To understand the difference in between traditional RAPD-PCR and real-time RAPD, it isimportant to understand what happens during a PCR reaction. A basic PCR run can be broken upinto three phases: Exponential, Linear and Plateau. In plateau phase, reaction has stopped, nomore products are being made and if left long enough, the PCR products will begin to degrade.Each tube or reaction will plateau at a different point, due to the different reaction kinetics for eachsample. The plateau phase is where traditional PCR and real-time HRM analysis take itsmeasurement, also known as end-point detection.

At the end of traditional and real-time PCR, amplification efficiency is not exponential because double-strandedDNA is an excellent inhibitor of DNA polymerase. The reason why duplex DNA inhibits DNA polymerase is that thepolymerase binds to the duplex rather than binding to the small quantity of duplex arising from the primersbinding to target strands during the early cycles of PCR.

Application of the Inhibition Principle to real-time HRM assisted RAPD-PCR Design

The concept of amplicon inhibition of PCR is particularly important for multiplex PCR design and in our real-timeRAPD-PCR approach we exploit this fact of real-time PCR. Consider a RAPD reaction in which there are plenty ofdNTPs available. It is expected that if one of the amplicons is produced more efficiently than the others, then it willreach saturation and inhibit the polymerase from subsequently amplifying the other amplicons. Here we do notnecessitate the amplification of different targets as it can be optimized in traditional RAPD-PCR, but we let thereal-time conditions choose the appropriate amplicon to amplify efficiently and reproducibly to further theevaluation of variation from this selected amplicon’s window with HRM analysis and real-time PCR’s selectiondynamic and even to produce SCAR markers. To achieve uniform amplification of the different targets, the RAPDprimers must be screened to bind with high efficiency to its preferred target. Binding with high efficiency meanswith compromise to the requirements of real-time machine, real-time detection criteria, real-time PCR chemistryand HRM chemistry and thermodynamic parameters and any accounting for the effects of competing equilibria.

HRM analysis represents a simple and cost-effective screening method for the detection of genetic variation withinRAPD-PCR amplicons. HRM-based post-screening assay could easily be integrated into the traditional RAPD-PCRroutine. Using HRM, a large number of RAPD primers can be screened simultaneously for identification of real-time selected amplicons of interest for further DNA sequencing analysis and SCAR marker production. As this is aproof of concept study, more work will be done to further test and prove the limits and the benefits of theprocedure especially in producing useful SCAR markers to detect further variation.

1. Atienzar, F.A., Jha, A.N., 2006. The random amplified polymorphic DNA (RAPD) assay and related techniques applied to genotoxicityand carcinogenesis studies: a critical review. Mutat. Res. 613, 76–102.

2. S. M. Tulsiani, S. B. Craig, G. C. Graham, R. C. Cobbold, M. F. Dohnt, M.-A. Burns, L. K.-P. Leung, H. E. Field And L. D. Smythe (2010).High-resolution melt-curve analysis of random-amplified-polymorphic-DNA markers, for the characterisation of pathogenicLeptospira. Annals of Tropical Medicine and Parasitology, 151-161.

3. http://www.gene-quantification.de/hrm.html4. Corbett Research, HRM Assay Design and Analysis CorprotocolTM 6000-1-July06, 1-24, 2006.

FROM TRADITIONAL RAPD-PCR TO NEXT GENERATIONHRM ASSISTED REAL-TIME RAPD PCR

Irem Uzonur; Zeynep Katmer; Esma Ozsoy; Fatih Koyuncu; Tugba SenelFatih University, Biology Department, İstanbul, TURKEYCorresponding author e-mail: iuzonur@fatih.edu.tr

High Resolution Melt (HRM) Analysis is a powerful technique for the detection of mutations,polymorphisms and epigenetic comparisons through differences in melting plots of amplicons bymeasuring themelting temperature of amplicons in real time.In this study, a novel RAPD-PCR based method will be discussed to detect various levels of geneticvariation exploiting the advantages of various next-generation qPCR applications such as new dyetechnologies and HRM analysis.

Traditional randomamplified polymorphic DNA (RAPD)technique is based on the amplification of DNA fragmentsusing a short arbitrary primer that anneals multiple locationson the genomic DNA. This is followed by separation ofamplified fragments based on their sizes usinggel electrophoresis. Samples are identified by comparingthe DNA bands of the end-point fingerprints, which areexpected to be consistent for the same primer, DNA andexperimental conditions used.

The real-time PCR system is based on the detection and quantitation of a fluorescent reporter. Thissignal increases in direct proportion to the amount of PCR product in a reaction. By recording theamount of fluorescence emission at each cycle, it is possible to monitor the PCR reaction duringexponential phase where the first significant increase in the amount of PCR product correlates tothe initial amount of target template. In addition, fluorescence data can be collected directly froma real-time PCR instrument avoiding the drawbacks of end-point detection.

High Resolution Melting (HRM) is a novel, homogeneous, close-tube, post-PCR method. HRM allows to study thethermal denaturation of a double-stranded DNA in much more detail and with much higher information yield thanever before. HRM characterizes nucleic acid samples based on their disassociation (melting) behavior. Samples canbe discriminated according to their sequence, length, GC content or strand complementarity. Even single basechanges such as SNPs (single nucleotide polymorphisms) can be readily identified.

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-endinstrumentation and sophisticated analysis software allows to detect these changes and to derive informationabout the underlying sequence constellation. With HRM, these and other applications are done using low-costgeneric dyes where previously custom labeled probes such as TaqMan® or fluorescence resonance energy transfer(FRET) probes were required. HRM is thus a simpler and much more cost-effective way to characterize samples.

Traditional random amplified polymorphic DNA (RAPD) techniqueWhen RAPD is used, it is necessary to be as careful as possible by (i) optimising RAPD reactions, (ii) knowing thepotential variation among control samples, (iii) using two DNA concentrations that differ at least by a factor 2(when variation is observed or not), and (iv) interpreting the changes in RAPD profiles (bearing in mind that mostof the time diverse factors can have an impact on the RAPD assay).

HRM analysis differs from standard melt curve analysis in three ways:1.Chemistry—HRM analysis uses brighter dyes at higher concentrations.2.Instruments—HRM analysis requires instruments that collect fluorescence data at finer temperatureresolution.3.Software—HRM analysis requires more sophisticated software which uses new fluorescent scaling algorithmsand plots.

HRM data normalization­shape & shift

There are two ways HRM curve plots can discriminate between samples; by “Shape”, i.e. using detail in the shapeof the melt curve itself and by “Shift”; i.e. the thermal offset of a curve from other curves. Before HRM curves areplotted, the raw data is first normalized. Melt curves are normally plotted with fluorescence on the Y axis andtemperature on the X axis. This is similar to real-time PCR amplification plots but with the substitution oftemperature for cycle number. As with real-time PCR plots, the fluorescence axis of HRM plots is normalized onto a0 to 100% scale.

Runs and HRM analysis was performed on a Rotor-Gene 6000, by using variable real time kits.The Rotor-Gene 6000 is the first of the multi-well instruments capable of both thermal cycling and HRM. This dualcapability enables pre-amplification and HRM analysis consecutively in the one run. A major advantage of this isthat amplification plots can be used to help interpret HRM results since aberrant amplification plots (i.e. those thatamplified differently to what was expected) also produce aberrant HRM data. In this way compromised samplescan be easily identified and removed from downstream HRM analysis. The main advantage of the Rotor-Gene forHRM stems from its rotary design, in which samples spin under centrifugal force past a common optical detector.This is seemingly ideal for HRM as thermal or optical variation between samples is insignificant. The result is thatthe Rotor-Gene HRM performance closely matches the HR-1 benchmark with the compromise that samples arenot arranged in a conventional array format (as they are in block-based instruments) but are instead arrangedaround the perimeter of a spinning rotor.

Optimized conditions to produce good quality HRM asisted Real-time RAPD PCR.

Real-time RAPD-PCR detection

TraditionalRAPD-PCR detection

This figure shows three replicate samples, which had same amount of DNA inthe beginning of the reaction but have different quantities of amplicons at theend of the reaction. Therefore, it will be more precise to take measurementsduring the exponential phase, where the replicate samples are amplifyingexponentially.

Real-time HRMRAPD-PCRdetection

REAL TIME PCR

PRECISION TYPE-IT TAKARA

RAPD

PRIMER

SPECIFIC

PRIMER

RAPD

PRIMER

SPECIFIC

PRIMER

RAPD

PRIMER

SPECIFIC

PRIMERPrimer length

(bp)10 20 10 20 10 20

Primer

concentration

(µM)

2.5 1.25 1 0.75 1.6 0.93

Annealing

temperature

( °C)

36 55 36 55 36 55

Number of

cycles48 45 48 45 48 45

Separation Agarose Agarose Agarose Agarose Agarose Agarose

Visualisation Safe View Safe View Safe View Safe View Safe View Safe View

Size range of

PCR products

(kb)

0.1-0.6 0.2-0.4 0.1-0.6 0.2-0.4 0.1-0.6 0.2-0.4

DREAMTAQ MASTER

MIX

TYPE-IT MASTER MIX

EVA GREEN

PRIMER DESIGN

CHROMOFY

TAKARA

SYBER GREEN

RAPD

PRIMER

SPECIFIC

PRIMER

RAPD

PRIMER

SPECIFIC

PRIMER

RAPD

PRIMER

SPECIFIC

PRIMER

RAPD

PRIMER

SPECIFIC

PRIMER

THERMAL

CYCLING

CONDITIONS

Initial

denaturation:

94 C - 5 min

45 cycle;

94 C - 30 sec

36 C - 1 min

72 C - 2 min

Final

extension;

72 C - 5 min

Initial

denaturation:

95 C - 5 min

45 cycle;

95 C - 10 sec

55 C - 20 sec

72 C - 30 sec

Final

extension;

72 C - 5 min

Initial

denaturation:

95 C - 10 min

48 cycle;

95 C - 25 sec

36 C - 30 sec

72 C - 30 sec

Hold 1;

95 C - 1 min

Hold 2;

55 C - 1 sec

Initial

denaturation:

95 C - 10 min

45 cycle;

95 C - 10 sec

55 C - 30 sec

72 C - 10 sec

Initial

denaturation:

95 C - 8 min

48 cycle;

95 C - 25 sec

36 C - 30 sec

72 C - 30 sec

Hold 1;

95 C - 1 min

Hold 2;

55 C - 1 sec

Initial

denaturation:

95 C - 5 min

45 cycle;

95 C - 10 sec

55 C - 30 sec

72 C - 10 sec

Initial

denaturation:

95 C - 3 min

48 cycle;

95 C - 20 sec

36 C - 20 sec

72 C - 30 sec

Hold 1;

95 C - 1 min

Hold 2;

55 C - 1 sec

Initial

denaturation:

95 C - 30 sec

45 cycle;

95 C - 5 sec

50 C - 20 sec

72 C - 40 sec

PCR

PARAMETERS

0.05 u/µl Taq DNA Polymerase

Reaction buffer

4 mM MgCl2

0.4 mM of each dNTP

Traditional RAPD PCR 10 replicate results withtwo different DNA samples and RAPD 10 merprimer OPB5.

Real-time RAPD with HRM analysis and agarose gelresults using the same 10-mer RAPD primer OPB5 andthe same DNA, SA1 as 3 replicates.

Further genetic variation detection by using real-time RAPD and HRM analysis.