Chloroplast DNA (cpDNA) has highly conserved regions and also intergenic spacers with relatively high levels ofpolymorphisms for the analysis of intraspecific to intergeneric levels of variation. Chloroplast DNA (cpDNA) is asource of original markers very useful in phylogeny and in population genetics. The degree of universality of cpDNAprimers within the plant kingdom varies. In our case primers (KLR-F: 5’-AGTTCGAGCCTGATTATCCC-3’and KLR-R: 5’-GCATGCCGCCAGCGTTCATC-3’) amplify a 298 bp highly conserved region that is virtually a conserved region fromany land plant and many alga with end-point detection. We further our end-point detection using qPCR assistedwith HRM analysis and sequencing to detect the genetic heterogeneity of cpDNA that is undetectable with end-point PCR and agarose gel electrophoresis from terrestrial plants: Urtica dioica, Zea mays, Phaselous vulgaris andbloom forming micro-algae species Prorocentrum minimum and Skeletonema costatum.In this context as approved by sequencing, HRM assisted qPCR is a very practical and sensitive tool that can beadapted to the different levels of genetic heterogeneity determinations.

Because tiny amounts of even degraded DNA can be enough for successful amplification, and also because cpDNA ispresent at many more copies per cell than most nuclear DNA sequences, cpDNA primers have been used tosuccessfully amplify sequences very efficiently.High Resolution Melt (HRM) analysis is a powerful technique for the detection of mutations, polymorphisms andepigenetic comparisons through differences in melting plots of amplicons by measuring the melting temperature ofamplicons in real time, using a fluorescent DNA-binding dye.

The most important HRM application is gene scanning - the search for the presence of unknown variations in PCRamplicons prior to or as an alternative to sequencing. Mutations in PCR products are detectable by HRM becausethey change the shape of DNA melting curves. A combination of new-generation DNA dyes, high-end instrumentationand sophisticated analysis software allows to detect these changes and to derive information about the underlyingsequence constellation. With HRM, these and other applications are done using low-cost generic dyes wherepreviously custom labeled probes such as TaqMan® or fluorescence resonance energy transfer (FRET) probes wererequired. HRM is thus a simpler and much more cost-effective way to characterize samples.

Criterias when designing HRM assays:• Obtaining a clear understand of the sequence targeted. Where possible, it is important to determine all thevariations present within the sequence of interest. Check for species homology, intron-exon boundaries, splice sites,known SNPs, etc.• Designing primers with anneal temperatures of 60°C amplify short products (100–250 bp). Longer products can beused, however, using products above 250 bp can reduce sensitivity due to the increased potential for multiple meltdomains with complicated melt curves.• Determining the folding characteristics of the product and primers at the annealing temperature (e.g. useDINAMelt) and test for specificity (BLAST search).• Keep post-amplification sample concentrations similar; the concentration of a DNA fragment affects its temperatureof melting (TM). For this reason sample DNA concentrations must be kept as similar as possible. When analyzingamplification products, ensure every reaction has amplified to the plateau phase.

•Runs and HRM analysis was performed on Rotor-Gene 6000, by using real time kits.•Tables provide an overview of conditions which were optimized to produce good quality HRM asisted products.•Following the PCR, melting-curve analysis of amplicons was conducted in the thermocycler (Rotor-Gene 6000) byincreasing the temperature from 65 C to 80 C at different ramping increments from 0.2 C/s, recording changes influorescence with changes in temperature (dF/dT) and plotting against changes in temperature. HRM analysis wasperformed using the software Rotor-Gene 1.7 (Build 94) with normalization regions between 65.15–65.65 C and79.50–80.00 C, and an average confidence threshold of 90%. Normalisation is a process for assigning attributes toentities using principles that will greatly reduce or eliminate data redundancies…HRM data normalization­shape & shift: There are two ways HRM curve plots can discriminate between samples;by “Shape” , i.e. using detail in the shape of the melt curve itself and by “Shift”; i.e. the thermal offset of a curvefrom other curves. Before HRM curves are plotted, the raw data is first normalized. Melt curves are normally plottedwith fluorescence on the Y axis and temperature on the X axis. This is similar to real-time PCR amplification plots butwith the substitution of temperature for cycle number. As with real-time PCR plots, the fluorescence axis of HRMplots is normalized onto a 0 to 100% scale.•1.7% agarose gel electrophoresis were applied to the products and results were observed with Gel DOC 2000(BIORAD) system.

In the present study, we critically evaluated and established a practical real-time PCR-coupled HRM analysis method,employing chloroplast DNA as the genetic marker, for the detection of the chloroplast DNA heterogeneity whichmight have further implications in taxonomic discussions and even in toxicologic evaluations such as genotoxicitydetections and target tissue toxicity monitorings.

CHLOROPLAST DNA HETEROGENEITY DETECTION WITH HRM ANALYSISIrem Uzonur, Esma Ozsoy, Zeynep Katmer, Fatih Koyuncu, Gamze Akdeniz, Ali Rıza AtasoyFatih University, Biology Department, Istanbul, TURKEYCorresponding author e-mail: zkatmer@yahoo.com

A genetic map of the tobacco chloroplast genome

Chloroplast DNA (cpDNA) is a source of original markers very useful inphylogeny and in population genetics. cpDNA is a circular molecule(155844 bp in Nicotiana tabaccum), which is highly conserved in sizeand structure. It usually possesses two long inverted repeats (IR) whichseparate a large single copy region (LSC) from a small single copy region(SSC). Nucleotide substitution rates of cpDNA sequences is quite high.The conservation of the arrangement of the genes in cpDNA has lead tothe design of numerous 'consensus' or 'universal' chloroplast primerswhich facilitate phylogenetic or population genetic studies. Theavailability of primers which allow the amplification and directsequencing of the gene rbcL has revolutionized plant taxonomy. Atlower taxonomic levels, consensus primers which amplify non-codingchloroplast DNA separating very conserved regions are also extremelyuseful. The sequence amplified can be small enough to allow directsequencing and the 'universality' of this type of cpDNA primers is veryhigh and they are conserved enough to amplify virtually any land plantsand many algae, that are also applicable in our work.

THERMAL

CYCLING

CONDITIONS

Initial

denaturation;

45 cycle; Hold 1; Hold 2;

95 C - 10 min 95 C - 10 sec

55 C - 30 sec

72 C - 10 sec

95 C - 1 min 55 C - 1 sec

PCR

PARAMETERS

2x HRM PCR Master Mix includes;

HotStartTaq Plus DNA Polymerase,

Type-it HRM PCR Buffer,

Q-Solution,

dNTP,

Fluorescent dye (EVA-GREEN)

TYPE-IT

HRM

(EVA-GREEN)

Primer

length

(bp)

Primer

concentration

(µM)

Annealing

temperature

( °C)

Number of

cycles Separation Visualisation

Size range of

PCR products

(kb)

20 0.75 55 45 Agarose Safe View 0.2-0.4

A measure of genetic variation is allelic variation which refers to

variation within a specific gene.

500 mutational sites

Interspecies cpDNA comparisons with HRManalysis and direct sequencing of pure sampleDNA P. minimum & pure sample DNAS.costatum

Intraspecies cpDNA comparisons with HRManalysis and direct sequencing of pure sampleDNA P.minimum & bloom sample DNA P.minimum

Intraspecies cpDNA comparisons with HRManalysis and direct sequencing of pure sampleDNA P.minimum & bloom sample DNA P.Minimum (Tm values differ 0,07oC)

Intraspecies cpDNA comparisons with HRManalysis and direct sequencing of bloomsample DNA S. costatum & pure sample DNAS. costatum (Tm values differ 0,12oC)

Interspecies cpDNA comparisons with HRManalysis and direct sequencing of bloomsample DNA P. minimum & bloom sampleDNA S.costatum. (Tm values differ 0,12oC.)

Intraspecies (S. costatum pure and bloomsamples 1 and 2 cpDNA comparisons withHRM analysis , three replicates from eachsample)

1. Petit RJ, Demesure B, Dumolin-Lapègue S (1996) CpDNA and plant mtDNA primers. In Molecular tools for screening

biodiversity: Plants and Animals. A Karp, PG Isaac, D Ingram eds, Chapman & Hall, full paper.

2. Schmitt JM, Bohnert HJ, Gordon KH, Herrmann R, Bernardi G, Crouse EJ. Compositional heterogeneity of the chloroplast

DNAs from Euglena gracilis and Spinacia oleracea. Eur J Biochem. 1981 Jul;117(2):375-82.

3. Alan M. Magee, Sue Aspinall, Danny W. Rice, Brian P. Cusack, Marie Sémon, Antoinette S. Perry, Saša Stefanović, Dan

Milbourne, Susanne Barth, Jeffrey D. Palmer, John C. Gray, Tony A. Kavanagh, and Kenneth H. Wolfe Localized

hypermutation and associated gene losses in legume chloroplast genomes Genome Res. December 2010 20: 1700-1710

4. Corbett Research, HRM Assay Design and Analysis CorprotocolTM 6000-1-July06, 1-24, 2006.

5. Qiagen, Type-it® HRM™ PCR Handbook,July 2009,6-12