Review Article Quantitative Assessment of Heteroplasmy of ...method using Surveyor nuclease [ ], and...

Review ArticleQuantitative Assessment of Heteroplasmy of MitochondrialGenome Perspectives in Diagnostics and Methodological Pitfalls

Igor A Sobenin12 Konstantin Y Mitrofanov2

Andrey V Zhelankin12 Margarita A Sazonova12 Anton Y Postnov1 Victor V Revin3

Yuri V Bobryshev345 and Alexander N Orekhov26

1 Laboratory of Medical Genetics Russian Cardiology Research and Production Complex Moscow 121552 Russia2 Laboratory of Cellular Mechanisms of Atherogenesis Institute of General Pathology and Pathophysiology Moscow 125315 Russia3 Biological Faculty NP Ogaryov Mordovian State University Republic of Mordovia Saransk 430005 Russia4 Faculty of Medicine School of Medical Sciences University of New South Wales Kensington Sydney NSW 2052 Australia5 School of Medicine University of Western Sydney Campbelltown NSW 2560 Australia6 Institute for Atherosclerosis Research Skolkovo Innovative Centre Moscow 143025 Russia

Correspondence should be addressed to Yuri V Bobryshev ybobryshevunsweduau

Received 16 December 2013 Accepted 14 March 2014 Published 10 April 2014

Academic Editor Marko Ljubkovic

Copyright copy 2014 Igor A Sobenin et alThis is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The role of alterations of mitochondrial DNA (mtDNA) in the development of human pathologies is not understood well Most ofmitochondrial mutations are characterized by the phenomenon of heteroplasmy which is defined as the presence of a mixture ofmore than one type of an organellar genomewithin a cell or tissueThe level of heteroplasmy varies inwide range and the expressionof disease is dependent on the percent of alleles bearing mutations thus allowing consumption that an upper threshold level mayexist beyond which the mitochondrial function collapses Recent findings have demonstrated that some mtDNA heteroplasmicmutations are associated with widely spread chronic diseases including atherosclerosis and cancer Actually each etiologicalmtDNA mutation has its own heteroplasmy threshold that needs to be measured Therefore quantitative evaluation of a mutantallele of mitochondrial genome is an obvious methodological challenge since it may be a keystone for diagnostics of individualgenetic predisposition to the diseaseThis review provides a comprehensive comparison of methods applicable to the measurementof heteroplasmy level of mitochondrial mutations associated with the development of pathology in particular in atherosclerosisand its clinical manifestations

1 Introduction

Analysis of genetic predisposition to the development ofchronic diseases in humans may be a significant and inno-vative addition to the existing approaches to evaluation ofpredisposition to different pathologies including atheroscle-rotic disease and cancer Mutations of mitochondrial genomerepresent the novel type of genetic markers to predict thedevelopment of disease Advisability of studies on pathogenicrole of such mutations is derived from molecular mech-anisms by which they can cause defects in the proteinstructure of some respiratory chain enzymes and in transferRNA (tRNA) synthesized directly in the mitochondriaThis

in turn leads to reduction in concentrations of these enzymesand tRNAor their complete dysfunction in themitochondriaAs a result oxidative stress occurs in the cells which is likelyto contribute to the emergence and development of pathol-ogy Typically mutations in mitochondrial genes codingtRNA and respiratory chain proteins lead to the disruptionof protein synthesis in mitochondria which decrease theefficiency of oxidative phosphorylation and ATP production

In contrast to the nuclear genome mitochondrial DNA(mtDNA) is inherited strictly through the maternal lineThe systems of mtDNA reparation act less efficiently thanthose of nuclear DNA [1] This results in mtDNA mutationrate 10ndash20 times higher than nuclear DNA [2] Mutations

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 292017 9 pageshttpdxdoiorg1011552014292017

2 BioMed Research International

of mtDNA may be accumulated in certain tissue withoutaffecting other tissue Such a mosaic type of distributionof mitochondrial mutations may lead to focal developmentof pathology in organs and tissue Most of mitochondrialmutations are characterized by the phenomenon of hetero-plasmy which is defined as the presence of a mixture ofmore than one type of an organellar genome within a cell ortissueThe level of heteroplasmy varies in wide range and theexpression of disease is dependent on the percent of allelesbearing mutations thus allowing consumption that an upperthreshold level may exist beyond which the mitochondrialfunction collapses Typically the emergence ofmitochondrialpathology requires high levels of mutant mtDNA allelesin specific tissue exceeding 50ndash60 [3] However there isevidence that lower levels of heteroplasmy of certain mtDNAmutations that do not exceed 30ndash40 already significantlyincrease the risk of age-related chronic diseases such asleft ventricular hypertrophy atherosclerosis coronary heartdisease and diabetes [4ndash6] Therefore both qualitative andquantitative estimations of mutant alleles in the mitochon-drial genome (presence or absence of a mutation and thelevel of heteroplasmy respectively) are necessary for studyingthe association betweenmitochondrialmutations and humandiseases For the quantitative determination of mutationalburden of mitochondrial genome there are a number ofchallenges and obstacles which significantly limit the abilityof the genetic diagnostics

To detect and quantify mtDNA heteroplasmy levels manymethodologies may be employed including Sanger DNAsequencing [7] high performance liquid chromatography[8] pyrosequencing [5] Snapshot [9] HRM (high resolu-tion melt profiling) [10] temporal temperature gradient gelelectrophoresis (TTGE) [11] invader assay [12] amplifica-tion refractory mutation system (ARMS) [13] endonucleasemethod using Surveyor nuclease [14] and the next genera-tion sequencing [15] This review provides a comprehensivecomparison of methods applicable to the measurement ofheteroplasmy level of mitochondrial mutations associatedwith the development of pathology in humans

2 Association betweenHeteroplasmy of MitochondrialMutations and Chronic Diseases

In general there are twomajor pathologies which provide theldquolionrsquos sharerdquo of morbidity and mortality in modern societyand represent the key challenge to healthcare systems theseare atherosclerosis and oncopathology The mitochondrialmutations may play mechanistic role in the development ofboth pathologies and nowadays there is a growing body ofevidence in support of a nonredundant role of mitochondrialfactors in the pathogenesis of these diseases

During the process of aging mtDNA increasingly accu-mulates somatic mutations through multiplication of dam-aged DNA molecules because of limited effectiveness of thereparative mechanisms In experiments in mitochondrialDNA polymerase-deficient mice (mtDNA mutator mice) itwas demonstrated that the premature aging is the result of the

increase of mtDNA mutations which primarily alter genesencoding respiratory chain proteins thereby reducing thefunctions of mitochondrial complexes I III and IV [16]Thus the failure of mitochondrial biogenesis (ie reductionof respiratory function) can be a major inducer of prematureaging in mtDNA [17] Due to the large number of themitochondrial genome copies in each cell the ratio of themutant and wild-type mtDNA is a significant factor in aphenotype formation [18] Moreover the accumulation ofalleles bearing mtDNA point mutations may be associatedwith mitochondrial dysfunction

Nowadays there is a limited number of studies onevaluation of mtDNA mutations in atherosclerosis Sinceseveral early studies have demonstrated the monoclonaltype of increase of vascular smooth muscle cells numberin atherosclerotic lesions [19ndash21] a possible mechanism ofthe focal origin of atherosclerotic plaques may be partiallyexplained by a local damage of cells with high level ofmutatedmtDNA

In two studies del4977mtDNA mutation (deletion ofsim5KB mtDNA region containing five tRNA genes andseven genes of respiratory chain enzymes) was analyzed butno significant differences in the heteroplasmy levels werefound between control subjects and patients with coronaryatherosclerosis [22 23] On the other hand the frequencyof this mutation was 5-fold higher in atherosclerotic patientsthan in controls [23]

A mitochondrial disorder can result from the substitu-tion deletion and duplication ofmtDNAbases and depletionof mtDNA copies Most of the data come from the studies onpoint substitutemutations As an example heteroplasmy levelof mutation A3243G in the mitochondrial tRNA-Leu 1 gene(UUAG) in diabetic patients with atherosclerosis was 4-foldhigher compared to those age-matched nondiabetic subjects[24]

Mutation T16189C located in the hypervariable D-loopregion of mtDNA has attracted attention because of itsprobable association with a variety of multifactorial dis-eases In the study which included 482 patients with coro-nary heart disease (CHD) 505 patients with type 2 dia-betes and 1481 healthy individuals it was shown that theprevalence of T16189C mutation was significantly higherin patients with coronary artery disease and with diabetes[25]

Recent studies have included a comprehensive analy-sis of mtDNA mutations in tissue of the atheroscleroticvascular wall In two studies 10 of 40 analyzed mutationsdiffered in heteroplasmy level between atherosclerotic andunaffected autopsy samples of human aortic intima [5 26]Mutations of mtDNA associated with lipofibrous plaqueswere found in genes encoding 12S rRNA tRNA-Leu NADH-dehydrogenase subunits 1 2 5 and 6 and cytochrome BFrom 29 to 86 of aortic intimal samples had significantdifferences between the atherosclerotic plaque and unaffectedtissue in relation to the level of heteroplasmy for each muta-tion [26] The homogenates of damaged and normal aorticintima were compared by the mean level of heteroplasmyfor these 10 mutations For five mutations (A1555G C3256TT3336C G13513A and G15059A) the mean heteroplasmy

BioMed Research International 3

level in atherosclerotic intimal homogenates differed signif-icantly from those in the intact tissue It was shown thatmtDNA mutation burden in intimal tissue explains at least14 of the variability of atherosclerosis [27] These resultssuggest that the mutations in human mitochondrial genomeplay a significant role in the development of atherosclerosis

In the cross-sectional clinical study the associationbetween the level of heteroplasmy for the mutation C3256Tin human white blood cells and the extent of carotidatherosclerosis as well as the presence of coronary heartdisease (CHD) was investigated [28] High-resolution B-mode ultrasonography of carotids was used to estimatethe extent of carotid atherosclerosis by measuring of thecarotid intima-media thickness (cIMT) Pyrosequencing wasused to estimate the level of C3256T heteroplasmy Thehighly significant relationship betweenC3256T heteroplasmylevel and predisposition to atherosclerosis was revealed Inindividuals with low predisposition to atherosclerosis themean level of C3256T heteroplasmy was 168 as comparedto 238 in moderately predisposed subjects and furtherto 252 and 283 in significantly and highly predisposedsubjects respectively [28]

An association of mitochondrial genetic variation withthe severity of carotid atherosclerosis as assessed by carotidintima-media thickness and the presence of coronary heartdisease was analyzed [29] Significant correlations werefound between cIMT and the levels of heteroplasmy forC3256T T3336CG12315AG13513A andG15059Amutationsof mtDNA Additionally the levels of heteroplasmy formutations C3256T T3336C G12315A G13513A G14459AG14846A and G15059A correlated significantly with the sizeof atherosclerotic plaques visualized in any segment of carotidarteries A regression analysis has been performed in whichthe positive or negative correlation was factored for derivingthe association betweenmtDNAmutations and atherosclero-sisThemodel which included both conventional risk factorsandmutations provided 14-fold better explanatory level thanthe model based on estimation of conventional risk factorsonly [29]

Taken together these findings indicate that mutationsof mitochondrial genome play a substantial role in thedevelopment of atherosclerosis However mechanistic expla-nations of this role are not available yet As an exampleC3256T mutation is located in coding sequence of the MT-TL1 gene (codon recognizing UUR) which encodes tRNAleucine and is expressed at the cellular level as a reducedamount of cellular organelles and impaired protein synthe-sis [30ndash32] Mutations G13513A G14459A G14846A andG15059A occur in coding regions of genes responsible forthe synthesis of respiratory chain enzymes (MT-ND5 andMT-ND6 genes encoding the subunits 5 and 6 of NADHdehydrogenase respectively and MT-CYB gene encodingcytochrome B) An impairment of NADH dehydrogenaseactivity can be expected to attenuate NADH oxidation andCoQ (ubiquinone) reduction and thus promote oxidativestress It is obvious that the current knowledge of themechanisms whereby mitochondrial mutations can induceand accelerate atherogenesis at the cellular and molecularlevel is insufficient The further important areas of research

should include the studies evaluating the mechanistic roleof mtDNA mutations in cellular and molecular mechanismsof atherogenesis the changes in levels of oxidative DNAdamage bymeans of specific drugs aswell as chemopreventiveagents (ie antioxidants) and the effects of such interven-tions on the initiation and progression of atherosclerosisThis understanding is of direct clinical relevance becauseincreased mtDNA damage can be an important pathogenicfactor an additional prognostic predictor and a potentialtarget of therapeutic strategies in atherosclerosis [33]

There is also the growing evidence of the role of mito-chondrialmutations in oncopathology As an example highlysignificant associations were found between mtDNA muta-tions and the risk of stomach cancer colon carcinoma lungcancer and breast cancer [34ndash36]

3 Applicable Methodologies forQuantitative Measurement of mtDNAHeteroplasmy Level

The above considerations raise an important methodologicalproblem since quantitative evaluation of a mutant allele ofmitochondrial genome may be a keystone for diagnosticsof individual genetic predisposition to the disease includ-ing atherosclerosis and oncopathology Routine methodsof direct sequencing like Sanger sequencing are generallyinappropriate due to erroneous results in the case of polynu-cleotide sequences very often observed in mtDNA althoughsuch method has been used earlier for the measurement ofheteroplasmy level [7] As a rule sequence analysis of het-eroplasmy around 50 provides clear results in terms of thepresence of heteroplasmy but not in a quantitative mannerLower level of mtDNA heteroplasmy is often undetectableby direct sequencing As an example Meierhofer et al haveused denaturing high performance liquid chromatographyto rapidly screen the entire mtDNA for mutations thisapproach yielded straightforward interpretation of resultswith a detection limit down to 1 mtDNA heteroplasmyHowever direct sequencing analysis has become informativeonly after collection and reamplification of low degree het-eroduplex peak fractions [8]

At present all existing approaches to the detection ofmtDNA allelic variants can be divided into several groups

(1) enzymatic approaches

(i) restriction fragment length polymorphism(RFLP)

(ii) amplified fragment length polymorphism(AFLP)

(iii) cleavase fragment length polymorphism(CFLP)

(iv) enzymatic mutation detection using the bacte-riophage resolvase (EMD)

(v) analysis based on the ligase reaction (LDR LCRpadlock)

(vi) invasive oligonucleotide cleavage (Invader)


(vii) random amplification of polymorphic DNA(RAPD AP-PCR)

(viii) PCR with direct termination (DT-PCR)(ix) allele-specific PCR (AS-PCR)

(2) chemical methods

(i) chemical cleavage of heteroduplexes(ii) chemical ligation

(3) methods based on the different electrophoreticmobility of DNA polymorphisms

(i) analysis of the conformation of single-strandedfragments (SSCP)

(ii) heteroduplex analysis(iii) DNA sequencing

(4) detection of the solid phase

(i) hybridization to oligonucleotide arrays(ii) fiberoptic DNA hybridization analysis(iii) elongation of immobilized primers (minise-

quencing)(iv) pyrosequencing

(5) chromatographic methods

(i) denaturing HPLC

(6) physical methods

(i) mass spectrometry(ii) resonance fluorescence quenching using fluo-

rescence resonance energy transfer (FRET)

Subdivision of these methods into above groups is rathernominal primarily because in most cases a combination ofdifferent methods is used (eg all of the physical methodsalso use enzymatic reactions)

The choice of the optimal approach depends on thespecific objective of the study Historically widely appliedmethods for the detection of DNA polymorphisms employedrestriction enzymes and were limited to detection of changesin the restriction sites Later other enzymatic approacheshave been developed Although they are currently imple-mented mainly in a high-performance design a good por-tion of data indicates the possibility of their expansionand automation The advantages of these methods are therelatively low cost of the analysis which is independent of thenumber of samples and relative easiness of implementationHowever such methods are time-consuming that should beregarded as obvious flaw

Each of the above-mentioned strategies has its ownadvantages and disadvantages For example HRM HPLCand endonuclease method may be excellent for qualitativedetection of heteroplasmy itself but they have insufficient res-olution for the quantitative measurement of mtDNA hetero-plasmy level Pyrosequencing and next generation sequenc-ing provide high accuracy for the quantitative measurements

of heteroplasmy levels but are extremely expensive thuslimiting the possibility of routine analysis of a large numberof samples

Table 1 provides an overview of the most informativemethods used to quantify heteroplasmy level of mtDNAmutations Among the quantitation methods based on thePCR Snapshot minisequencing has insufficient resolutionin measuring the content of the mutant allele of less than5 [37] ARMS strategy (amplification refractory mutationsystem) also implies the use of fluorescent dye for thequantitative determination of the mutant allele by real-timePCR Experiments using allele-specific PCR and real-timedetection of heteroplasmy level by incorporating of CYBRGreen intercalating fluorescent dye have demonstrated highreproducibility of measurement (with correlation coefficientof 0994) for the analysis of picogram amounts of totalDNA obtained from different sources and the possibility ofquantifying mtDNA heteroplasmy levels up to 05 [38]Furthermore ARMS strategies unlike pyrosequencing andnext generation sequencing are not very expensive whichmakes them ideal for the analysis of small samples (Table 1)

Currently the most versatile methodology for the quan-titative measurement of mtDNA heteroplasmy level is qPCRemploying fluorescence detection To detect the presence ofspecific nucleotide sequences using fluorescence detectionin real time there are several techniques the most knownof which are the ones with intercalating dye SYBR Green Iandmethods with specific degradable oligonucleotide probes(TaqMan) and hairpin structures (Scorpions) (Table 2)

Summarizing the advantages and disadvantages of thisapproach it should be noted that the use of DNA probesin some specific form is the most preferable to increasethe specificity of analysis of mtDNA heteroplasmy Howeverthe disadvantages of the above described approach relateto a high cost of probes and accordingly the primers andamplification process are expensive as well At the same timethe use of intercalating agents is very simple and cheap Theneed for selection of specific primers and probes seems tobe a limitation factor but now the number of commerciallyavailable primers steadily increases and their efficiency hasalready been validated Thus the use of intercalating agentsbecomes highly attractive

However the use of intercalating dye SYBR Green I andhairpin structures (Scorpions) has certain limitations thatprevent thewide use for quantitativemeasurement ofmtDNAheteroplasmy such as

(i) low specificity and frequent false-positive results forthe intercalating dye SYBRGreen I the inconvenienceof job associated with the need to carry out the test intwo separate tubes

(ii) low efficiency of reaction and consequently lowsensitivity of hairpin structures method (Scorpions)

A widely recognized method of mutations detectionis the use of the energy transfer through the fluorescentresonance phenomenon (FRET) which is used for examplein analysis systems based on TaqMan-type and similarprobes [39] TaqMan analysis is based on the 51015840-nuclease


Table 1 Comparison of methods for the quantitative analysis of mtDNA heteroplasmy

Method Advantages Disadvantages

Invasive cleavage ofoligonucleotide probe(invader assay)

(1) Allows SNP-analysis in a single reaction tube(2) Allows the use of automatic setup of the reaction

(1) Requires a long incubationtime (3-4 h)(2) Big amount of genomicDNA (20ndash100 ng) in a singlereaction(3) The laborious selection ofallele-specific probes

Pyrosequencing

(1) Allows to estimate the exact nucleotide sequence and its changes(2) The possibility of automation(3) Quick performance(4) Can be used for SNP-analysis(5) The ability to analyze complex secondary structures

(1) Requires special equipment(2) Limited read length(3) The quality andeffectiveness of four enzymesused in reactions are criticalfor the accuracy ofmeasurements(4) Enzymes can lose activity(5) Sample dilution mayreduce the read length

Real-time amplificationrefractory mutationsystem for quantitativePCR analysis

(1) Simple(2) Single-stage(3) Fast(4) Nonradioactive(5) Sensitive(6) Specific

(1) It is difficult to find theoptimal allele-specific primers(2) It is difficult to find theoptimal mode for RT PCR toensure proper hybridization ofallele-specific primers

454 sequencing (Roche)(1) High read length(2) Relatively low cost of the instrument and reagents for the single run of GSjunior titanium

(1) High cost of the instrumentand reagents for the single runof GS FLX titanium(2) High cost per 1Mb read(3) High consumption offluids passed through the flowcell

Illumina sequencingwith Illumina MiSeq

(1) Relatively low cost of the instrument and reagents for the single run(2) The lowest cost per 1Mb read among small platforms(3) The fastest Illumina run time

(1) Relatively few reads(2) Higher cost per 1Mb readas compared to other Illuminaplatforms

Illumina HiScanSQ (1) Allows versatile genomic research and is scalable in future(1) Higher cost per 1Mb readthan HiSeq for large amountsof data

Illumina GAIIx (1) Lower instrument cost than HiSeq(2) The large number of publications using this instrument

(1) Higher cost per 1Mb readthan HiSeq

Illumina HiSeq 1000 and2000

(1) The largest number of reads(2) Maximum sequencing output per 1 day and 1 run(3) The lowest price for 1 Gb read among all NGS platforms

(1) High instrument cost(2) High computation needs

Applied BiosystemsSOLiD sequencing

(1) Each lane of Flow-Chip can be run independently(2) The highest sequencing accuracy(3) The ability to rescue failed sequencing cycles(4) 96 validated barcodes per lane(5) Throughput of 10ndash15Gbday for SOLiD-5500 and 20ndash30Gbday for SOLiD5500XL

(1) High-performance deviceshave become available onlyfrom 2011(2) Relatively short reads(3) Increased number of gapsin the assembly of genomes(4) High instrument cost forSOLiD-5500XL

HRM analysis

(1) Low consumption of reagents with minor losses it takes 20 120583L of PCR mixfor the analysis of each sample eliminating the need for HPLC-solvents orDGGE-gels(2) The stage of highly sensitive analysis of the melting curves can be added inthe end of the PCR for immediate analysis(3) Unlike DHPLC it does not require thermal optimization(4) Low consumption of samples after HRM-analysis PCR products can beused for Sanger sequencing(5) High resolution for accurate and reproducible results

(1) Method is not adapted forthe quantitative analysis ofmitochondrial mutations


Table 1 Continued


TGGE(1) Does not require denaturing agents(2) High reproducibility(3) The ability to analyze fragments with a size of up to 1000 bp

The exact nature of thenucleotide changes isunknown

HPLC

(1) High accuracy(2) The ability to analyze very small amounts of DNA(3) High separation efficiency(4) Flexibility to alter the conditions of separation(5) Rapid analysis(6) Comparative simplicity of instrumentation(7) Automation ability

(1) High cost of columns(2) The exact nature ofchanges in nucleotidesequence remains unknown(3) Sequencing is required todetermine the nature of themutation(4) Difficult identification ofheterozygotes

Endonuclease methodusing Surveyor nuclease

(1) Simplicity(2) Special kits for mutations detection (1) Qualitative estimation

Sanger sequencing(1) Using dideoxynucleotides with fluorescent labels with different emissionwavelengths allows to carry out the reaction in a single tube(2) The ability to analyze 500ndash1000 bp sequence in a single run

(1) Complexity of theelectrophoretic separation offragments(2) Low throughput comparedwith NGS methods(3) The loss of accuracy whenreading large fragments

Snapshot

(1) A reliable method to identify uncharacterized nucleotide damage(2) Allows to subsequently determine the untimely stop codons missense orsilent mutations(3) Can be used for SNP analysis(4) Automation ability

(1) Relativeness of the results(2) Low accuracy of the data(3) Requires valuation ofresults

activity of Taq-polymerase which cleaves nucleotides of theprobes hybridized to DNA oligonucleotide Two TaqManprobes are required one probe should be complementary tothe wild allele and the other one to the polymorphic variantThese probes have different fluorescent dyes at the 51015840-endand fluorescence quenchers at the 31015840-end When the probesare inactive the quenchers interact with the dye by FRETmechanism inhibiting fluorescence activity At the primersannealing step during qPCR TaqMan probes hybridize withtheDNAmolecule In the untwisting step 51015840-end of the dye iscleaved by Taq-polymerase 51015840-nuclease activity which leadsto an increase in fluorescence of the dye Base mismatchesin the probes lead to the cleavage of a probe without anydye release The genotype of the sample is determined bythe intensities of the emission of two different dyes ratiothus allowing to calculate the proportion of mutant allele inheteroplasmic sample

Considering the presence of two alleles (normal andmutant) within the mitochondrial genome in the samesample in an unknown proportion the most appropriateapproach for the development of RT-PCRmethod of mtDNAheteroplasmy quantitativemeasurement is the use of TaqManprobes As for SYBR Green method it can be used for thecases when the multiplex measurement of heteroplasmy levelof some mutations would be impossible

4 Conclusion

At present it is clear that mitochondrial factor may play apivotal role in susceptibility induction and progression of

chronic diseases including atherosclerosis and oncopathol-ogy However the impact of certain mtDNAmutations to thedevelopment of pathology is widely unknown More studiesfocused on the assessment of new heteroplasmic mtDNAmutations affecting mitochondrial function in relation toatherosclerosis and oncopathology are definitely requiredThe deep knowledge about the pathophysiological relevanceof each disease-associated mtDNA mutations is of greatimportance

Among the various modern methods for quantitativeassessment of mtDNA heteroplasmy level the qPCR methodbased on TaqMan technologies seems to be the most promis-ing for the purposes of clinical diagnostics The simplicity ofsequences design low price wide availability of equipmentand the simplicity of the analysis itself can make it availablefor clinical practice In parallel an informative pyrosequenc-ing method may be further considered as the ldquogold standardrdquofor the measurements of mtDNA heteroplasmy for gainingreference values

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Igor A Sobenin and Konstantin Y Mitrofanov equally con-tributed to this paper


Table2Th

ebasicmetho

dssuitablefor

quantitativem

easuremento

fmtD

NAheteroplasmybasedon

qPCR

with

fluorescenced

etectio

n

Principleo

foperatio

nAd

vantages

Disa

dvantages

Non

specificm

etho

ds

Intercalatingdyes

(SYB

RGreen

Iand

others)

Thefl

uorescence

signalincreases

duetoan

intercalatingdyeb

inding

todo

uble-stranded

DNA

Highlevelofsignalthes

ame

approach

tothed

esignof

vario

usexperim

ents

False

positiver

esultsarep

ossib

ledu

eto

prim

er-dim

erso

rnon

specific

amplicon

sso

thesem

etho

dsaren

otim

mediatelysuitablefor

diagno

stic

purposes

Amplifluo

rAllele-specific

PCRrevealsa

hairp

instructureo

ffluo

rescently

labeled

oligon

ucleotides

Lux

Thefl

uorescence

signalchanges

whenthe

fluorescentlylabeledprim

erisinclu

dedin

theP

CRprod

uct

DzyNA-

PCR

Bind

ingof

thep

robe

with

theP

CRprod

ucts

generatesrestrictio

nenzymerecognitio

nsite

Specificm

etho

ds

TaqM

anTh

edye

andtheq

uenchera

reseparatedby

51015840-exonu

cleasea

ctivity

ofTaq-po

lymerase

Highsig

nallevel

simplicity

ofdesig

nandsynthesis

Prob

lemsw

iththed

iscrim

inationof

somes

equences

Molecular

beacon

sDye

andqu

encher

ares

eparated

after

bind

ingof

thep

robe

tothea

mplicon

and

disclosure

oftheh

airpin

Goo

dsig

nal-to-no

iseratio

Thelow

levelsignaldue

toreactio

nkinetic

s

Eclip

se

SimilartoTaqM

anbut

isresis

tant

toexon

ucleasea

ctivity

ofthep

olym

erasea

signalisg

enerated

after

bind

ingof

thep

robe

tothea

mplicon

Highsig

nallevel

simplicity

ofdesig

nandsynthesis

Poor

signal-to-no

iseratio

problem

swith

thed

iscrim

inationof

some

sequ

ences

Hyb

prob

esTw

odyes

arec

onvergingaft

erbind

ingof

the

prob

esto

thea

mplicon

the

signalis

generatin

gby

theF

RETmechanism

Und

efined

Poor

signal-to-no

iseratio

not

suitable

fora

lldevicesther

eactionrequ

iresa

trim

olecular

interaction

Scorpion

s

Thep

robe-prim

erisinclu

dedin

theP

CRprod

uctthes

ignalisg

enerated

after

disclosure

oftheh

airpin

andbind

ingof

the

freee

ndof

thep

robe

tothea

mplicon

Goo

dsig

nal-to-no

iseratio

mon

omolecular

reactio

nRe

latively

expensives

ynthesis


Acknowledgments

The authors wish to thank the Ministry of Education andScience of the Russian Federation and the School of MedicalSciences University of New SouthWales Sydney for supportof our work

References

[1] P Boesch F Weber-Lotfi N Ibrahim et al ldquoDNA repairin organelles pathways organization regulation relevance indisease and agingrdquo Biochimica et Biophysica Acta vol 1813 no1 pp 186ndash200 2011

[2] C Haag-Liautard N Coffey D Houle M Lynch BCharlesworth and P D Keightley ldquoDirect estimationof the mitochondrial DNA mutation rate in Drosophilamelanogasterrdquo PLoS Biology vol 6 no 8 p e204 2008

[3] JMW van denOuweland H H P J LemkesW Ruitenbeek etal ldquoMutation inmitochondrial tRMALeu(UUR) gene in a largepedigree with maternally transmitted type II diabetes mellitusand deafnessrdquo Nature Genetics vol 1 no 5 pp 368ndash371 1992

[4] K Majamaa-Voltti K Peuhkurinen M-L Kortelainen I EHassinen and K Majamaa ldquoCardiac abnormalities in patientswithmitochondrial DNAmutation 3243AgtGrdquoBMCCardiovas-cular Disorders vol 2 article 12 2002

[5] M Sazonova E Budnikov Z Khasanova I Sobenin APostnov and A Orehov ldquoStudies of the human aortic intima bya direct quantitative assay ofmutant alleles in themitochondrialgenomerdquo Atherosclerosis vol 204 no 1 pp 184ndash190 2009

[6] S Zhang A-L Tong Y Zhang M Nie Y-X Li and H WangldquoHeteroplasmy level of the mitochondrial tRNALeu(UUR)A3243G mutation in a chinese family is positively associatedwith earlier age-of-onset and increasing severity of diabetesrdquoChinese Medical Sciences Journal vol 24 no 1 pp 20ndash25 2009

[7] J A Irwin J L Saunier H Niederstatter et al ldquoInvestigationof heteroplasmy in the human mitochondrial DNA controlregion a synthesis of observations from more than 5000 globalpopulation samplesrdquo Journal of Molecular Evolution vol 68 no5 pp 516ndash527 2009

[8] D Meierhofer J A Mayr S Ebner W Sperl and B KoflerldquoRapid screening of the entiremitochondrial DNA for low-levelheteroplasmic mutationsrdquoMitochondrion vol 5 no 4 pp 282ndash296 2005

[9] D Cassandrini M G Calevo A Tessa et al ldquoA new methodfor analysis of mitochondrial DNA point mutations and assesslevels of heteroplasmyrdquo Biochemical and Biophysical ResearchCommunications vol 342 no 2 pp 387ndash393 2006

[10] S F Dobrowolski A T Hendrickx B J van den Bosch etal ldquoIdentifying sequence variants in the human mitochondrialgenome using high-resolution melt (HRM) profilingrdquo HumanMutation vol 30 pp 891ndash898 2009

[11] L-J C Wong T-J Chen and D-J Tan ldquoDetection of mito-chondrial DNAmutations using temporal temperature gradientgel electrophoresisrdquo Electrophoresis vol 25 no 15 pp 2602ndash2610 2004

[12] Y Mashima M Nagano T Funayama et al ldquoRapid quantifi-cation of the heteroplasmy of mutant mitochondrial DNAs inLeberrsquos hereditary optic neuropathy using the Invader technol-ogyrdquo Clinical Biochemistry vol 37 no 4 pp 268ndash276 2004

[13] R-K Bai and L-J C Wong ldquoDetection and quantification ofheteroplasmic mutant mitochondrial DNA by real-time ampli-fication refractory mutation system quantitative PCR analysis

a single-step approachrdquo Clinical Chemistry vol 50 no 6 pp996ndash1001 2004

[14] S Bannwarth V Procaccio and V Paquis-Flucklinger ldquoSur-veyor nuclease a new st rategy for a rapid identification ofheteroplasmic mitochondrial DNA mutations in patients withrespiratory chain defectsrdquo Human Mutation vol 25 no 6 pp575ndash582 2005

[15] M V Zaragoza J Fass M Diegoli D Lin and E Arbus-tini ldquoMitochondrial DNA variant discovery and evaluation inhuman cardiomyopathies throughnext generation sequencingrdquoPLoS ONE vol 5 no 8 Article ID e12295 2010

[16] A Trifunovic A Hansson A Wredenberg et al ldquoSomaticmtDNA mutations cause aging phenotypes without affectingreactive oxygen species productionrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 102 no50 pp 17993ndash17998 2005

[17] D Edgar and A Trifunovic ldquoThe mtDNA mutator mousedissecting mitochondrial involvement in agingrdquo Aging vol 1no 12 pp 1028ndash1032 2009

[18] K J Krishnan A K Reeve D C Samuels et al ldquoWhat causesmitochondrial DNAdeletions in human cellsrdquoNatureGeneticsvol 40 no 3 pp 275ndash279 2008

[19] I-M Chung S M Schwartz and C E Murry ldquoClonal archi-tecture of normal and atherosclerotic aorta implications foratherogenesis and vascular developmentrdquoTheAmerican Journalof Pathology vol 152 no 4 pp 913ndash923 1998

[20] S M Schwartz and C E Murry ldquoProliferation and the mon-oclonal origins of atherosclerotic lesionsrdquo Annual Review ofMedicine vol 49 pp 437ndash460 1998

[21] E R Andreeva I M Pugach and A N Orekhov ldquoCollagen-synthesizing cells in initial and advanced atherosclerotic lesionsof human aortardquo Atherosclerosis vol 130 pp 133ndash142 1997

[22] M Bogliolo A Izzotti S de Flora C Carli A Abbondandoloand P Degan ldquoDetection of the 4977 bp mitochondrial DNAdeletion in human atherosclerotic lesionsrdquoMutagenesis vol 14no 1 pp 77ndash82 1999

[23] N Botto S Berti S Manfredi et al ldquoDetection of mtDNA with4977 bp deletion in blood cells and atherosclerotic lesions ofpatients with coronary artery diseaserdquo Mutation Research vol570 no 1 pp 81ndash88 2005

[24] T Nomiyama Y Tanaka L Piao et al ldquoAccumulation ofsomatic mutation inmitochondrial DNA and atherosclerosis indiabetic patientsrdquo Annals of the New York Academy of Sciencesvol 1011 pp 193ndash204 2004

[25] E E Mueller W Eder S Ebner et al ldquoThe mitochondrialT16189C polymorphism is associated with coronary arterydisease inMiddle European populationsrdquo PLoS ONE vol 6 no1 Article ID e16455 2011

[26] I A Sobenin M A Sazonova A Y Postnov Y V Bobryshevand A N Orekhov ldquoMitochondrial mutations are associatedwith atherosclerotic lesions in the human aortardquo Clinical andDevelopmental Immunology vol 2012 Article ID 832464 5pages 2012

[27] I A Sobenin M A Sazonova A Y Postnov Y V Bobryshevand A N Orekhov ldquoChanges of mitochondria in atheroscle-rosis possible determinant in the pathogenesis of the diseaserdquoAtherosclerosis vol 227 pp 283ndash288 2013

[28] I A Sobenin M A Sazonova M M Ivanova et al ldquoMutationC3256T of mitochondrial genome in white blood cells novelgenetic marker of atherosclerosis and coronary heart diseaserdquoPLoS ONE vol 7 no 10 Article ID e46573 2012


[29] I A Sobenin M A Sazonova A Y Postnov J T Salonen Y VBobryshev and A N Orekhov ldquoAssociation of mitochondrialgenetic variation with carotid atherosclerosisrdquo PLoS ONE vol8 no 7 Article ID e68070 2013

[30] C T Moraes F Ciacci E Bonilla et al ldquoTwo novel pathogenicmitochondrial DNAmutations affecting organelle number andprotein synthesis Is the tRNA(Leu(UUR)) gene an etiologic hotspotrdquo Journal of Clinical Investigation vol 92 no 6 pp 2906ndash2915 1993

[31] W Rossmanith and R M Karwan ldquoImpairment of tRNA pro-cessing by point mutations in mitochondrial tRNALeu(UUR)associated with mitochondrial diseasesrdquo FEBS Letters vol 433no 3 pp 269ndash274 1998

[32] L Levinger M Morl and C Florentz ldquoMitochondrial tRNA3∘ and metabolism and human diseaserdquo Nucleic Acids Researchvol 32 no 18 pp 5430ndash5441 2004

[33] I A Sobenin D A Chistiakov Y V Bobryshev A Y Postnovand A N Orekhov ldquoMitochondrial mutations in atherosclero-sis new solutions in research and possible clinical applicationsrdquoCurrent Pharmaceutical Design vol 19 pp 5942ndash5953 2013

[34] D C Wallace ldquoMitochondria and cancerrdquo Nature ReviewsCancer vol 12 pp 685ndash698 2012

[35] M Yu ldquoSomatic mitochondrial DNA mutations in humancancersrdquo Advances in Clinical Chemistry vol 57 pp 99ndash1382012

[36] L Pereira P Soares V Maximo and D C Samuels ldquoSomaticmitochondrial DNA mutations in cancer escape purifyingselection and high pathogenicity mutations lead to the onco-cytic phenotype pathogenicity analysis of reported somaticmtDNA mutations in tumorsrdquo BMC Cancer vol 12 article 532012

[37] J Naue T Sanger U Schmidt R Klein and S Lutz-BonengelldquoFactors affecting the detection and quantification ofmitochon-drial point heteroplasmy using Sanger sequencing and SNaP-shot minisequencingrdquo International Journal of Legal Medicinevol 125 no 3 pp 427ndash436 2011

[38] S Biffi B Bortot M Carrozzi and G M Severini ldquoQuantifica-tion of heteroplasmic mitochondrial DNA mutations for DNAsamples in the low picogram range by nested real-time ARMS-qPCRrdquo Diagnostic Molecular Pathology vol 20 no 2 pp 117ndash122 2011

[39] T Chavakis A Bierhaus and P P Nawroth ldquoRAGE (receptorfor advanced glycation end products) a central player in theinflammatory responserdquo Microbes and Infection vol 6 no 13pp 1219ndash1225 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


of mtDNA may be accumulated in certain tissue withoutaffecting other tissue Such a mosaic type of distributionof mitochondrial mutations may lead to focal developmentof pathology in organs and tissue Most of mitochondrialmutations are characterized by the phenomenon of hetero-plasmy which is defined as the presence of a mixture ofmore than one type of an organellar genome within a cell ortissueThe level of heteroplasmy varies in wide range and theexpression of disease is dependent on the percent of allelesbearing mutations thus allowing consumption that an upperthreshold level may exist beyond which the mitochondrialfunction collapses Typically the emergence ofmitochondrialpathology requires high levels of mutant mtDNA allelesin specific tissue exceeding 50ndash60 [3] However there isevidence that lower levels of heteroplasmy of certain mtDNAmutations that do not exceed 30ndash40 already significantlyincrease the risk of age-related chronic diseases such asleft ventricular hypertrophy atherosclerosis coronary heartdisease and diabetes [4ndash6] Therefore both qualitative andquantitative estimations of mutant alleles in the mitochon-drial genome (presence or absence of a mutation and thelevel of heteroplasmy respectively) are necessary for studyingthe association betweenmitochondrialmutations and humandiseases For the quantitative determination of mutationalburden of mitochondrial genome there are a number ofchallenges and obstacles which significantly limit the abilityof the genetic diagnostics

To detect and quantify mtDNA heteroplasmy levels manymethodologies may be employed including Sanger DNAsequencing [7] high performance liquid chromatography[8] pyrosequencing [5] Snapshot [9] HRM (high resolu-tion melt profiling) [10] temporal temperature gradient gelelectrophoresis (TTGE) [11] invader assay [12] amplifica-tion refractory mutation system (ARMS) [13] endonucleasemethod using Surveyor nuclease [14] and the next genera-tion sequencing [15] This review provides a comprehensivecomparison of methods applicable to the measurement ofheteroplasmy level of mitochondrial mutations associatedwith the development of pathology in humans

2 Association betweenHeteroplasmy of MitochondrialMutations and Chronic Diseases

In general there are twomajor pathologies which provide theldquolionrsquos sharerdquo of morbidity and mortality in modern societyand represent the key challenge to healthcare systems theseare atherosclerosis and oncopathology The mitochondrialmutations may play mechanistic role in the development ofboth pathologies and nowadays there is a growing body ofevidence in support of a nonredundant role of mitochondrialfactors in the pathogenesis of these diseases

During the process of aging mtDNA increasingly accu-mulates somatic mutations through multiplication of dam-aged DNA molecules because of limited effectiveness of thereparative mechanisms In experiments in mitochondrialDNA polymerase-deficient mice (mtDNA mutator mice) itwas demonstrated that the premature aging is the result of the

increase of mtDNA mutations which primarily alter genesencoding respiratory chain proteins thereby reducing thefunctions of mitochondrial complexes I III and IV [16]Thus the failure of mitochondrial biogenesis (ie reductionof respiratory function) can be a major inducer of prematureaging in mtDNA [17] Due to the large number of themitochondrial genome copies in each cell the ratio of themutant and wild-type mtDNA is a significant factor in aphenotype formation [18] Moreover the accumulation ofalleles bearing mtDNA point mutations may be associatedwith mitochondrial dysfunction

Nowadays there is a limited number of studies onevaluation of mtDNA mutations in atherosclerosis Sinceseveral early studies have demonstrated the monoclonaltype of increase of vascular smooth muscle cells numberin atherosclerotic lesions [19ndash21] a possible mechanism ofthe focal origin of atherosclerotic plaques may be partiallyexplained by a local damage of cells with high level ofmutatedmtDNA

In two studies del4977mtDNA mutation (deletion ofsim5KB mtDNA region containing five tRNA genes andseven genes of respiratory chain enzymes) was analyzed butno significant differences in the heteroplasmy levels werefound between control subjects and patients with coronaryatherosclerosis [22 23] On the other hand the frequencyof this mutation was 5-fold higher in atherosclerotic patientsthan in controls [23]

A mitochondrial disorder can result from the substitu-tion deletion and duplication ofmtDNAbases and depletionof mtDNA copies Most of the data come from the studies onpoint substitutemutations As an example heteroplasmy levelof mutation A3243G in the mitochondrial tRNA-Leu 1 gene(UUAG) in diabetic patients with atherosclerosis was 4-foldhigher compared to those age-matched nondiabetic subjects[24]

Mutation T16189C located in the hypervariable D-loopregion of mtDNA has attracted attention because of itsprobable association with a variety of multifactorial dis-eases In the study which included 482 patients with coro-nary heart disease (CHD) 505 patients with type 2 dia-betes and 1481 healthy individuals it was shown that theprevalence of T16189C mutation was significantly higherin patients with coronary artery disease and with diabetes[25]

Recent studies have included a comprehensive analy-sis of mtDNA mutations in tissue of the atheroscleroticvascular wall In two studies 10 of 40 analyzed mutationsdiffered in heteroplasmy level between atherosclerotic andunaffected autopsy samples of human aortic intima [5 26]Mutations of mtDNA associated with lipofibrous plaqueswere found in genes encoding 12S rRNA tRNA-Leu NADH-dehydrogenase subunits 1 2 5 and 6 and cytochrome BFrom 29 to 86 of aortic intimal samples had significantdifferences between the atherosclerotic plaque and unaffectedtissue in relation to the level of heteroplasmy for each muta-tion [26] The homogenates of damaged and normal aorticintima were compared by the mean level of heteroplasmyfor these 10 mutations For five mutations (A1555G C3256TT3336C G13513A and G15059A) the mean heteroplasmy






























(i) denaturing HPLC





















Pyrosequencing




















HRM analysis




Table 1 Continued




HPLC







Snapshot





4 Conclusion









Table2Th

ebasicmetho

dssuitablefor

quantitativem

easuremento

fmtD


qPCR

with

fluorescenced

etectio

n

Principleo

foperatio

nAd

vantages

Disa

dvantages

Non

specificm

etho

ds

Intercalatingdyes

(SYB

RGreen

Iand

others)

Thefl

uorescence

signalincreases

duetoan

intercalatingdyeb

inding

todo

uble-stranded

DNA


ame

approach

tothed

esignof

vario

usexperim

ents

False

positiver

esultsarep

ossib

ledu

eto

prim

er-dim

erso

rnon

specific

amplicon

sso

thesem

etho

dsaren

otim


diagno

stic

purposes

Amplifluo

rAllele-specific

PCRrevealsa

hairp

instructureo

ffluo

rescently

labeled

oligon

ucleotides

Lux

Thefl

uorescence

signalchanges

whenthe


erisinclu

dedin

theP

CRprod

uct

DzyNA-

PCR

Bind

ingof

thep

robe

with

theP

CRprod

ucts

generatesrestrictio

nenzymerecognitio

nsite

Specificm

etho

ds

TaqM

anTh

edye

andtheq

uenchera

reseparatedby

51015840-exonu

cleasea

ctivity

ofTaq-po

lymerase

Highsig

nallevel

simplicity

ofdesig

nandsynthesis

Prob

lemsw

iththed

iscrim

inationof

somes

equences

Molecular

beacon

sDye

andqu

encher

ares

eparated

after

bind

ingof

thep

robe

tothea

mplicon

and

disclosure

oftheh

airpin

Goo

dsig

nal-to-no

iseratio

Thelow

levelsignaldue

toreactio

nkinetic

s

Eclip

se

SimilartoTaqM

anbut

isresis

tant

toexon

ucleasea

ctivity

ofthep

olym

erasea

signalisg

enerated

after

bind

ingof

thep

robe

tothea

mplicon

Highsig

nallevel

simplicity

ofdesig

nandsynthesis

Poor

signal-to-no

iseratio

problem

swith

thed

iscrim

inationof

some

sequ

ences

Hyb

prob

esTw

odyes

arec

onvergingaft

erbind

ingof

the

prob

esto

thea

mplicon

the

signalis

generatin

gby

theF

RETmechanism

Und

efined

Poor

signal-to-no

iseratio

not

suitable

fora

lldevicesther

eactionrequ

iresa

trim

olecular

interaction

Scorpion

s

Thep

robe-prim

erisinclu

dedin

theP

CRprod

uctthes

ignalisg

enerated

after

disclosure

oftheh

airpin

andbind

ingof

the

freee

ndof

thep

robe

tothea

mplicon

Goo

dsig

nal-to-no

iseratio

mon

omolecular

reactio

nRe

latively

expensives

ynthesis


Acknowledgments


References

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






























(i) denaturing HPLC





















Pyrosequencing




















HRM analysis




Table 1 Continued




HPLC







Snapshot





4 Conclusion









Table2Th

ebasicmetho

dssuitablefor

quantitativem

easuremento

fmtD


qPCR

with

fluorescenced

etectio

n

Principleo

foperatio

nAd

vantages

Disa

dvantages

Non

specificm

etho

ds

Intercalatingdyes

(SYB

RGreen

Iand

others)

Thefl

uorescence

signalincreases

duetoan

intercalatingdyeb

inding

todo

uble-stranded

DNA


ame

approach

tothed

esignof

vario

usexperim

ents

False

positiver

esultsarep

ossib

ledu

eto

prim

er-dim

erso

rnon

specific

amplicon

sso

thesem

etho

dsaren

otim


diagno

stic

purposes

Amplifluo

rAllele-specific

PCRrevealsa

hairp

instructureo

ffluo

rescently

labeled

oligon

ucleotides

Lux

Thefl

uorescence

signalchanges

whenthe


erisinclu

dedin

theP

CRprod

uct

DzyNA-

PCR

Bind

ingof

thep

robe

with

theP

CRprod

ucts

generatesrestrictio

nenzymerecognitio

nsite

Specificm

etho

ds

TaqM

anTh

edye

andtheq

uenchera

reseparatedby

51015840-exonu

cleasea

ctivity

ofTaq-po

lymerase

Highsig

nallevel

simplicity

ofdesig

nandsynthesis

Prob

lemsw

iththed

iscrim

inationof

somes

equences

Molecular

beacon

sDye

andqu

encher

ares

eparated

after

bind

ingof

thep

robe

tothea

mplicon

and

disclosure

oftheh

airpin

Goo

dsig

nal-to-no

iseratio

Thelow

levelsignaldue

toreactio

nkinetic

s

Eclip

se

SimilartoTaqM

anbut

isresis

tant

toexon

ucleasea

ctivity

ofthep

olym

erasea

signalisg

enerated

after

bind

ingof

thep

robe

tothea

mplicon

Highsig

nallevel

simplicity

ofdesig

nandsynthesis

Poor

signal-to-no

iseratio

problem

swith

thed

iscrim

inationof

some

sequ

ences

Hyb

prob

esTw

odyes

arec

onvergingaft

erbind

ingof

the

prob

esto

thea

mplicon

the

signalis

generatin

gby

theF

RETmechanism

Und

efined

Poor

signal-to-no

iseratio

not

suitable

fora

lldevicesther

eactionrequ

iresa

trim

olecular

interaction

Scorpion

s

Thep

robe-prim

erisinclu

dedin

theP

CRprod

uctthes

ignalisg

enerated

after

disclosure

oftheh

airpin

andbind

ingof

the

freee

ndof

thep

robe

tothea

mplicon

Goo

dsig

nal-to-no

iseratio

mon

omolecular

reactio

nRe

latively

expensives

ynthesis


Acknowledgments


References

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







Pyrosequencing




















HRM analysis




Table 1 Continued




HPLC







Snapshot





4 Conclusion









Table2Th

ebasicmetho

dssuitablefor

quantitativem

easuremento

fmtD


qPCR

with

fluorescenced

etectio

n

Principleo

foperatio

nAd

vantages

Disa

dvantages

Non

specificm

etho

ds

Intercalatingdyes

(SYB

RGreen

Iand

others)

Thefl

uorescence

signalincreases

duetoan

intercalatingdyeb

inding

todo

uble-stranded

DNA


ame

approach

tothed

esignof

vario

usexperim

ents

False

positiver

esultsarep

ossib

ledu

eto

prim

er-dim

erso

rnon

specific

amplicon

sso

thesem

etho

dsaren

otim


diagno

stic

purposes

Amplifluo

rAllele-specific

PCRrevealsa

hairp

instructureo

ffluo

rescently

labeled

oligon

ucleotides

Lux

Thefl

uorescence

signalchanges

whenthe


erisinclu

dedin

theP

CRprod

uct

DzyNA-

PCR

Bind

ingof

thep

robe

with

theP

CRprod

ucts

generatesrestrictio

nenzymerecognitio

nsite

Specificm

etho

ds

TaqM

anTh

edye

andtheq

uenchera

reseparatedby

51015840-exonu

cleasea

ctivity

ofTaq-po

lymerase

Highsig

nallevel

simplicity

ofdesig

nandsynthesis

Prob

lemsw

iththed

iscrim

inationof

somes

equences

Molecular

beacon

sDye

andqu

encher

ares

eparated

after

bind

ingof

thep

robe

tothea

mplicon

and

disclosure

oftheh

airpin

Goo

dsig

nal-to-no

iseratio

Thelow

levelsignaldue

toreactio

nkinetic

s

Eclip

se

SimilartoTaqM

anbut

isresis

tant

toexon

ucleasea

ctivity

ofthep

olym

erasea

signalisg

enerated

after

bind

ingof

thep

robe

tothea

mplicon

Highsig

nallevel

simplicity

ofdesig

nandsynthesis

Poor

signal-to-no

iseratio

problem

swith

thed

iscrim

inationof

some

sequ

ences

Hyb

prob

esTw

odyes

arec

onvergingaft

erbind

ingof

the

prob

esto

thea

mplicon

the

signalis

generatin

gby

theF

RETmechanism

Und

efined

Poor

signal-to-no

iseratio

not

suitable

fora

lldevicesther

eactionrequ

iresa

trim

olecular

interaction

Scorpion

s

Thep

robe-prim

erisinclu

dedin

theP

CRprod

uctthes

ignalisg

enerated

after

disclosure

oftheh

airpin

andbind

ingof

the

freee

ndof

thep

robe

tothea

mplicon

Goo

dsig

nal-to-no

iseratio

mon

omolecular

reactio

nRe

latively

expensives

ynthesis


Acknowledgments


References

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table 1 Continued




HPLC







Snapshot





4 Conclusion









Table2Th

ebasicmetho

dssuitablefor

quantitativem

easuremento

fmtD


qPCR

with

fluorescenced

etectio

n

Principleo

foperatio

nAd

vantages

Disa

dvantages

Non

specificm

etho

ds

Intercalatingdyes

(SYB

RGreen

Iand

others)

Thefl

uorescence

signalincreases

duetoan

intercalatingdyeb

inding

todo

uble-stranded

DNA


ame

approach

tothed

esignof

vario

usexperim

ents

False

positiver

esultsarep

ossib

ledu

eto

prim

er-dim

erso

rnon

specific

amplicon

sso

thesem

etho

dsaren

otim


diagno

stic

purposes

Amplifluo

rAllele-specific

PCRrevealsa

hairp

instructureo

ffluo

rescently

labeled

oligon

ucleotides

Lux

Thefl

uorescence

signalchanges

whenthe


erisinclu

dedin

theP

CRprod

uct

DzyNA-

PCR

Bind

ingof

thep

robe

with

theP

CRprod

ucts

generatesrestrictio

nenzymerecognitio

nsite

Specificm

etho

ds

TaqM

anTh

edye

andtheq

uenchera

reseparatedby

51015840-exonu

cleasea

ctivity

ofTaq-po

lymerase

Highsig

nallevel

simplicity

ofdesig

nandsynthesis

Prob

lemsw

iththed

iscrim

inationof

somes

equences

Molecular

beacon

sDye

andqu

encher

ares

eparated

after

bind

ingof

thep

robe

tothea

mplicon

and

disclosure

oftheh

airpin

Goo

dsig

nal-to-no

iseratio

Thelow

levelsignaldue

toreactio

nkinetic

s

Eclip

se

SimilartoTaqM

anbut

isresis

tant

toexon

ucleasea

ctivity

ofthep

olym

erasea

signalisg

enerated

after

bind

ingof

thep

robe

tothea

mplicon

Highsig

nallevel

simplicity

ofdesig

nandsynthesis

Poor

signal-to-no

iseratio

problem

swith

thed

iscrim

inationof

some

sequ

ences

Hyb

prob

esTw

odyes

arec

onvergingaft

erbind

ingof

the

prob

esto

thea

mplicon

the

signalis

generatin

gby

theF

RETmechanism

Und

efined

Poor

signal-to-no

iseratio

not

suitable

fora

lldevicesther

eactionrequ

iresa

trim

olecular

interaction

Scorpion

s

Thep

robe-prim

erisinclu

dedin

theP

CRprod

uctthes

ignalisg

enerated

after

disclosure

oftheh

airpin

andbind

ingof

the

freee

ndof

thep

robe

tothea

mplicon

Goo

dsig

nal-to-no

iseratio

mon

omolecular

reactio

nRe

latively

expensives

ynthesis


Acknowledgments


References

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table2Th

ebasicmetho

dssuitablefor

quantitativem

easuremento

fmtD


qPCR

with

fluorescenced

etectio

n

Principleo

foperatio

nAd

vantages

Disa

dvantages

Non

specificm

etho

ds

Intercalatingdyes

(SYB

RGreen

Iand

others)

Thefl

uorescence

signalincreases

duetoan

intercalatingdyeb

inding

todo

uble-stranded

DNA


ame

approach

tothed

esignof

vario

usexperim

ents

False

positiver

esultsarep

ossib

ledu

eto

prim

er-dim

erso

rnon

specific

amplicon

sso

thesem

etho

dsaren

otim


diagno

stic

purposes

Amplifluo

rAllele-specific

PCRrevealsa

hairp

instructureo

ffluo

rescently

labeled

oligon

ucleotides

Lux

Thefl

uorescence

signalchanges

whenthe


erisinclu

dedin

theP

CRprod

uct

DzyNA-

PCR

Bind

ingof

thep

robe

with

theP

CRprod

ucts

generatesrestrictio

nenzymerecognitio

nsite

Specificm

etho

ds

TaqM

anTh

edye

andtheq

uenchera

reseparatedby

51015840-exonu

cleasea

ctivity

ofTaq-po

lymerase

Highsig

nallevel

simplicity

ofdesig

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ynthesis


Acknowledgments


References

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Acknowledgments


References

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Review Article Quantitative Assessment of Heteroplasmy of ...method using Surveyor nuclease [ ], and...

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