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Clinical Chemistry 53: 1046-1052, 2007. First published April 19, 2007; 10.1373/clinchem.2006.083303
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(Clinical Chemistry. 2007;53:1046-1052.)
© 2007 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Quantitative Mitochondrial DNA Mutation Analysis by Denaturing HPLC

Kok Seong Lim1,a, Robert K. Naviaux2,3 and Richard H. Haas1,3

Departments of1 Neurosciences, 2 Medicine, and 3 Pediatrics, School of Medicine, University of California San Diego, La Jolla, CA.

aAddress 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{at}ucsd.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: In recent years, denaturing HPLC (DHPLC) has been widely used to screen the whole mitochondrial genome or specific regions of the genome for DNA mutations. The quantification and mathematical modeling of DHPLC results is, however, underexplored.

Methods: We generated site-directed mutants containing some common mutations in the mitochondrial DNA (mtDNA) tRNA(leu) region with different mutation loads and used PCR to amplify the gene segment of interest in these mutants. We then performed restriction digestion followed by slow reannealing to induce heteroduplex formation and analyzed the samples by use of DHPLC.

Results: We observed a quadratic relationship between the heteroduplex peak areas and mutant loads, consistent with the kinetics of heteroduplex formation reported by others. This was modeled mathematically and used to quantify mtDNA mutation load. The method was able to detect a mutation present in a concentration as low as 1% and gave reproducible measurements of the mutations in the range of 2.5%–97.5%.

Conclusion: The quantitative DHPLC assay is well suited for simultaneous detection and quantification of DNA mutations.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
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)1 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)(2)(3)(4)(5)(6), single-strand conformation polymorphism (7)(8)(9), denaturing gradient gel electrophoresis (10)(11)(12), temperature gradient gel electrophoresis (13), temporal temperature gradient gel electrophoresis (14)(15)(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)(2)(3)(4)(5)(6). In these studies, DHPLC has been used either to screen the whole mitochondrial genome (1)(2)(4)(5)(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
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
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 µmol/L uridine, and 1 mmol/L pyruvate (Sigma-Aldrich) in 5% CO2 at 37 °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). PCRs were performed in 70 µL Optimase reaction buffer (Transgenomic) containing 50 ng plasmid DNA or 150 ng total DNA, 200 µmol/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 iCycler Thermal Cycler (Bio-Rad). The conditions for PCR were as follows: 95 °C for 2 min; 35 cycles of 95 °C for 30 s, 56 °C for 30 s, and 72 °C for 3 min; and a final extension step of 72 °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 °C for 6 h. Digested PCR products were denatured at 95 °C for 5 min and then slowly cooled to 25 °C at a rate of 1 °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 °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 µL) 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 nm. 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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
restriction enzyme digestion
Incubation of the PCR products with restriction enzyme DdeI results in the generation of 5 fragments of different sizes: 442, 342, 278, 210, and 126 bp (1). The mutation sites of interest lie in the 342-bp fragment containing the tRNA (leu1) 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 °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 DdeI for 6, 12, and 24 h and analyzed by DHPLC at 50 °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 °C). We found the optimal temperature (the temperature that results in the best resolution of the heteroduplex and homoduplex peak) to be 59 °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 signal:noise ratio >1.3 for the mutants studied was 1%, whereas the quantification limit with a signal:noise ratio >2 was 2.5% (Fig. 1B ).


Figure 1
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  		Figure 1. Detection of A3243G mutations by DHPLC in a series of samples with increasing mutant loads.

(A), various amounts of PCR products generated from mutant and wild-type DNA were mixed to produce samples of different mutant loads ranging from 0% to 100%, and these samples were subjected to heteroduplex formation. DHPLC analysis at 59 °C showed that the heteroduplex species (eluted earlier) resulting from the mutation at A3243G were well separated from the homoduplex species (eluted later). (B), enlarged image of the heteroduplex peaks in samples with mutant loads from 0% to 5% A3243G. The small peak preceding the homoduplex peak in the 0% mutant is labeled peak X. The detection limit in DHPLC analysis with a signal:noise ratio >1.3 for the A3243G mutants studied was 1%.

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. 1BUp ), 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. 1BUp ) 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 2
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  		Figure 2. Parabolic relationships between proportion of measured peak areas of heteroduplexes and levels of heteroplasmy.

PCR products generated from mutant and wild-type plasmids were mixed in different proportions to give different levels of heteroplasmy, and the mixtures were subjected to heteroduplex formation and DHPLC analysis. A parabolic relationship with a correlation coefficient of ~0.99 was observed in the A3234G mutant. Each data point represents mean ± SD for 3 separate samples. Graphs for other mutants can be found in Fig. 3 in the online Data Supplement.

quantitative analysis of mutation by dhplc
We measured peak areas of both heteroduplex and homoduplex for samples containing different levels of A3243G mutation (Fig. 1AUp ). 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. 2Up ). 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.

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. 2Up ). 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:

Formula 1(1)

Formula 2(2)

Formula 3(3)

On the basis of Eqs. 1–3Up Up Up , 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. 2Up ; see Fig. 3 in the online Data Supplement).


Figure 3
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  		Figure 3. Measurement of levels of heteroplasmy in plasmid DNA samples with known mutant loads.

Digested PCR products generated from wild-type plasmid DNAs and A3243G mutant plasmid DNA were mixed to generate samples of various levels of heteroplasmy (0%–100%), subjected to heteroduplex formation, and analyzed by DHPLC. Measured levels consistently corresponded to the actual levels in samples tested, hence validating the quantitative method used in our analysis. Each level represents the mean of 3 separate samples with SD.

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

Formula 4(4)
Solving x in a quadratic equation:

Formula 4

Formula 4

Formula 5(5)
It follows from Eqs. 4Up and 5Up that:

Formula 6(6)
In an experiment, y can be obtained with Eq. 7 :

Let AHET = measured peak area of heteroduplex peak; AHOM = measured peak area of homoduplex peak

Formula 7(7)

Eq. 6Up 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. 1–3Up Up Up , 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 y1 = predicted/measured proportion of heteroduplex (%) after mixing with 100% wild-type:

Formula 8(8)

Formula 9(9)
As heteroplasmy must be <100%:

Formula 10(10)
Therefore, we hypothesized that Eqs. 6Up and 10Up 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 y1 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 x1 will have undefined values. In these cases, the level of heteroplasmy can be estimated to be ~50% ± 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. 3Up ).

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 ).


Figure 4
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  		Figure 4. Effects of nuclear DNA and DNA amount on the quantitative DHPLC analysis.

(A), parabolic relationships between measured proportion of heteroduplexes and levels of heteroplasmy in the presence of nuclear DNA. Samples of different levels of heteroplasmy were subjected to PCR in the presence or absence of nuclear DNA isolated from rho-0 cells and then analyzed by DHPLC after heteroduplex formation. Each data point represents the mean ± SD for 3 separate samples. (B), determination of mutant loads in various amounts of DNA isolated from whole blood of 1 patient. Increasing amounts of total DNA (10–300 ng) were subjected to PCR, restriction enzyme digestion, and analysis by DHPLC after heteroduplex formation. The level of heteroplasmy measured was independent of the sample amount used and remained at ~34%. Each data point represents the mean ± SD for 3 separate samples.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
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).

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.


   Acknowledgments
 
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.


   Footnotes
 
1 Nonstandard abbreviations: RFLP, restriction fragment length polymorphism; DHPLC, denaturing HPLC; mtDNA, mitochondrial DNA.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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K. S. Lim, R. K. Naviaux, S. Wong, and R. H. Haas
Pitfalls in the Denaturing High-Performance Liquid Chromatography Analysis of Mitochondrial DNA Mutation
J. Mol. Diagn., January 1, 2008; 10(1): 102 - 108.
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