In this article we will discuss about the rapid analysis to detect base changes in human mitochondrial DNA by restriction enzyme analysis followed by Denaturing High Performance Liquid Chromatography (DHPLC).
Contents
Introduction to Mitochondrial Genome Analysis:
Mitochondria are DNA-containing organelles found within the cytoplasm of eukaryotic cells. Their main function is to provide energy for cellular activity in the form of ATP through the process of oxidative phosphorylation. Sequence analysis of the mitochondrial genome revealed this 16.5-kb circular molecule encodes 37 genes necessary for mitochondrial function.
Mitochondrial DNA molecules are maternally inherited and can be present at up to 10,000 copies per cell. It is estimated that the mutation rate in mtDNA is 10-times higher than that for nuclear DNA due to the combined effects of exposure to reactive oxygen species, the lack of protective histones, and the lack of efficient repair mechanisms.
Molecular analysis of the mitochondrial genome has applications in a wide range of study areas. Since 1988 defects in the human mitochondrial genome have been reported for several degenerative diseases, cancer, and aging.
Mitochondrial DNA analysis is increasingly being utilized in forensic cases where only limited amounts of degraded material are available. In addition, the study of mitochondrial DNA has also provided insights into human evolution and genetic population studies.
Several approaches have been undertaken to analyze the mitochondrial genome including direct sequence analysis, SSCP, TGGE, and DHPLC. An efficient method to rapidly screen mtDNA for base changes requires an extremely sensitive system capable of detecting low levels of heteroplasmy. This is the coexistence of normal and altered/mutant mtDNA molecules within an individual and can be maternally inherited or arise through somatic mutation.
For many degenerative diseases, the proportion and distribution of this heteroplasmy often determines the severity of clinical symptoms. DHPLC has been shown by several researchers to be a highly sensitive method for detecting low levels of heteroplasmy and somatic mutations in cases where other methods failed. An efficient assay is necessary because disorders resulting from mtDNA mutations often present with a wide range of clinical symptoms and have complex inheritance patterns.
The recent identification of mutations in nuclear genes that cause disease by introducing mutation in mtDNA further complicates the molecular diagnosis of mitochondrial disorders.
We present a rapid, sensitive DHPLC method to screen the human mitochondrial genome for base changes. Results from this method will contribute to the growing understanding of the complexities of mitochondrial pathology and can be used for a variety of different applications involving mtDNA.
Materials and Methods:
i. Amplification of Mitochondrial DNA:
Primer sets were designed to amplify the entire human mitochondrial genome in eighteen overlapping segments. The mitochondrial regions amplified, and the size of the polymerase chain reaction (PCR) products are listed in Table 5-1. Mitochondrial base pair designations are consistent with the MITO- MAP sequence version.
Genomic DNA from cell lines CHR and 9947A were obtained from the National Institutes of Standards and Technologies (NIST; SRM 2392), and genomic DNA from the K562 cell line was purchased from Promega.The mitochondrial fragments were amplified using the Expand’ Long Template PCR System in 1X PCR buffer 1 with 1.75 mM MgCl2, 350 µM each dNTP , 300 nM forward primer, 300 nM reverse primer, 1U Expand’ Taq polymerase mix, and 50 – 100 ng of genomic DNA according to the manufacturer’s instructions. The components were denatured at 93°C for 2 minutes, followed by 30 cycles of 93°C for 10 seconds; 58°C for 30 seconds; 68°C for 1.5 minutes, with a final extension step of 68°C for 7 minutes.
ii. Restriction Enzyme Digestion:
Restriction endonucleases were selected that cleave the mtDNA PCR products into ~ 100- to 600-bp fragments. The restriction enzymes, recommended 10X buffers, fragment sizes, and the corresponding mtDNA regions are listed in Table 5-2. PCR products amplified in mitochondrial sets 1, 2, 3, and 15 do not require a restriction enzyme digestion, and can be set-aside until the next step in the procedure.
Each restriction enzyme digestion was performed in a final volume of 100 µl with 88.5 µl of mtDNA PCR product, 10µl of the recommended 10X restriction enzyme buffer, and 1.5µl of the appropriate enzyme(s). The reactions were incubated at 37°C for a minimum of 2 hours, and 9pl were analyzed on the Transgenomic WAVE® DNA Fragment Analysis System at 50°C to check for complete digestion of the PCR products. A 10-µl aliquot of the pUC18/HaeIII digest was run as a size standard.
iii. Heteroduplex Detection by Denaturing High Performance Liquid Chromatography (DHPLC):
The WAVE System has a DNASep column with a stationary phase consisting of 2 µm nonporous, alkylated poly(styrene-divinylbenzene) particles, a UV detector set at 260 nm, and two 96-well autosamplers. The mobile phase consists of Solvent A: 0.1M triethylammonium acetate and Solvent B: 0.1M triethylammonium acetate, 25% (v/v) acetonitrile.The gradient for elution of the restriction enzyme fragments is shown in Table 5-3.
Digested mtDNA PCR products and MT sets 1, 2, 3, and 15 were analyzed by DHPLC using the same gradient conditions as shown in Table 5-3 for the restriction enzyme fragments. Products from each cell line were analyzed individually, and an equal amount of product from each cell line was mixed together and analyzed by DHPLC to detect the presence of single nucleotide polymorphisms (SNPs).
The samples were heated to 95°C for 5 minutes and cooled slowly to room temperature (-0.1°C/sec) to allow the formation of heteroduplex molecules. The column temperatures for each fragment were predicted by Wavemaker Software version 4.0 with minor adjustments (Table 5-4). An injection volume of 12µl was sufficient for each sample at each temperature, but this may need to be increased if the PCR product yield is low.
Results of Mitochondrial Genome Analysis:
i. Restriction Enzyme Analysis:
Mitochondrial PCR fragments amplified from CHR and 9947A cell lines were digested with the appropriate restriction enzymes and run on the WAVE System at 50°C. DNA fragments are separated on the basis of size at this non-denaturing temperature. This step is critical to ensure the expected number of peaks is produced and that no partial digestion products are present.
All PCR products and restriction enzyme fragments from the CHR and 9947A cell lines had the predicted number of peaks with elution times consistent with their expected sizes. Figure 5-1 shows the products of an AluI restriction enzyme digestion of MT set 18 amplified from K562, CHR, and 9947A cell lines. This example illustrates the potential problems that can result when a restriction fragment length polymorphism (RFLP) is present in one of the samples.
The expected peaks with elution times of 11.5, 14, and 15 minutes were present in the CHR and 9947A cell lines. However, the peak with the elution time of 14 minutes is absent in the K562 cell line (shown as an arrow with a dotted line in Figure 5- 1) and two additional peaks are present with elution times of 10 and 10.5 minutes (shown as arrows with solid lines). These results indicate that an additional recognition site for AluI is present in the K562 mitochondrial DNA sequence; therefore this cell line should not be used as a control for MT set 18.
ii. Heteroduplex Detection by DHPLC:
An equal amount of PCR product or restriction enzyme digested material from the CHR and 9947A cell lines was mixed, heated to 95°C to denature the DNA strands, and cooled slowly to allow the formation of heteroduplex molecules. Comparison of the peak areas on the WAVE® System at a column temperature of 50°C was a convenient method to determine the relative concentration of each sample.
In most cases, the yield of the PCR products was similar, so those samples were mixed in equal volumes. Each cell line was analyzed separately and run under the same conditions as the mixed samples as a control. Wavemaker(tm) software version 4.0 was used to predict the column temperature to detect the presence of heteroduplex molecules in each fragment. Comparison of the published mitochondrial DNA base pair changes between the CHR and 9947A cell lines resulted in 32 single nucleotide polymorphisms that map to 21 separate PCR or restriction enzyme fragments (see Table 5-5).
DHPLC was able to detect the SNPs in 21/21 (100%) of the fragments in the CHR and 9947A mixed samples. In most cases, the SNP was easily detected because the heteroduplex molecules eluted earlier than the homoduplex molecules and generated a new peak in the elution profile compared to the CHR and 9947A individual control samples. Figure 5-2 shows an example of a SNP in the MT set 9 CHR and 9947A mixed sample.
Genomic DNA from CHR and 9947A cell lines was amplified with MT Set 9 primers and digested with the restriction enzyme HaeIII. The individual CHR and 9947A digested PCR products and a 50:50 mixture of the two products were denatured at 95°C for 5 minutes and cooled slowly to room temperature. The size of the original PCR product is 1036 bp, and digestion with HaeIII results in fragments of 123 bp, 190 bp, 233 bp, and 490 bp.
Sequence analysis of CHR and 9947A mitochondrial DNA detected a single base change between these two cell lines (6221C/T) that maps within the 233 bp fragment eluting at 12 minutes (indicated by an arrow in Figure 5-2). The CHR and 9947A mixed sample clearly shows the formation of a heteroduplex peak at a column temperature of 58°C compared to the single 233- bp peak in the individual samples. This example demonstrates the sensitivity of DHPLC to detect this SNP, and interpretation of the result is straightforward based on heteroduplex detection by peak shape.
Since the multiple fragments analyzed in the mitochondrial sets are derived from a restriction enzyme digestion, the relative ratio of the peak heights within a sample can be used to detect the presence of SNPs by DHPLC. Figures 5-3A and 5-3B show two examples in which the peak height was used in combination with the peak shape to detect SNPs in the mixed samples.
The individual CHR and 9947A digested PCR products and a 50:50 mixture of the two products were denatured at 95°C for 5 minutes and cooled slowly to room temperature. DHPLC analysis was performed at a column temperature of 56°C for MT Set 8 and 59°C for MT Set 12. Sequence analysis of the mitochondrial DNA detected base changes between these two cell lines (5186G/A in the 396-bp fragment of MT set 8; 9315T/C in the 188-bp fragment of MT set 12).
Comparison of the peak heights in Figure 5-3A reveals the mV intensities of the peaks eluting at 14 and 14.6 minutes are similar in the CHR and 9947A cell lines. However, the relative ratio of the peak heights in the CHR and 9947A mixed sample are different from the individual samples.
The peak that elutes at 14.6 minutes has approximately half the mV intensity as the peak that elutes at 14 minutes. These examples demonstrate that although heteroduplex peaks can be identified in the CHR and 9947A mixed samples containing these SNPs, interpretation of the results is most sensitive when both peak shape and peak height (mV intensity) are evaluated.
Figure 5-4 shows the chromatograms of MT set 10 at column temperatures from 56 – 59°C. Heteroduplex peaks can be clearly identified in each CHR and 9947A mixed sample containing a SNP, but interpretation of the results is more complex. The size of the MT set 10 PCR product is 1390 bp, and digestion with Mspl results in fragments of 117 bp, 162 bp, 252 bp, 354 bp, and 505 bp.
Sequence analysis of CHR and 9947A mitochondrial DNA detected base changes between these two cell lines (6371T/C – 252-bp fragment or peak 3; 6791G/A – 162-bp fragment or peak 2; 7028T/C – 354-bp fragment or peak 4; 7645T/C – 505-bp fragment or peak 5).
The arrow in each chromatogram indicates the optimal screening temperature recommended for each fragment. Analysis of the elution profiles is more complex because the 505-bp fragment (peak 5) has a lower melting temperature than the 354bp fragment (peak 4), thus peak 5 elutes before peak 4 at 58°C and 59°C.
Finally, CHR and 9947A products were mixed in different ratios to determine if heteroplasmic base changes in the mitochondrial genome could be detected using this approach. Genomic DNA from CHR and 9947A cell lines was amplified with MT set 10 primers and digested with Mspl at 37°C for 2 hours.
The individual CHR and 9947A digested PCR products and 50:50, 80:20, and 90:10 mixtures of the two products were denatured at 95°C for 5 minutes and cooled slowly to room temperature. DHPLC analysis was performed at a column temperature of 56°C.
The CHR and 9947A samples show a single peak, while a heteroduplex peak is detected in the CHR:9947A-mixed samples (See Figure 5-5 arrow). This example demonstrates the feasibility of scanning for variable heteroplasmic base changes in the mitochondrial genome using this method.
Conclusion:
Molecular studies of the mitochondrial genome are of interest to members of the medical community determining links between mtDNA base changes and disease, the forensics community for identification purposes, and individuals studying human evolution. The goal of this study was to develop a protocol to efficiently screen the 16.5-kb mitochondrial genome for mutations and polymorphisms by DHPLC.
The mitochondrial genome was amplified in 18 over lapping sets and 14/18 products were digested with restriction enzymes that generate fragments between 100 – 600 bp. Restriction enzymes were selected that cleave the PCR products at 37°C into suitable fragments for DHPLC analysis, with consideration for the total number of enzymes required and the cost per unit of enzyme. Mitochondrial sets 1-3 span the hypervariable region and were analyzed by DHPLC without a restriction enzyme digestion step because this region is highly polymorphic.
A Standard Reference Material (SRM 2392) was established to provide researchers with a well-characterized source of mitochondrial DNA for sequencing, forensic identifications, medical diagnostics, and mutation detection studies. CHR and 9947A DNA samples were extracted from human lymphoblast cell culture lines and are commercially available.
Sequence comparison of these two mtDNA molecules revealed several polymorphic base changes dispersed throughout the 16.5-kb genome. Each cell line analyzed individually represents a homo- plasmic mitochondrial DNA sample, while a mixture of the two in equal proportions produces a sample that is 50% heteroplasmic for each base change.
The mixed CHR and 9947A mtDNAs provided an ideal standard to validate the feasibility and sensitivity of this approach to detect base changes in the mitochondrial genome. Twenty-one of the total 62 fragments analyzed had at least a single base change present in the mixed sample, and this method was able to detect heteroduplex peaks in all 21 positive control fragments.
Thus, the CHR and 9947A mixed sample should be routinely included as a positive control for this mitochondrial genome scanning procedure to detect unknown base changes in samples under investigation. Samples suspected of harboring homoplasmic mtDNA alterations/mutations should be mixed with normal template prior to DHPLC analysis.
Evaluation of the restriction enzyme products at a non-denaturing column temperature of 50°C is an important step in this method for two reasons. Firstly, the highly polymorphic nature of the mtDNA sequence can result in base changes that cause the gain or loss of an enzyme recognition site.
Figure 5-1 illustrates an example of the gain of an AluI site in MT set 18 in the K562 cell line. It would be virtually impossible to correctly interpret the elution profile of this sample at elevated column temperatures. Secondly, this step is important to determine that no partial restriction enzyme products are present that would also lead to misinterpretation of the results.
One clear advantage of this technique is that once a heteroduplex peak is detected, the corresponding region of the mitochondrial genome can be immediately located. For example, a heteroduplex peak was detected in the mixed MT set 9 CHR and 9947A sample (Figure 5-2; elution time 12 minutes). Table 5-2 indicates that this 233-bp peak corresponds to mitochondrial base pairs 6029-6261.
The mitochondrial genome scan can be used to rapidly identify regions in mtDNA that contain putative base changes, so only a limited number of sequencing reactions are needed to confirm the DHPLC results. This technique does require careful examination of the elution order of the peaks at different temperatures to ensure the proper peak is identified for further analysis, since each fragment has different melting characteristics.
The elution profiles can be complex for some fragments (Figure 5-4C-D), but the CHR and 9947A positive control fragments provide a well-characterized reference to guide the identification of specific peaks, and thus the location in the mitochondrial genome for further analysis.
This report describes a rapid, highly sensitive DHPLC method to scan for genetic alterations in the human mitochondrial genome. Commercially available cell lines were used to validate the accuracy of this procedure to detect polymorphic changes dispersed throughout the 16.5-kb sequence. This approach can be used to detect the presence of both inherited or somatic mtDNA base changes. Identification and characterization of these base changes is an important first step in the challenging task of assigning functional consequences to alterations in the mitochondrial genome.