methods Appl 1994;3:268-71.

(11.) Kuypers AWHM, Willems PMW, van der Schans MJ, Linssen PCM, Wessels HMC, de Bruijn CHMM, et al. Detection of point mutation in DNA using capillary electrophoresis in a polymer network. J Chromatogr 1993;621:149-56.

(12.) Arakawa H, Nakashiro S, Maeda M, Tsuji A. Analysis of single-strand DNA conformation polymorphism by capillary electrophoresis. J Chromatogr A 1996;722:359-68.

(13.) Kuypers AWHM, Linssen PCM, Willems PMW, Mensink EJBM. On-line melting double strand DNA for analysis of single-stranded DNA using capillary electrophoresis. J Chromatogr B 1996;675: 205-11.

(14.) Hebenbrock K, Williams PM, Karger BL. Single strand conformational polymorphism using capillary electrophoresis with two-dye laser-induced fluorescence detection. Electrophoresis 1995;16: 1429-36.

(15.) St Claire RL. Capillary electrophoresis. Anal Chem 1996;68: 569R-86R.

(16.) Grossman P. Electrophoretic separation of DNA sequencing extension products using low-viscosity entangled polymer network. J Chromatogr 1994;663:219-27.

(17.) Gelfi C, Orsi A, Leoncini F, Righetti PG. Fluidified polyacrylamide as molecular sieves in capillary zone electrophoresis of DNA fragments. J Chromatogr A 1995;689:97-105.

(18.) Ren J, Ulvik A, Ueland PM, Refsum H. Analysis of single-strand conformation polymorphism by capillary electrophoresis with laser-induced fluorescent detection using short-chain polyacrylamide as sieving medium. Anal Biochem 1997;245:79-84.

(19.) van der Schans MJ, Kuypers AWHM, Kloosterman AD, Janssen HJT, Everaerts FM. Comparison of resolution of double-stranded and single-stranded DNA in capillary electrophoresis. J Chromatogr A 1997;772:255-64.

(20.) Kraus JP, Kim L, Swaroop M, Ohuro T, Tahara T, Rosenberg LE, et al. Human cystathionine R-synthase cDNA: sequence, alternative splicing and expression in cultured cells. Hum Mol Genet 1993; 2:1633-8.

(21.) Hu FL, Gu Z, Kozich V, Kraus JP, Ramesh V, Shih VE. Molecular basis of cystathionine R-synthase deficiency in pyridoxine responsive and nonresponsive. Hum Mol Genet 1993;1:1857-60.

(22.) Tsai MY, Bignell M, Schwichtenberg K, Hanson NQ. High prevalence of a mutation in the cystathionine R- synthase gene. Am J Hum 1996;59:1262-7.

(23.) Tsai MY, Hanson NQ, Bignell M, Schwichtenberg KA. Simultaneous detection and screening of T833C and G919A mutation R-synthase gene by single-strand conformational polymorphism. Clin Biochem 1996;29:473-7.

(24.) Kim CE, Gallagher PM, Guttormsen AB, Refsum H, Ueland PM, Ose L, et al. Functional modelling of the cystathionine R-synthase in yeast: a common pyridoxine-responsive mutation in homocystinuria. Hum Mol Genet 1997;6:2213-27.

(25.) Zarrin F, Dovichi NJ. Sub-picoliter detection with the sheath flow cuvette. Anal Chem 1985;57:2690-2.

(26.) Jorgenson JW, Lukacs KD. Zone electrophoresis in open-tubular glass capillaries. Anal Chem 1981;53:1298-302.

(27.) Ren J, Liu H. Chiral separation of dioxypromethazine enantiomers by capillary electrophoresis using R-cyclodextrin as a Chiral selector. J Chromatogr A 1996;732:175-81.

(28.) Kraus JP. Molecular basis of phenotype expression in homocystinuria. J Inherit Metab Dis 1994;17:383-90.

(29.) Dawson PA, Cox AJ, Emmerson BT, Dudman NPB, Kraus JP, Gordon RB. Characterisation of five missense mutation in the cystathionine beta-synthase gene from three patients with B6 nonresponsive homocystinuria. Eur J Hum Genet 1997;5:15-21.

(30.) Rubin CM, Schmid CW. Pyrimidine-specific chemical reactions useful for DNA sequencing. Nucleic Acids Res 1980;8:4613-9.

(31.) Saleeba JA, Ramus SJ, Cotton RGH. Complete mutation detection using unlabeled chemical cleavage. Hum Mut 1992;1:63-9.

(32.) Guttman A, Nelson RJ, Cooke N. Prediction of migration behaviour of oligonucleotides in capillary gel electrophoresis. J Chromatogr 1992; 593:297-303.

(33.) Youil R, Kemper BW, Cotton RGH. Screening for mutations by enzyme mismatch cleavage with T4 endonuclease VII. Proc Natl Acad Sci U S A 1995;92:87-91.

(34.) Smith J, Modrich P. Mutation detection with Mutes, Mutt, and MutS mismatch repair proteins. Proc Natl Acad Sci U S A 1996;93:4374-9.


Department of Pharmacology, University of Bergen, Armauer Hansens Hus, 5021 Bergen, Norway.

* Author for correspondence. Fax 47-55-974605; e-mail

[1] Nonstandard abbreviafions: CMC, chemical mismatch cleavage; ds, double-stranded; ss, single-stranded; CE, capillary electrophoresis; LIF, laser-induced fluorescence; SLPA, short-chain linear polyacrylamide; CBS, cystathionine [beta]-synthase; and nt, nucleotide.
Table 1. The real and estimated sizes of the original PCR product and
cleaved products after CMC (n = 8).

Genotype Real size, nt Estimated size, nt RSD, (a) %

1st peak (PCR) 186 176 0.16
2nd peak (PCR) 186 184 0.16
T833C 40 36 0.12
G919A 61 60 0.11

(a) RSD, relative standard deviation.
COPYRIGHT 1998 American Association for Clinical Chemistry, Inc.
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 1998 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Title Annotation:Molecular Diagnostics and Genetics
Author:Ren, Jicun; Ulvik, Arve; Refsum, Helga; Ueland, Per Magne
Publication:Clinical Chemistry
Date:Oct 1, 1998
Previous Article:K-ras mutations in stools and tissue samples from patients with malignant and nonmalignant pancreatic diseases.
Next Article:The precursor form of the human kallikrein 2, a kallikrein homologous to prostate-specific antigen, is present in human sera and is increased in...

Related Articles
Capillary electrophoresis-based heteroduplex analysis with a universal heteroduplex generator for detection of point mutations associated with...
Rapid detection of [beta]-globin gene mutations and polymorphisms by temporal temperature gradient gel electrophoresis.
Capillary and microchip electrophoresis for rapid detection of known mutations by combining allele-specific DNA amplification with heteroduplex...
Use of constant denaturant capillary electrophoresis of pooled blood samples to identify single-nucleotide polymorphisms in the genes (Scnn1a and...
Genotyping of eight thiopurine methyltransferase mutations: three-color multiplexing, "two-color/shared" anchor, and fluorescence-quenching...
Validation of double gradient denaturing gradient gel electrophoresis through multigenic retrospective analysis.
C677T mutation of methyl enetetrahydrofolate reductase gene determined in blood or plasma by multiple-injection capillary electrophoresis and...
Simultaneous determination of methylenetetrahydrofolate reductase C677T and factor V G1691A genotypes by mutagenically separated pcr and...
Automated fluorescent analysis procedure for enzymatic mutation detection.
Rapid capillary zone electrophoresis in isoelectric histidine buffer: high resolution of the poly-T tract allelic variants in intron 8 of the CFTR...

Terms of use | Privacy policy | Copyright © 2020 Farlex, Inc. | Feedback | For webmasters