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Quantitative mitochondrial DNA mutation analysis by denaturing HPLC.

Most methods for detecting point mutations are either simple with low sensitivity or laborious and overly sensitive. Relatively simple, inexpensive, and conventional methods include PCR-restriction fragment length polymorphism (RFLP) [4] analysis and PCR/allele-specific oligonucleotide dot blot analysis. Radiolabeled PCR-RFLP is more sensitive but radioactive. Other methods that have been developed to study mutations include denaturing HPLC (DHPLC) (1-6), single-strand conformation polymorphism (7-9), denaturing gradient gel electrophoresis (10-12), temperature gradient gel electrophoresis (13), temporal temperature gradient gel electrophoresis (14-16), and pyrosequencing (2,17). A mutation detection method recently used in the identification of heteroplasmy relies on the use of Surveyor nuclease, which cleaves DNA at sites of base-substitution mismatch and short insertion/ deletion (18,19). Highly sensitive but time-consuming methods include ligation-mediated PCR (20), real-time fluorescent PCR (21, 22), and peptide nucleic acid binding assays (23). A recently developed method is based on reversed-phase HPLC coupled to electrospray ionization time-of-flight mass spectrometry (24).

DHPLC has become a popular technology in the study of mutation detection. It relies on the use of a hydrophobic column based on reversed-phase liquid chromatography for the separation of heteroduplex and homoduplex DNA at specific optimized temperatures and has recently been used by several research groups to study mitochondrial DNA (mtDNA) mutation (1-6). In these studies, DHPLC has been used either to screen the whole mitochondrial genome (1, 2, 4-6) or to study specific regions of the genome for mutation (3, 4, 25), followed by DNA sequencing to identify the mutation sites. Several of these studies have also measured the percentage of heteroplasmy in the mtDNA by use of PCR-RFLP-PAGE (4) and pyrosequencing (2).

The use of DHPLC in quantitative analysis of DNA mutation, however, has not been popular. Earlier studies on heteroduplex analysis suggested that the kinetics of heteroduplex formation would fit a quadratic model (26, 27). Using DHPLC technology, we investigated the relationship between heteroduplex peak area (which is more commonly used in quantitative chromatographic analysis) and level of heteroplasmy, finding a parabolic rather than a linear relationship between peak area and mutant load. We developed a method to accurately quantify the mutant load in mtDNA; using this quantitative DHPLC analysis method, we measured the levels of heteroplasmy in several DNA samples with known mutation loads.

Materials and Methods

CELL CULTURE

We cultured 143B osteosarcoma rho-0 cells (gift from Dr. Keshav Singh of Roswell Park Cancer Institute, Buffalo, NY) in DMEM with 4.5 g/L D-glucose and L-glutamine (Invitrogen) containing 10% (vol/vol) fetal bovine serum, 1% (vol/vol) penicillin/ streptomycin (Invitrogen), 200 [micro]mol/L uridine, and 1 mmol/L pyruvate (Sigma-Aldrich) in 5% C[O.sub.2] at 37 [degrees]C. We maintained the cells in logarithmic growth phase by routine passage every 1-2 days. Uridine and pyruvate added to the media were always from fresh stock solution.

DNA EXTRACTION

We used a blood DNA sample from a healthy individual as the template for the generation of site-directed mutants and 143B osteosarcoma rho-0 cells as the source of pure nuclear DNA. In 1 experiment involving the investigation of the effect of DNA amount on heteroplasmy, we used patient blood DNA; the sample was obtained under a University of California San Diego Institutional Review Board-approved protocol, and informed consent was obtained from the patient. We extracted total DNA from whole blood samples and nuclear DNA from rho-0 cells with the Puregene DNA Purification reagent set (Gentra Systems) according to the manufacturer's instructions. Briefly, we collected the leukocytes from whole blood by centrifugation after removal of erythrocytes by mild lysis, whereas we collected rho-0 cells at 80%-90% confluence. The cells were lysed and incubated with RNase A, and protein was removed by high-salt precipitation. DNA was precipitated by use of isopropanol.

SITE-DIRECTED MUTAGENESIS

All mutant clones used in this study have the genome segment containing mitochondrial tRNA Leu (UUR; 2415-3812 bp), which is inserted into pCRII (Invitrogen). We generated point mutations by use of the QuikChange XL Site-Directed Mutagenesis reagent set (Stratagene) according to the manufacturer's instructions. Forward and reverse primers used to create point mutations are listed in Table 1 in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol53/issue6. We confirmed target mutations by sequencing with 5'-CTA CTT CAA ATT CCT CCC TGT AC-3' (3104-3126 bp) and 5'-CATTAGGAATGCCATTGC-3' (3352-3369 bp).

PCR PRIMERS AND CONDITIONS

Primers (Invitrogen) used for the amplification of the region of interest in the mitochondrial genome (J01415.2) before DHPLC analysis were 5'-CTCACTGTCAACCCAACACAGG-3' (2415-2436 bp) and 5'-TGTGTTGTGATAAGGGTGGAGAG-3' (3790-3812 bp) for detection of A3243G, C3256T, A3260G, T3271C, T3291C, and T3308C. These primers can be obtained from the MitoScreen reagent set (Transgenomic) and have been used in a published study (1). PCBs were performed in 70 p,L Optimase reaction buffer (Transgenomic) containing 50 ng plasmid DNA or 150 ng total DNA, 200 [micro]mol/L of each dNTP (Transgenomic), 21 pmol of the forward and reverse primer, and 3.5 units Optimase DNA polymerase (Transgenomic). We performed PCR by use of the CCycler Thermal Cycler (Bio-Rad). The conditions for PCR were as follows: 95 [degrees]C for 2 min; 35 cycles of 95 [degrees]C for 30 s, 56 [degrees]C for 30 s, and 72 [degrees]C for 3 min; and a final extension step of 72 [degrees]C for 5 min.

RESTRICTION ENZYME DIGESTION AND HETERODUPLEX FORMATION

PCR products generated were digested with 1 unit of DdeI (New England Biolabs) at 37 [degrees]C for 6 h. Digested PCR products were denatured at 95 [degrees]C for 5 min and then slowly cooled to 25 [degrees]C at a rate of 1 [degrees]C /min to induce heteroduplex formation before DHPLC analysis. These conditions produce maximal heteroduplex formation--~50% heteroduplex (see Results) in samples containing ~50% heteroplasmy, indicating that the slow reannealing procedure is optimal. We confirmed stability of these complexes by comparison of samples injected immediately after reannealing and 2 days later, finding no change in heteroduplex peak areas (data not shown).

DHPLC ANALYSIS

We analyzed samples by use of WAVE 3500 equipped with a Hitachi D-7000 Interface, L-7100 Pump, L-7200 Autosampler, L-7300 Oven, and L-7400 UV Detector (Transgenomic). We separated nucleic acids by use of a DNASep cartridge (4.6 mm x 50 mm; Transgenomic). We predicted temperatures for sample analysis by use of Navigator software (version 1.6.2) (28) (Transgenomic) and confirmed them experimentally to be 59 [degrees]C. Alternatively, prediction of melting temperatures could be performed by use of the DHPLC Melt program created by Stanford Genome Technology (29). The gradient mobile phase consists of buffer A [0.1 mol/L triethylammonium acetate, pH 7 (Transgenomic)] and buffer B [0.1 mol/L triethylammonium acetate, pH 7, and 25% acetonitrile (Chromasolv; Sigma-Aldrich)]. Samples (5 [micro]L) were injected for analysis. Fragments were eluted with a linear acetonitrile gradient of 2% /min from 45% to 67% buffer B at a flow rate of 0.9 mL/min. UV detection was set at 260 run. After each run, we washed the column with 75% acetonitrile for 1 min and equilibrated it for 1.5 min before the next sample injection. The peak areas were determined by use of Navigator software, and quadratic curve fitting was performed with Microsoft Excel.

Results

RESTRICTION ENZYME DIGESTION Incubation of the PCR products with restriction enzyme Ddel results in the generation of 5 fragments of different sizes: 442, 342, 278, 210, and 126 by (1). The mutation sites of interest lie in the 342-bp fragment containing the tRNA (leul) sequence. We studied the amount of DNA and incubation time required for complete restriction enzyme digestion of PCR products. Various amounts of the total and plasmid DNA were used separately in the PCR, and amplicons were analyzed by DHPLC at 50 [degrees]C after restriction enzyme digestion (see Fig. 1 in the online Data Supplement). PCR products generated from plasmid DNA were more abundant than those from total DNA, suggesting a difference in kinetics of DNA amplification between these 2 types of DNA templates. The concentrations of both types of PCR products began to reach steady state after use of ~25 ng (for plasmid DNA) and 50 ng (for total DNA; see Fig. 1 in the online Data Supplement).

In the time course experiment, PCR products were digested by Ddel for 6, 12, and 24 h and analyzed by DHPLC at 50 [degrees]C. Neither an increase in restriction fragment peak area nor appearance of additional fragment peaks occurred, suggesting that the restriction enzyme digestion was complete (data not shown). This was also confirmed by gel electrophoresis of the digestion mixture, which showed the absence of undigested PCR products after 6 h of restriction enzyme digestion (data not shown).

DETECTION OF MUTATION BY DHPLC

We experimentally confirmed the DHPLC temperature predicted by the Navigator software by analyzing the 50% heteroplasmic A3243G mutant samples at various temperatures (57-61 [degrees]C). We found the optimal temperature (the temperature that results in the best resolution of the heteroduplex and homoduplex peak) to be 59 [degrees]C (data not shown). We generated serial dilutions of the 100% mutant with 100% wild-type PCR products, resulting in samples with mutation present at levels of 0%,1%, 2.5%, 5%, 10%, 25%, 40%, 50%, 60%, 75%, 90%, 95%, 97.5%, 99%, and 100%. We subjected these samples to reannealing to induce heteroduplex formation before DHPLC analysis. We determined peak areas of both heteroduplex and homoduplex peaks (Fig. 1A). The

detection limit in DHPLC analysis with a signalmoise ratio >1.3 for the mutants studied was 1%, whereas the quantification limit with a signalmoise ratio >2 was 2.5% (Fig. 113).

Optimase is a proofreading thermostable polymerase that has been shown to effectively minimize the misincorporation of nucleotides during DNA amplification by PCR compared with other DNA polymerase, e.g., AmpliTaq and Pfu polymerase (5,30). However, we consistently observed a small peak that precedes the homoduplex peak in all the 100% wild-type and mutant DNA (Fig. 113), a finding similar to what has been reported in the 2 abovementioned studies. Such a low-level peak with a consistent retention time (referred to as peak X in the 0% mutant in Fig. 1B) can affect the correct identification of real mutations, especially in DNA with low levels of mutation. We investigated whether peak X is affected by the amount of DNA used in the PCR. We observed no increase in the ratio of peak X area to total peak area, and this ratio remained constant at ~3% or less over the range of DNA concentrations studied (see Fig. 2 in the online Data Supplement). Further increase in the total DNA amount to 600 ng did not increase the ratio measured (not shown). Such a background level may vary from column to column (but the maximum we have observed is 3%), and we always used a wild-type DNA (DNA from a healthy volunteer that has been confirmed by sequencing to be 100% identical in the region studied to the published mitochondrial genome sequence J01415.2) in the DHPLC analysis as a negative control.

[FIGURE 1 OMITTED]

QUANTITATIVE ANALYSIS OF MUTATION BY DHPLC

We measured peak areas of both heteroduplex and homoduplex for samples containing different levels of A3243G mutation (Fig. 1A). Regression analysis of the measured proportion of heteroduplex (ratio of heteroduplex peak area to total peak area, %) and the level of heteroplasmy (%) yielded a quadratic plot with a correlation coefficient of ~0.99 (Fig. 2). Such a relationship appeared to be consistent with the kinetics of heteroduplex formation (27). To confirm this, we generated 5 other site-directed mutants and performed the regression analysis with different levels of mutations. The plots generated on the basis of these mutants confirmed our finding with A3243G (see Fig. 3 in the online Data Supplement), and thus such a calibration curve could be used for the measurement of level of heteroplasmy.

[FIGURE 2 OMITTED]

DETERMINATION OF EXACT PERCENTAGE OF MUTATION

Given the parabolic relationship between the measured proportion of heteroduplex and levels of heteroplasmy, only the vertex of the parabola at 50% heteroduplex has a single, unique solution for the level of heteroplasmy, all other proportions of heteroduplex formation having 2 positive solutions by virtue of the quadratic function that governs DNA reannealing. For example, a measured proportion of 30% heteroduplex would suggest 2 possible heteroplasmy levels at ~20% and 80% (Fig. 2). By use of PCR products with known mutant loads (10%, 25%,40%, 60%, 75%, and 90%), we determined the correct levels by mixing these samples with equal concentrations of 100% wild-type PCR products and subjected them to additional slow reannealing process before DHPLC analysis. Analysis of the proportions of measured heteroduplex peak area and the level of heteroplasmy gave a quadratic plot that peaks at 50% when a 100% mutant DNA is mixed with 100% wild-type DNA (see Fig. 4A in the online Data Supplement). This quadratic equation allows confirmation of the heteroplasmy level in mtDNA. Such a mixing experiment produces samples that have a measured proportion of heteroduplex that may be higher or lower than the measured proportion before mixing, depending on the level of heteroplasmy.

MODELING OF QUANTITATIVE DHPLC ANALYSIS

Because all 6 mutants that we studied showed a similar quadratic relationship between the measured proportion of heteroduplex and level of heteroplasmy in plasmid DNA, and because an independent group also reported a similar observation with genomic DNA (31), we speculated that there might be a universal equation that is applicable to most if not all types of quantitative heteroduplex analysis--hence quantitative DHPLC analysis. Theoretically, the proportion of a given heteroduplex or homoduplex can be calculated from the product of the stoichiometric fraction of each DNA species (27).

Let m = fraction of mutant DNA species; 1 - m = fraction of wild-type DNA species; [therefore] after heteroduplex formation:

Proportion of mutant homoduplex = [m.sup.2] (Eq. 1)

Proportion of wild-type homoduplex = [(1 - m).sup.2] (Eq. 2)

Proportion of each type of heteroduplex = m (1 - m) (Eq. 3)

On the basis of Eqs. 1-3, we calculated the predicted proportion (in percentage) of the heteroduplexes formed after slow reannealing for DNA with mutant load of 0%, 1%, 2.5%, 5%,10%, 25%, 40%, 50%, 60%, 75%, 90%, 95%, 97.5%, 99%, and 100% and plotted a graph on the basis of these predicted values (see Fig. 4B in the online Data Supplement). We observed a quadratic relationship (Eq. 4) between the predicted proportions of heteroduplex (%) and the level of heteroplasmy, consistent with our experimental data (Fig. 2; see Fig. 3 in the online Data Supplement).

Let x = level of heteroplasmy (%); y = predicted/ measured proportion of heteroduplex (%):

[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] (Eq. 5)

It follows from Eqs. 4 and 5 that:

[therefore]x [approximately equal to] 50 [+ or -] [square root of (2500 - 50y)] (Eq. 6)

In an experiment, y can be obtained with Eq. 7:

Let [A.sub.HET] = measured peak area of heteroduplex peak; [A.sub.HOM] = measured peak area of homoduplex peak

y = [A.sub.HET] / [A.sub.HET] + [A.sub.HOM] x 100% (Eq. 7)

Eq. 6 showed that there would be 2 values for each x. As mentioned earlier, we can confirm the levels by mixing samples with 100% wild-type samples. Using Eqs. 1-3, we calculated the predicted proportions of the heteroduplexes (in percentage) formed after slow reannealing for DNA (with initial mutant load of 0%, 1%, 2.5%,5%, 10%, 25%, 40%, 50%, 60%, 75%, 90%, 95%, 97.5%, 99%, and 100%) that are later mixed in equal concentrations with 100% wild-type PCR products. Fig. 4C in the online Data Supplement shows a graph plotted on the basis of these predicted values. We also obtained a quadratic equation (Eq. 8) consistent with our experimental data (see Fig. 4A in the online Data Supplement).

Let [y.sub.1] = predicted /measured proportion of heteroduplex (%) after mixing with 100% wild-type:

[y.sub.1] = -0.005[x.sub.2] + x (Eq. 8)

[therefore]x [approximately equal to] 100 [+ or -] [square root of (10 000 - 200[y.sub.1])] (Eq. 9)

As heteroplasmy must be <100%:

[therefore]x [approximately equal to] 100 - [square root of (10 000 - 200[y.sub.1])] (Eq. 10)

Therefore, we hypothesized that Eqs. 6 and 10 can be applied to the generic problem of DHPLC analysis to determine the correct level of heteroplasmy in mtDNA. It is important to note that if y or [y.sub.1] in these equations has a value >50 (which is likely to occur during the analysis because of variations in the measurements of DNAs with mutant loads at ~50%), x or [x.sub.1] will have undefined values. In these cases, the level of heteroplasmy can be estimated to be -50% [+ or -] 10%.

We tested this model by generating several samples with various degrees of mutation in A3243G (0%-100%), analyzed them by DHPLC, and determined the mutant load with these equations. Measured levels of heteroplasmy based on this equation were highly consistent with actual levels of heteroplasmy (Fig. 3).

EFFECT OF THE DNA AMOUNT AND THE PRESENCE OF NUCLEAR DNA

Because the calibration curves that we generated by use of plasmid DNA fitted our parabolic equation, we examined whether the presence of nuclear DNA from rho-0 cells has any effect on the measured level of heteroplasmy. Fig. 4A shows a parabola that is highly consistent between the measurements obtained from samples amplified in the presence and absence of nuclear DNA. Therefore, the presence of nuclear DNA does not affect the quantification of heteroplasmy. We also measured the level of heteroplasmy with various amounts of DNA (obtained from a single patient with a known level of heteroplasmy). We observed a similar level of mutation with differing DNA amounts in analysis, concluding that quantification by DHPLC is robust with various DNA amounts used in the analysis, at least in the interval of 10 to 300 ng (Fig. 4B).

Discussion

DHPLC exploits the differential melting properties of homoduplex and heteroduplex DNA to achieve the separation of these 2 species. Since its introduction in 1995 (32), DHPLC has gained tremendous popularity in the field of DNA mutation detection, especially over the last 5 years, owing to its high degree of automation and high sensitivity (33). It has been used widely as a mutation screening tool, but little has been done to explore its use as a quantitative tool. In this study, by analyzing plasmid DNA of various mutant loads, we demonstrated a quadratic relationship between the proportion of the peak area of heteroduplex and the level of heteroplasmy. Such a relationship agrees well with the kinetics of heteroduplex formation (see Fig. 4, B and C, in the online Data Supplement) and may well be applicable to all types of heteroduplex-based assays. Consistent with this observation is a recent report by Palais et al. (31), who observed a similar quadratic relationship in genomic DNA by high-resolution melting analysis. In an attempt to identify the optimal proportion of reference homozygous DNA to be added to a DNA sample to differentiate heterozygous, homozygous, and wild-type genotypes, Palais et al. (31) studied the dependence of heteroduplex proportion on genotype and the fraction of added wild-type reference DNA by the use of a series of samples containing different fractions of reference DNA. They concluded that optimal mixing with reference DNA permits genotyping of all single nucleotide polymorphisms (31).

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

On the basis of these observations, we developed a quantitative DHPLC method to study mtDNA mutation load. We validated this method by determining the heteroplasmy level in several plasmid DNA samples of known mutant loads. We found levels of heteroplasmy in DNA to be highly consistent with the expected mutant load of DNA. Sequencing can be carried out to identify the sites and types of these mutations. One main caveat in quantitative DHPLC analysis, however, which is common to all quantitative chromatographic analyses (e.g., gas chromatography--mass spectrometry, liquid chromatography-mass spectrometry, HPLC/UV), is that the resolution of the heteroduplex and homoduplex peaks is critical to allow accurate quantification. Good peak separation can be achieved with the selection of optimal temperature and acetonitrile gradient. Theoretically, 4 peaks are resolved in a reannealed heteroplasmic DNA mixture, which produces 2 homoduplex peaks and 2 heteroduplex peaks. Unfortunately, in most cases the homoduplex peaks elute at almost the same time, preventing quantification of heteroplasmy level from the homoduplex peaks (data not shown).

Several reports have used DHPLC for mutation detection but have relied on alternative assays to measure the level of heteroplasmy (2, 4). This report is the first validated quantitative method based on DHPLC, and with the increasingly widespread applications of DHPLC in the study of mtDNA mutation, it will be useful to rely on DHPLC for both mutation screening and quantification in the study of mtDNA. Similarly, the quantitative DHPLC method can also be applied to the study of nuclear DNA mutation. For instance, cancerous tissues are heterogeneous in nature (tumor cells are often aneuploidy and surrounded by normal cells), and the tissue specimens obtained may contain both wild-type and mutant DNAs. Determination of the mutant loads in such tissues may be useful in the genetic characterization of cancer.

Grant/funding support: We are grateful to the United Mitochondrial Disease Foundation for a grant to R.H.H. in support of this project and to the Wright Family Foundation and Rita and Steven Achard for generous gifts in support of this work. R.K.N. was supported by the University of California San Diego Foundation Christini Fund and by generous gifts from the Scott Pawlowski Memorial Fund and Betty Gleeson. Financial disclosures: None declared.

Acknowledgements: We thank Scott Wong for technical assistance with instrumentation and cloning and Dr. Keshav Singh for the gift of the 143B osteosarcoma rho-0 cells.

Received November 17, 2006; accepted March 27, 2007. Previously published online at DOI: 10.1373/clinchem.2006.083303

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[4] Nonstandard abbreviations: RFLP, restriction fragment length polymorphism; DHPLC, denaturing HPLC; mtDNA, mitochondrial DNA.

KOK SEONG LIM, [1] * ROBERT K. NAVIAUX, [2,3] and RICHARD H. HAAS [1,3]

Departments of [1] Neurosciences, [2] Medicine, and [3] Pediatrics, School of Medicine, University of California San Diego, La Jolla, CA.

* Address correspondence to this author at: University of California San Diego School of Medicine, 9500 Gilman Dr. B112, La Jolla, CA 92093-0935. Fax 619-543-7868; e-mail kslim@ucsd.edu.
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Title Annotation:Molecular Diagnostics and Genetics
Author:Lim, Kok Seong; Naviaux, Robert K.; Haas, Richard H.
Publication:Clinical Chemistry
Date:Jun 1, 2007
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