We would like to acknowledge Silvia Marchet and Constanza Lamperti (Istituto Neurologico &#34;Carlo Besta&#34;, Fondazione IRCCS, Milan) for preparing and providing the m.3243A&#62;G cybrid cells used as illustrative examples for this protocol. We would also like to acknowledge the members of the Mitochondrial Genetics Group (MRC-MBU, University of Cambridge) for useful discussion during the course of this research. This work was supported by core funding from the Medical Research Council UK (MC_UU_00015/4 and MC_UU_00028/3). P.A.N. and P.S.-P. are additionally supported by The Lily Foundation and The Champ Foundation, respectively.

Informed consent was provided for the use of the human 3243A&#62;G cybrid cells and the immortalized m.5024C&#62;T MEFs used in this study. Ethical approval was not required in this instance as the patient cells were not collected at the University of Cambridge. The use of human fibroblasts may, however, require ethical approval. It is highly recommended to follow best practices for PCR setup when preparing the sample DNA for pyrosequencing. Frequent amplification using identical primers can lead to amplicon contamination and introduce bias to the subsequent genotyping if strict separation between the pre-PCR and post-PCR areas is not observed. The pipeline presented here uses specific equipment from a sole manufacturer; the details can be found in the Table of Materials. The primer design for the PCR can be performed manually if so desired; however, it is recommended to use existing software for this purpose (see the Table of Materials).
1. Pyrosequencing primer design and assay selection
<ol>
	<li>Obtaining primer candidates with software
	<ol>
		<li>Obtain an mtDNA sequence file for the species being genotyped, and identify the position of the SNP. Ensure that the reference sequence used employs the appropriate base numbering for the mutation studied so that the position of the SNP can be easily identified.</li>
		<li>Copy 1,000 base pairs upstream and downstream of the SNP site, and paste the truncated sequence into the software intended for the pyrosequencing primer design (see the Table of Materials).</li>
		<li>Set the analyzed SNP base as the target of the pyrosequencing assay in the software by highlighting the polymorphic base and right-clicking on and then selecting set target region.</li>
		<li>Press the play icon in the top right-hand corner of the interface to launch primer selection.</li>
		<li>Wait for the software to now automatically generate primer trios: two primers for preamplification of the template DNA and a third primer for sequencing by synthesis in the pyrosequencing machine. Retain all the primer sets by right-clicking on and selecting copy all primer sets.</li>
	</ol>
	</li>
	<li>Selecting the optimal primer sets from the generated list
	<ol>
		<li>Retain the primer sets with the highest quality score based on the software output, which provides primer sets in descending order based on the quality score. If possible, omit primer sets with a score below 80.</li>
		<li>For the retained primer sets, identify and copy the amplicon produced by the amplification primers.</li>
		<li>Align the amplicons produced with the entire genome of the organism to be genotyped using NCBI Blast by using the online submission portal at&#160;https://blast.ncbi.nlm.nih.gov/Blast.cgi. From the dropdown menus during submission, select the following:
		<ol>
			<li>Select Database | Genomic + Transcript databases.</li>
			<li>Select Human or Mouse in the dropdown menu, depending on the SNP analyzed.</li>
			<li>Select Optimize for | Somewhat similar sequences (Blastn).</li>
		</ol>
		</li>
		<li>(Optional) If genotyping an organism other than a mouse or human, select the following during submission:
		<ol>
			<li>Select Database | Standard database.</li>
			<li>Select RefSeq Representative genomes in the dropdown menu</li>
			<li>Select Optimize for | Somewhat similar sequences (Blastn)</li>
		</ol>
		</li>
		<li>When possible, omit any amplicons that have perfect homology to the mtDNA amplicon, particularly if the primer binding regions are perfectly homologous (see the discussion). 
		NOTE: The BLAST alignment tool will return any sequence with considerable homology to the pre-amplification amplicons. This allows for the detection of the NUMTs described in the introduction, which if not accounted for, can induce a bias as they are co-amplified with the mitochondrial DNA.</li>
		<li>Order the oligos obtained via this pipeline. Make sure that 5&#39; biotin modification is added to the correct amplification primer during the synthesis order, such that the sequencing primer will be complementary to the biotinylated strand. 
		&#8203;NOTE: The choice of sequencing primer is more flexible than the amplification primers, and it is recommended to have at least one base separating the 3&#39; end of the sequencing primer and the variable position.</li>
	</ol>
	</li>
</ol>
2. Preamplification PCR optimization
<ol>
	<li>Prepare the DNA samples by extracting the total genomic DNA using an appropriate method. 
	NOTE: This will largely depend on the number of cells being genotyped and their origin. If isolating DNA from single cells using a lysis buffer containing proteinase K, the sample must be denatured at 95 &#176;C for 10 min so as not to disrupt the polymerase during the subsequent PCR.</li>
	<li>PCR setup and thermal block settings
	<ol>
		<li>Set up the PCR with a high-fidelity polymerase of choice. Prepare a Master Mix for four reactions. As only 10 &#181;L of PCR reaction are required for a pyrosequencing run, prepare 25 &#181;L of PCR reactions (to allow for a technical repeat if necessary). Use 40 cycles of PCR with approximately 10 ng of genomic DNA as the starting material with 1 &#181;M of both the forward and reverse primers.</li>
		<li>Set the extension time according to the manufacturer&#39;s specifications for the polymerase used, taking into consideration the length of the chosen preamplification primers.</li>
		<li>Use a thermal cycler with variable annealing temperature settings. As the annealing temperature can differ depending on the primer sequence, the salt content of the polymerase buffer, and other factors, program four different annealing temperatures into the thermal cycling program. 
		NOTE: The worked example in the representative results used the following temperatures: 55 &#176;C, 60 &#176;C, 65 &#176;C, and 70 &#176;C.</li>
		<li>Split the Master Mix into four 200 &#181;L PCR tubes, and set them to run at each of the four selected annealing temperatures in the thermal cycler for 40 cycles.</li>
	</ol>
	</li>
	<li>Preamplification band visualization
	<ol>
		<li>During the PCR run, prepare a 2% (w/v) agarose gel in 1x TBE with SYBR Safe or ethidium bromide as a visualizing agent. 
		NOTE: A higher percentage gel is recommended for visualization as the amplified fragments are usually short, typically ranging from 100 to 500 base pairs.</li>
		<li>After the PCR in step 2.2 is complete, mix 10 &#181;L of the reaction with an appropriate amount of DNA loading buffer, and run on the 2% agarose gel at 7 V/cm for approximately 45 min.</li>
		<li>Visualize the resulting DNA fragments on a UV transilluminator. 
		&#8203;NOTE: The thermal cycling conditions selected for the preamplification step should produce a single clean band of the expected size. Lower annealing temperatures can occasionally result in off-target amplification, which can introduce biases to the subsequent pyrosequencing.</li>
		<li>Select the lowest annealing temperature that produces a clean band of the correct size for subsequent amplifications.</li>
		<li>(Optional) Excise a band of the expected size, purify using a gel extraction kit of choice, and analyze by Sanger sequencing to confirm the approximate heteroplasmy of the sample as a reference.</li>
	</ol>
	</li>
</ol>
3. Instrument setup and run
NOTE: Once the PCR step in the previous section is optimized, the next step involves programming the pyrosequencer with the correct nucleotide sequence to analyze for the specific SNP. This involves entering 10 bases directly downstream of the 3&#39; end of the sequencing primer. This is detailed in the following section.
<ol>
	<li>Assay configuration
	<ol>
		<li>Open the run software provided with the pyrosequencer, and select New Assay in the top-left corner of the interface.</li>
		<li>Select the Allele quantification assay template.</li>
		<li>Input the Sequence to be analyzed into the corresponding box by typing in the nucleotides to be incorporated directly downstream of the sequencing primer, which should be upstream of the SNP of interest. For the variable position, denote the two possible bases separated by a slash (e.g., A/T).</li>
		<li>Press Generate dispensation order, and let the software automatically determine a suitable order for the nucleotides to be dispensed in the sequencing-by-synthesis reaction.</li>
		<li>Save and provide a name for the assay.</li>
	</ol>
	</li>
	<li>Reagent preparation and storage
	<ol>
		<li>Dilute the sequencing primer ordered to 4 &#181;M in the Annealing Buffer provided in pyrosequencer reagent kit. 
		NOTE: The sequencing primer can first be diluted in water to 100 &#181;M as a stock solution and subsequently further diluted in Annealing Buffer to 4 &#181;M as needed.</li>
		<li>Enzyme and substrate preparation and handling
		<ol>
			<li>When first unboxing, redissolve the lyophilized enzyme and substrate as per the manufacturer&#39;s recommendations. Store at &#8722;20 &#176;C when not in use.</li>
			<li>Upon subsequent use, thaw the enzyme and substrate vials from &#8722;20 &#176;C. 
			NOTE: All the other reagents in the provided kit can be kept refrigerated at 4 &#176;C. Diluted sequencing primers can also be kept at 4 &#176;C.</li>
		</ol>
		</li>
		<li>Place the required amount of each reagent on ice.</li>
		<li>Keep the remaining components required, namely the streptavidin-coated magnetic beads, absorber strips, and pyrosequencing disks, at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Run execution 
	NOTE: When an assay is first designed, it must be calibrated using PCR products of known heteroplasmy, which ensures that the assay can accurately distinguish heteroplasmies. Researchers can use mixtures of PCR products of known heteroplasmy as standards. Samples can also be verified by other methods mentioned in the introduction and the discussion, notably NGS.
	<ol>
		<li>Dilute the DNA to be analyzed to 5 ng/&#181;L. Particularly in the first run, be sure to include samples of known heteroplasmy as a reference and/or wild-type samples.</li>
		<li>Perform a presequencing PCR of 5 &#181;L of diluted DNA in 25 &#181;L reactions using the amplification primers and parameters identified in section 1 and section 2. Perform technical PCR replicates for each sample. 
		NOTE: The preamplification PCR can be stored short term at 4 &#176;C or long term at &#8722;20 &#176;C before proceeding to sequencing.</li>
		<li>Run file setup
		<ol>
			<li>Select New Run in the top-left corner of pyrosequencer software.</li>
			<li>The pyrosequencer can simultaneously sequence 48 separate preamplified reactions, with an empty square representing a single sequencing well on a pyrosequencing disk. Load the assays configured in section 3.1 by right-clicking on a square and selecting load assay. If required, sequence up to four separate assays with different sequencing primers.</li>
			<li>Set the primer dispensation mode to Automatic to automatically assign an injection chamber for each sequencing primer used for the run.</li>
			<li>Set Run mode to Standard unless running four different types of assays on one sequencing disk.</li>
			<li>Ensure the number of assays on the run template file matches the number of PCR reactions being amplified. 
			NOTE: If running more than 48 samples, additional run files will need the be set up.</li>
			<li>Save the run files to a USB drive.</li>
		</ol>
		</li>
		<li>Priming the pyrosequencer
		<ol>
			<li>Press the Cleaning button on the main touch screen of the device, and clean all injectors with high-purity water following the instructions on the screen. When inserting the absorber strip into the machine, ensure that the ends meet in the &#34;9 o&#39;clock&#34; position (directly left of the center).</li>
			<li>Plug in the USB stick with the runs set up in step 3.3.3, and load the run files defined in step 3.3.3. 
			NOTE: The run files must be saved outside any directory or folder on the USB or they cannot be read by the machine.</li>
			<li>Follow the instructions on the device to load and prime the reagents as required into the corresponding injectors on the machine.</li>
		</ol>
		</li>
		<li>Sample disk preparation and assay launch
		<ol>
			<li>Allow the streptavidin-coated magnetic beads to equilibrate to room temperature. 
			NOTE: These beads will enable the device to capture the biotinylated strand from the preamplification PCR step. They need to be purchased separately from the kit.</li>
			<li>Load 3 &#181;L of beads into the wells defined in the run file from step 3.3.3.</li>
			<li>Load 10 &#181;L of each PCR reaction to be sequenced into the corresponding sample, and pipette up and down to mix the sample with the magnetic beads. Avoid bubbles if possible.</li>
			<li>Look for the indication in the primed pyrosequencer that the disk can be loaded into the machine. Unscrew the plate holding nut before aligning the loaded sample plate with the metal pin in the disk compartment.</li>
			<li>Once the plate is firmly screwed into the plate compartment, launch the sequencing run by pressing the start button on the touchscreen interface.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. Result acquisition
<ol>
	<li>Once the run is complete, remove the USB drive from the machine, and plug it back into the computer running the pyrosequencer software. While proceeding with the following steps, proceed with cleaning the pyrosequencer following the prompts if it is the final run of the day.</li>
	<li>Wait for the newly generated run file to appear on the USB drive, and double-click on the run result file. This automatically analyzes the luciferase output of each well upon nucleotide incorporation and quantifies the mitochondrial allele of interest defined during the assay configuration in section 3.1.</li>
	<li>Look for the following color scores shown by the software based on the quality of the reads. Blue indicates an optimal run, yellow a run with a warning, and red a failed run. To save the results, select Reports in the top window | Full report to generate a .pdf file containing the pyrograms and results from each well of the run. Alternatively, download the results in other formats under the Reports tab.</li>
</ol>

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M.M. is a co-founder, shareholder, and member of the Scientific Advisory Board of Pretzel Therapeutics, Inc. P.S.-P. and P.A.N. provide consultancy services for Pretzel Therapeutics, Inc.

A critical aspect for the success of the protocol is avoiding contaminations, particularly when using low amounts of starting material. It is recommended to use a UV hood and filtered pipette tips when preparing the samples wherever possible, as well as to keep the preamplification and post-amplification areas separate. Blank measurements and samples of known heteroplasmy (such as wild-type DNA) should always be included to be used as benchmarks to check for technical or biological bias. 
A no...

The mitochondrial genome exists in the form of small (16.5 kb) circular molecules (mtDNA) present in the innermost compartment of the mitochondria named the matrix and encodes 13 subunits of the mitochondrial respiratory chain, as well as the tRNAs and rRNAs necessary for their translation in situ by the mitochondrial ribosome1. This genome represents approximately 1% of all the proteins necessary for mitochondrial function, the remainder of which are encoded by the nuclear DNA (nDNA). It is commonly assumed that mitochondria are derived from an endosymbiotic fusion event between an alpha-proteobacterial ancestor and an ancestral eukaryotic cell. Once this hypothetical symbiosis took place, the genetic information of the mitochondria was gradually transferred to the nucleus over eons, which explains the aforementioned compactness of the mtDNA when compared to the genomes of modern cyanobacteria2. Such a transfer of genes is most strongly evidenced by the existence of long stretches of nDNA that are highly homologous to the sequences found in mtDNA. These nuclear mitochondrial sequences (NUMTs) are a common source of misinterpretation during genotyping, and certain precautions must be taken to avoid nuclear biases when genotyping mtDNA3 (Figure 1A).
Another distinctive feature of mtDNA is that its copy number varies depending on the cell type, numbering anywhere from tens to thousands of copies per cell4. Owing to this multi-copy nature, mtDNA can harbor a wide range of genotypes within a single cell, which can result in a more continuous distribution of alleles in contrast to the discrete alleles associated with nuclear genes when considering the zygosity of chromosomes. This heterogeneity of mitochondrial alleles is referred to as mitochondrial heteroplasmy, which is typically expressed in the percent prevalence of a given mutation as a proportion of the total mtDNA in a given cell. Heteroplasmy can be contrasted with homoplasmy, which refers to a unique species of mtDNA being present across a cell.
Measuring mitochondrial heteroplasmy is of particular interest when quantifying the proportion of mtDNA molecules harboring pathogenic variants. Such variants come in the form of single nucleotide polymorphisms (SNPs), small indels, or large-scale deletions5. Most humans are heteroplasmic for pathogenic variants; however, they do not exhibit any clinical phenotypes, which often only manifest at higher heteroplasmy levels of pathogenic mtDNA in a phenomenon referred to as the threshold effect6. While the values associated with pathogenicity are highly dependent on the nature of the pathogenic mutation and the tissue in which it occurs, they typically lie above 60% heteroplasmy7.
There are several research areas in which mitochondrial genotyping is common. In the medical field, testing for or quantifying mtDNA mutations can serve as a diagnostic criterion for mitochondrial diseases, many of which have mtDNA aberrations as their origin5. In addition to the study of human pathogenic mutations, the prevalence of animal models harboring pathogenic SNPs in the mtDNA is likely to increase, given the recent advent of mitochondrial base editing enabled by mitochondrially targeted DddA-derived cytosine base editors (DdCBEs)8 and TALE-based deaminases (TALEDs) for adenine base editing9. This approach will be instrumental in understanding the interplay between aberrant mitochondrial genotypes and the resulting dysfunctions. There is also ongoing scientific research into remodeling the mitochondrial genome for ultimate use as a therapeutic strategy in human mitochondrial diseases via an approach known as heteroplasmy shifting. This field of research primarily involves directing mutation-specific nucleases to the mitochondrial matrix; this results in the preferential degradation of pathogenic mtDNA, leading to rescues in phenotype10,11,12,13. Any experiments involving the remodeling of the mitochondrial genotype require a robust quantitative method to assess heteroplasmy shifts.
A wide variety of methods are used to genotype mtDNA, and these vary according to the nature of the mutation. Next-generation sequencing (NGS) methods are more precise when it comes to quantifying SNPs in mtDNA; however, these methods remain prohibitively expensive for the routine quantification of mitochondrial heteroplasmy, particularly if the number of samples is small. Sanger sequencing can also allow for the detection of SNPs; however, this approach is not quantitative and often fails to detect low levels of heteroplasmy or can be inaccurate when estimating high heteroplasmies. Pyrosequencing, as an assay that involves minimal preparation and enables the rapid quantification of heteroplasmy for any mtDNA sample, is proposed as an apt compromise between these two extremes. This method has been used routinely to quantify mitochondrial SNPs by numerous researchers in various contexts, including forensic analysis14,15, clinical diagnosis16, or the genotyping of mtDNA from single cells17.
This assay involves a first PCR preamplification step of a region flanking the SNP in the mtDNA, which is followed by a sequencing-by-synthesis assay using one strand of the previously generated amplicon. One of the two primers used in the preamplification step must be biotinylated on the 5&#39; end, which will enable the pyrosequencing apparatus to isolate the single strand of DNA to be used as template for the sequencing reaction. A third sequencing primer is then annealed onto the retained biotinylated strand, which allows for nascent DNA synthesis as deoxynucleotides to be dispensed in a predefined order into the reaction chamber. The pyrosequencer records the amount of each base incorporated based on a luminescent readout, allowing the relative quantification of mutant and wild-type mitochondrial alleles upon DNA synthesis (Figure 1B). The luminescence is generated by a luciferase enzyme, which emits light in the presence of ATP that an ATP sulfurylase synthesizes de novo at each incorporation event from the pyrophosphates released by each nucleotide. These two reactions can be summarized as follows:
1. PPi (from nucleotide incorporation) + APS &#8594; ATP + sulphate (ATP sulfurylase)
2. ATP + luciferin + O2 &#8594; AMP + PPi + oxyluciferin + CO2 + light (luciferase)
Detecting adenine bases by the pyrosequencer without ATP cross-reacting with the luciferase in the second reaction is a challenge. However, this is solved by using an adenine analog for DNA synthesis, namely dATP&#945;S. Despite not being a perfect substrate for luciferase, it produces a stronger luminescence compared with the three other nucleotides, which is digitally adjusted by the pyrosequencer and set to a factor of 0.9. Due to this inherent variability, it is suggested to avoid sequencing adenine at the SNP position (see the discussion for further details).
The following protocol details the method of mtDNA heteroplasmy assessment by pyrosequencing and outlines the necessary precautions in designing the amplification primers to avoid biological or technical bias when genotyping SNPs in mtDNA. The latter involves digitally surveying and selecting the primer sets, optimizing the preamplification PCR, and finally, sequencing and refining the assay. Two applied example assays are demonstrated: first, the optimization of the most common human pathogenic variant m.3243A&#62;G18, and second, the genotyping of mouse embryonic fibroblast (MEF) cells that have undergone heteroplasmy shifting using technologies developed at the Minczuk laboratory in Cambridge10,11,12,19,20,21,22.

<table><tbody><tr><td>KOD Hot Start DNA Polymerase</td><td>Sigma-Aldrich</td><td>71086</td><td></td></tr><tr><td>PyroMark Assay Design&amp;nbsp;2.0</td><td>QIAGEN</td><td></td><td></td></tr><tr><td>Pyromark Q48 Absorber Strips&amp;nbsp;</td><td>QIAGEN</td><td>974912</td><td></td></tr><tr><td>PyroMark Q48 Advanced CpG Reagents (4 x 48)</td><td>QIAGEN</td><td>974022</td><td></td></tr><tr><td>Pyromark Q48 Autoprep&amp;nbsp;</td><td>QIAGEN</td><td>9002470</td><td></td></tr><tr><td>PyroMark Q48 Cartridge Set</td><td>QIAGEN</td><td>9024321</td><td></td></tr><tr><td>Pyromark Q48 Disks</td><td>QIAGEN</td><td>974901</td><td></td></tr><tr><td>Pyromark Q48 Magnetic beads&amp;nbsp;</td><td>QIAGEN</td><td>974203</td><td></td></tr><tr><td>PyroMark Q48 Software License (1)</td><td>QIAGEN</td><td>9024325</td><td></td></tr></tbody></table>

genotyping single nucleotide polymorphisms in the mitochondrial genome by pyrosequencing

Mutations in the mitochondrial genome (mtDNA) have been associated with maternally inherited genetic diseases. However, interest in mtDNA polymorphisms has increased in recent years due to the recently developed ability to produce models by mtDNA mutagenesis and a new appreciation of the association between mitochondrial genetic aberrations and common age-related diseases such as cancer, diabetes, and dementia. Pyrosequencing is a sequencing-by-synthesis technique that is widely employed across the mitochondrial field for routine genotyping experiments. Its relative affordability when compared to massive parallel sequencing methods and ease of implementation make it an invaluable technique in the field of mitochondrial genetics, allowing for the rapid quantification of heteroplasmy with increased flexibility. Despite the practicality of this method, its implementation as a means of mtDNA genotyping requires the observation of certain guidelines, specifically to avoid certain biases of biological or technical origin. This protocol outlines the necessary steps and precautions in designing and implementing pyrosequencing assays for use in the context of heteroplasmy measurement.

This section presents an example optimization of a pyrosequencing assay for a human pathogenic mtDNA mutation, as well as sequencing data from the genotyping of heteroplasmic (m.5024C&#62;T) mouse embryonic fibroblasts (MEFs) treated with mitochondrial zinc finger nucleases (mtZFNs). Optimizing the assay for human cells and comparing two different assays demonstrates how to select the most accurate one, whereas genotyping genetically modified MEF cells in the second example serves as an applied example of detecting heter...

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Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing

Pyrosequencing assays enable the robust and rapid genotyping of mitochondrial DNA single nucleotide polymorphisms in heteroplasmic cells or tissues.

Mutations in the mitochondrial genome (mtDNA) have been associated with maternally inherited genetic diseases. However, interest in mtDNA polymorphisms ...

genotyping-single-nucleotide-polymorphisms-mitochondrial-genome

Research

JoVE Journal

Medicine

1.4K Views.  University of Cambridge, Cambridge Biomedical Campus. Pyrosequencing is een sequencing door synthesetechniek die kan worden gebruikt om single nucleotide polymorfismen in mitochondriaal DNA te genotyperen. Deze methode is voordelig in zijn gemak van implementatie en kosteneffectiviteit in vergelijking met andere sequencingmethoden. Het kan gemakkelijk worden gebruikt als een diagnostische methode voor bepaalde mitochondriale ziekten door het bepalen van heteroplasmiewaarden van pathogene varianten in mitochondriaal DNA.Verkrijg om te begi...

Video: Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

Biology

Methodology for Accurate Detection of Mitochondrial DNA Methylation

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert unmethylated cytosine into uracil, in a single-stranded DNA context. Bisulfite sequencing can be targeted (using PCR) or performed on the whole genome and provides absolute quantification of cytosine methylation at the single base-resolution. Given the distinct nature of nuclear- and mitochondrial DNA, notably in the secondary structure, adaptions of bisulfite sequencing methods for investigating cytosine methylation in mtDNA should be made. Secondary and tertiary structure of mtDNA can indeed lead to bisulfite sequencing artifacts leading to false-positives due to incomplete denaturation poor access of bisulfite to single-stranded DNA. Here, we describe a protocol using an enzymatic digestion of DNA with BamHI coupled with bioinformatic analysis pipeline to allow accurate quantification of cytosine methylation levels in mtDNA. In addition, we provide guidelines for designing the bisulfite sequencing primers specific to mtDNA, in order to avoid targeting undesirable NUclear MiTochondrial segments (NUMTs) inserted into the nuclear genome.

Here, we present a protocol to allow accurate quantification of mitochondrial DNA (mtDNA) methylation. In this protocol, we describe an enzymatic digestion of DNA with BamHI coupled with a bioinformatic analysis pipeline which can be used to avoid overestimation of mtDNA methylation levels caused by the secondary structure of mtDNA.

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert ...

Genetics

Optimized Analysis of DNA Methylation and Gene Expression from Small, Anatomically-defined Areas of the Brain

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to the display of physiological and behavioural phenotypes. Such environmental programming is likely to increase the susceptibility to metabolic, cardiovascular and mental diseases 3,4.
DNA methylation and histone modifications are considered key processes in the mediation of the gene-environment dialogue and appear also to underlay environmental programming 5. In mammals, DNA methylation typically comprises the covalent addition of a methyl group at the 5-position of cytosine within the context of CpG dinucleotides.
CpG methylation occurs in a highly tissue- and cell-specific manner making it a challenge to study discrete, small regions of the brain where cellular heterogeneity is high and tissue quantity limited. Moreover, because gene expression and methylation are closely linked events, increased value can be gained by comparing both parameters in the same sample.
Here, a step-by-step protocol (Figure 1) for the investigation of epigenetic programming in the brain is presented using the 'maternal separation' paradigm of early life adversity for illustrative purposes. The protocol describes the preparation of micropunches from differentially-aged mouse brains from which DNA and RNA can be simultaneously isolated, thus allowing DNA methylation and gene expression analyses in the same sample.

A streamlined workflow to study DNA methylation and gene expression changes upon early-life stress is shown. Starting from maternal separation of newborn mice and isolation of discrete brain tissues, we represent a protocol to simultaneously isolate DNA and RNA from brain tissue punches for subsequent bisulfite sequencing and RT-PCR analysis.

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to ...

Neuroscience

An In Vitro Approach to Study Mitochondrial Dysfunction: A Cybrid Model

Deficiency of the mitochondrial respiratory chain complexes that carry out oxidative phosphorylation (OXPHOS) is the biochemical marker of human mitochondrial disorders. From a genetic point of view, the OXPHOS represents a unique example because it results from the complementation of two distinct genetic systems: nuclear DNA (nDNA) and mitochondrial DNA (mtDNA). Therefore, OXPHOS defects can be due to mutations affecting nuclear and mitochondrial encoded genes.
The groundbreaking work by King and Attardi, published in 1989, showed that human cell lines depleted of mtDNA (named rho0) could be repopulated by exogenous mitochondria to obtain the so-called &#34;transmitochondrial cybrids.&#34; Thanks to these cybrids containing mitochondria derived from patients with mitochondrial disorders (MDs) and nuclei from rho0 cells, it is possible to verify whether a defect is mtDNA- or nDNA-related. These cybrids are also a powerful tool to validate the pathogenicity of a mutation and study its impact at a biochemical level. This paper presents a detailed protocol describing cybrid generation, selection, and characterization.

An In Vitro Approach to Study Mitochondrial Dysfunction: A Cybrid Model

Transmitochondrial cybrids are hybrid cells obtained by fusing mitochondrial DNA (mtDNA)-depleted cells (rho0 cells) with cytoplasts (enucleated cells) derived from patients affected by mitochondrial disorders. They allow the determination of the nuclear or mitochondrial origin of the disease, evaluation of biochemical activity, and confirmation of the pathogenetic role of mtDNA-related variants.

Deficiency of the mitochondrial respiratory chain complexes that carry out oxidative phosphorylation (OXPHOS) is the biochemical marker of human ...

Transmitochondrial Cybrid Generation From Suspension-Growing Cancer Cells

Transmitochondrial Cybrid Generation Using Cancer Cell Lines

In recent years, the number of studies dedicated to ascertaining the connection between mitochondria and cancer has significantly risen. However, more efforts are still needed to fully understand the link involving alterations in mitochondria and tumorigenesis, as well as to identify tumor-associated mitochondrial phenotypes. For instance, to evaluate the contribution of mitochondria in tumorigenesis and metastasis processes, it is essential to understand the influence of mitochondria from tumor cells in different nuclear environments. For this purpose, one possible approach consists of transferring mitochondria into a different nuclear background to obtain the so-called cybrid cells. In the traditional cybridization techniques, a cell line lacking mtDNA (&#961;0, nuclear donor cell) is repopulated with mitochondria derived from either enucleated cells or platelets. However, the enucleation process requires good cell adhesion to the culture plate, a feature that is partially or completely lost in many cases in invasive cells. In addition, another difficulty found in the traditional methods is achieving complete removal of the endogenous mtDNA from the mitochondrial-recipient cell line to obtain pure nuclear and mitochondrial DNA backgrounds, avoiding the presence of two different mtDNA species in the generated cybrid. In this work, we present a mitochondrial exchange protocol applied to suspension-growing cancer cells based on the repopulation of rhodamine 6G-pretreated cells with isolated mitochondria. This methodology allows us to overcome the limitations of the traditional approaches, and thus can be used as a tool to expand the comprehension of the mitochondrial role in cancer progression and metastasis.

Author Spotlight: Transmitochondrial Cybrid Generation Using Cancer Cell Lines  

This protocol describes a technique for cybrid generation from suspension-growing cancer cells as a tool to study the role of mitochondria in the tumorigenic process.

In recent years, the number of studies dedicated to ascertaining the connection between mitochondria and cancer has significantly risen. However, more ...

Cancer Research

Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been accompanied by increased genomic information about Plasmodium parasite species responsible for malaria, including characteristics of geographical populations as well as variations associated with reduced susceptibility to anti-malarial drugs.
One common genetic variation, single-nucleotide polymorphisms (SNPs), offers attractive targets for parasite genotyping. These markers are useful not only for tracking drug resistance markers but also for tracking parasite populations using markers not under drug or other selective pressures.
SNP genotyping methods offer the ability to track drug resistance as well as to fingerprint individual parasites for population surveillance, particularly in response to malaria control efforts in regions nearing elimination status.
While informative SNPs have been identified that are agnostic to specific genotyping technologies, high-resolution melting (HRM) analysis is particularly suited to field-based studies. Compared to standard fluorescent-probe based methods that require individual SNPs in a single labeled probe and offer at best 10% sensitivity to detect SNPs in samples that contain multiple genomes (polygenomic), HRM offers 2-5% sensitivity. Modifications to HRM, such as blocked probes and asymmetric primer concentrations as well as optimization of amplification annealing temperatures to bias PCR towards amplification of the minor allele, further increase the sensitivity of HRM. While the sensitivity improvement depends on the specific assay, we have increased detection sensitivities to less than 1% of the minor allele.
In regions approaching malaria eradication, early detection of emerging or imported drug resistance is essential for prompt response. Similarly, the ability to detect polygenomic infections and differentiate imported parasite types from cryptic local reservoirs can inform control programs.
This manuscript describes modifications to high resolution melting technology that further increase its sensitivity to identify polygenomic infections in patient samples.

While high resolution melting analysis offers the ability to differentiate between single nucleotide polymorphisms in a heterogeneous population, mutant allele amplification bias can increase its ability to detect alleles present at relatively low percentages within a sample. This protocol describes improvements that improve the sensitivity of high resolution melting analysis.

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been ...

Immunology and Infection

High-Throughput Quantification of the Synthesis and Distribution of mtDNA

High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells produce most of their ATP in the mitochondria by oxidative phosphorylation. Mitochondria are unique organelles because they have their own genome that is replicated and passed on to the next generation of cells. In contrast to the nuclear genome, there are multiple copies of the mitochondrial genome in the cell. The detailed study of the mechanisms responsible for the replication, repair, and maintenance of the mitochondrial genome is essential for understanding the proper functioning of mitochondria and whole cells under both normal and disease conditions. Here, a method that allows the high-throughput quantification of the synthesis and distribution of mitochondrial DNA (mtDNA) in human cells cultured in vitro is presented. This approach is based on the immunofluorescence detection of actively synthesized DNA molecules labeled by 5-bromo-2'-deoxyuridine (BrdU) incorporation and the concurrent detection of all the mtDNA molecules with anti-DNA antibodies. Additionally, the mitochondria are visualized with specific dyes or antibodies. The culturing of cells in a multi-well format and the utilization of an automated fluorescence microscope make it easier to study the dynamics of mtDNA and the morphology of mitochondria under a variety of experimental conditions in a relatively short time.

Author Spotlight: High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution  

A procedure for studying the dynamics of mitochondrial DNA (mtDNA) metabolism in cells using a multi-well plate format and automated immunofluorescence imaging to detect and quantify mtDNA synthesis and distribution is described. This can be further used to investigate the effects of various inhibitors, cellular stresses, and gene silencing on mtDNA metabolism.

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells ...

Biochemistry

Pyrosequencing for Microbial Identification and Characterization

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains and detect genetic mutations that confer resistance to anti-microbial agents. The advantages of pyrosequencing for microbiology applications include rapid and reliable high-throughput screening and accurate identification of microbes and microbial genome mutations. Pyrosequencing involves sequencing of DNA by synthesizing the complementary strand a single base at a time, while determining the specific nucleotide being incorporated during the synthesis reaction. The reaction occurs on immobilized single stranded template DNA where the four deoxyribonucleotides (dNTP) are added sequentially and the unincorporated dNTPs are enzymatically degraded before addition of the next dNTP to the synthesis reaction. Detection of the specific base incorporated into the template is monitored by generation of chemiluminescent signals. The order of dNTPs that produce the chemiluminescent signals determines the DNA sequence of the template. The real-time sequencing capability of pyrosequencing technology enables rapid microbial identification in a single assay. In addition, the pyrosequencing instrument, can analyze the full genetic diversity of anti-microbial drug resistance, including typing of SNPs, point mutations, insertions, and deletions, as well as quantification of multiple gene copies that may occur in some anti-microbial resistance patterns.

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains, and detect genetic mutations that confer resistance to anti-microbial agents. In this video, the procedure for microbial amplicon generation, amplicon pyrosequencing, and DNA sequence analysis will be demonstrated.

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate ...

Measuring Single-Cell Mitochondrial DNA Copy Number and Heteroplasmy Using Digital Droplet Polymerase Chain Reaction

The mammalian mitochondrial (mt)DNA is a small, circular, double-stranded, intra-mitochondrial DNA molecule, encoding 13 subunits of the electron transport chain. Unlike the diploid nuclear genome, most cells contain many more copies of mtDNA, ranging from less than 100 to over 200,000 copies depending on cell type. MtDNA copy number is increasingly used as a biomarker for a number of age-related degenerative conditions and diseases, and thus, accurate measurement of the mtDNA copy number is becoming a key tool in both research and diagnostic settings. Mutations in the mtDNA, often occurring as single nucleotide polymorphisms (SNPs) or deletions, can either exist in all copies of the mtDNA within the cell (termed homoplasmy) or as a mixture of mutated and WT mtDNA copies (termed heteroplasmy). Heteroplasmic mtDNA mutations are a major cause of clinical mitochondrial pathology, either in rare diseases or in a growing number of common late-onset diseases such as Parkinson's disease. Determining the level of heteroplasmy present in cells is a critical step in the diagnosis of rare mitochondrial diseases and in research aimed at understanding common late-onset disorders where mitochondria may play a role. MtDNA copy number and heteroplasmy have traditionally been measured by quantitative (q)PCR-based assays or deep sequencing. However, the recent introduction of ddPCR technology has provided an alternative method for measuring both parameters. It offers several advantages over existing methods, including the ability to measure absolute mtDNA copy number and sufficient sensitivity to make accurate measurements from single cells even at low copy numbers. Presented here is a detailed protocol describing the measurement of mtDNA copy number in single cells using ddPCR, referred to as droplet generation PCR henceforth, with the option for simultaneous measurement of heteroplasmy in cells with mtDNA deletions. The possibility of expanding this method to measure heteroplasmy in cells with mtDNA SNPs is also discussed.

Here we present a protocol for measuring absolute mitochondrial (mt)DNA copy number and mtDNA deletion heteroplasmy levels in single cells.

The mammalian mitochondrial (mt)DNA is a small, circular, double-stranded, intra-mitochondrial DNA molecule, encoding 13 subunits of the electron ...

Simultaneous Mapping and Quantitation of Ribonucleotides in Human Mitochondrial DNA

Established approaches to estimate the number of ribonucleotides present in a genome are limited to the quantitation of incorporated ribonucleotides using short synthetic DNA fragments or plasmids as templates and then extrapolating the results to the whole genome. Alternatively, the number of ribonucleotides present in a genome may be estimated using alkaline gels or Southern blots. More recent in vivo approaches employ Next-generation sequencing allowing genome-wide mapping of ribonucleotides, providing the position and identity of embedded ribonucleotides. However, they do not allow quantitation of the number of ribonucleotides which are incorporated into a genome. Here we describe how to simultaneously map and quantitate the number of ribonucleotides which are incorporated into human mitochondrial DNA in vivo by Next-generation sequencing. We use highly intact DNA and introduce sequence specific double strand breaks by digesting it with an endonuclease, subsequently hydrolyzing incorporated ribonucleotides with alkali. The generated ends are ligated with adapters and these ends are sequenced on a Next-generation sequencing machine. The absolute number of ribonucleotides can be calculated as the number of reads outside the recognition site per average number of reads at the recognition site for the sequence specific endonuclease. This protocol may also be utilized to map and quantitate free nicks in DNA and allows adaption to map other DNA lesions that can be processed to 5&#180;-OH ends or 5&#180;-phosphate ends. Furthermore, this method can be applied to any organism, given that a suitable reference genome is available. This protocol therefore provides an important tool to study DNA replication, 5&#180;-end processing, DNA damage, and DNA repair.

Here we describe a method amenable to simultaneously quantitate and genome-wide map ribonucleotides in highly intact DNA at single-nucleotide resolution, combining enzymatic cleavage of genomic DNA with its alkaline hydrolysis and subsequent 5&#180;-end sequencing.

Established approaches to estimate the number of ribonucleotides present in a genome are limited to the quantitation of incorporated ribonucleotides using ...

Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing

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