Molecular nature of RAPD markers from Haemophilus influenzaeRd genome

Elena Moria, Pietro Liòb, Simona Dalya, Giuseppe Damianic, Brunella Peritoa,Renato Fania*

aDipartimento di Biologia Animale e Genetica, Via Romana 17-19, 50125 Firenze, ItalybGenetics Dept. Cambridge University, Downing Street, Cambridge, UK

cI.D.V.G.A.-C.N.R., Via Celoria 10, 20133 Milano, Italy

(Submitted 21 July 1998; accepted 6 November 1998)

Abstract — Despite the widespread application of the random amplified polymorphic DNA (RAPD)technique, there is no experimental evidence of the molecular mechanism of random amplification startingfrom a complex template. To investigate this mechanism, we cloned and sequenced 23 selected RAPD bandsamplified from Haemophilus influenzae Rd genomic DNA using eight decamer primers different in GC contentand/or nucleotide sequence. As the whole genome sequence of H. influenzae Rd has been reported, the exactnucleotide sequence of each primer-template annealing site was identified. Results showed that, on anaverage, a homology of eight base pairs was involved in priming events and that the number ofnonhomologous base pairings declined exponentially from the 5’ end of the primer to its 3’ end. Theinteraction between the primer and the template DNA was stabilized by the formation of secondarystructures, and a perfect match of the 3’ terminal region of the primer was not necessary for successfulamplification. The complexity of the annealing process suggested that, in the studied reaction conditions,many primer-template annealing sites were extended in the first cycles and that differences in the efficiencyof priming and replication processes led to amplification of RAPD fragments. Moreover, the distribution ofthe amplified regions on the H. influenzae chromosome was analyzed. © Elsevier, Paris

RAPD amplification / RAPD sequence / primer-template annealing site / map position

1. Introduction

The polymerase chain reaction (PCR) hasbecome one of the most important tools inbiological research. The detection of a specificDNA sequence starting from small amounts ofDNA requires the design of primers flankingthe sequence. To overpass this limitation, twoPCR-based techniques have been introduced:the arbitrarily primed PCR (AP-PCR) [24] andthe random amplified polymorphic DNA

(RAPD) [23, 25] which use one or more arbi-trarily chosen primers to amplify unknownregions from a given genome. These techniqueshave become very popular due to their ability toeasily and rapidly generate polymorphic mark-ers using very small amounts of starting DNA,independently of any prior knowledge of thetarget DNA sequence. These features make ran-dom amplification technology a tool useful inmany areas of genetic research such as genemapping, individual and strain identification,population genetics and phylogenetics [15]. Ahuge amount of literature concerning the use ofthese techniques has been produced, but notmany steps forward have been made in under-standing how they work at the molecular level.This represents an important issue, since ran-

* Correspondence and reprintsTel.: 39 552288244; fax: 39 552288250;r_fani@dbag.unifi.it

Abbreviations: nt, nucleotide; RAPD, random amplifiedpolymorphic DNA; AP-PCR, arbitrarily primed PCR

Res. Microbiol. 150 (1999), 83−93© Elsevier, Paris

dom amplification techniques strongly dependon the reaction parameters, such as primers,MgCl2, and template concentrations, type ofDNA polymerase, DNA template quality, andcycling profiles [15] (and references therein).

At the molecular level, the minimal numberof perfect base pairings at the 3’ of the primerand the relationship between GC content of theprimer and that of the target genome are stillbeing debated. There are some common as-sumptions underlying several important theo-retical considerations; one of these notions isthat priming events start at all genomic siteswhich precisely match the primer sequencesand that one or more mismatches within theprimer binding site precludes successful ampli-fication [4, 5]. Results reported in the literaturecan be summarized as follows: i) single nucle-otide substitutions in decamer primers result indramatic differences in the pattern pro-duced [25, 26]; ii) a partial homology at the 3’end of primers may be sufficient for amplifica-tion when mini-hairpin primers are em-ployed [3]; and iii) A + T-rich primers should beat least 11 base pairs in length, whereas GC-richprimers may be 8 base pairs in length [18].Other results showed that a region of 8 nucle-otides in the 3’ terminal domain of the primerwas involved in priming events leading toRAPD amplification and that mismatcheswithin these eight bases are permitted [2, 11].

The characteristics of primer-template inter-actions cannot be deduced from sequencingregions of cloned amplification products thathave complementary termini to the primer se-quence [22]. The correct approach to testing thevarious hypotheses concerning the nature ofpriming events necessitate a knowledge of thenucleotide sequence of several primer-templateannealing sites.

The analysis of primer interactions withsimple templates (mitochondrial and plasmidDNA) under nonstringent conditions demon-strated that a complementary base pairing of atleast 5–6 nucleotides at the 3’ end of the ana-lyzed primers was required for amplification [2,17].

The sequencing of entire bacterial genomesprovides the groundwork for understandinghow RAPD amplification works. The aim of thepresent work was to investigate the relation-ships between primer sequences and amplifica-tion patterns in H. influenzae, the genome se-quence of which is known, by using the strategydescribed in figure 1.

2. Materials and methods

2.1. Bacterial strains

Escherichia coli INVαF’ endA1, recA1, hsdR17(r-

k, m-k), supE44, thi-1, gyrA96, relA1, U80

lacZ∆M15 ∆(lacZYA-argF), U169 deoR k- compe-tent cells were used in transformation experi-ments as recipients. These cells were providedwith the TA Cloning Kit (Invitrogen Inc., USA).Transformant clones were selected on Luria-Bertani agar medium 1% tryptone, 0.5% yeastextract, 1% NaCl, 1.5% agar with 50 µg/mLampicillin and 30 µg/mL of 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-gal).

2.2. H. Influenzae DNA

H. influenzae Rd DNA, used as template inRAPD amplification experiments, was kindlyprovided to us by H.O. Smith, John HopkinsUniversity School of Medicine, Baltimore, MD21205, USA.

2.3. Primers

Eight 10-nt oligonucleotides designated CD1,1247, 1281, 1290, 1253, 1283, RF2 and AP12(table I) were used as primers in RAPD experi-ments. Primers employed in this work were10-nt long, because these oligonucleotides arethe most frequently utilized in RAPD experi-ments. The primers were synthesized by stan-dard phosphoramidite chemistry, deprotected,dried, dissolved in TE (10 mM Tris-HCl pH 7.5,1 mM EDTA), and used without further purifi-cation. The primers do not contain palindromicsequences longer than 6 bases.

2.4. Generation of RAPD profiles

Amplification reactions were performed in a25 µL volume containing 3 mM MgCl2, 500 ng

84 Mori et al.

of primer, 200 µM of each dNTP, 1 ng of ge-nomic DNA and 0.5 U of AmpliTaq DNA poly-merase (Perkin-Elmer). After incubation at90 °C for 1 min and at 95 °C for 1.5 min, thereaction mixtures were cycled 45 times through

the following temperature profile: 95 °C for 30 s,36 °C for 1 min, and 75 °C for 2 min. Sampleswere then incubated at 75 °C for 10 min, at60 °C for 10 min, at 5 °C for 10 min, and thenheld at 4 °C. Amplification products were storedat -20 °C.

This cycling profile was used because, in ourexperience, it gives the most reproducible re-sults. A Perkin-Elmer 9600 thermocycler wasused with the fastest available transition timesbetween each temperature. An aliquot of 6 µL ofeach amplification mixture was loaded on a

Figure 1. Outline of the strategy for cloning RAPD bands.

Table I. Nucleotide sequence of primer-template annealingsites within the H. influenzae Rd genomic DNA compared withthat of the corresponding primer. ∆G values of each primer-template duplex are also reported. Small letters indicate mismat-ched bases. Asterisks indicate a missing nucleotide in the tem-plate sequence.

Molecular nature of H. influenzae RAPD markers 85

1.5% (w/v) agarose gel with TAE buffer [19]containing 0.5 µg/mL (w/v) of ethidium bro-mide and electrophoresed at 10 V/cm for 2 h.

Amplification patterns were scanned withUVP GDS2000 (UVP products) to determineboth the size and the intensity of each RAPDband.

2.5. Cloning of RAPD bands

RAPD products were cloned into the plasmidvector pCRTMII, supplied by the Invitrogen TAcloning kit (Invitrogen Inc., USA), according tothe protocol provided by the supplier. The clon-ing step was carried out directly on the RAPDyield or by using RAPD bands purified fromagarose gel using the Qiaquick Gel extractionkit (Qiagen Inc., USA). Analysis of recombinantplasmids was performed by extracting plasmidDNA from small volume cultures grown over-night at 37 °C by using the Qiaprep Spin plas-mid kit (Qiagen Inc. USA).

2.6. Southern hybridization

The probes, consisting of cloned RAPDbands, were labeled by the random primingmethod with digoxigenin-11-dUTP using theDNA labeling and chemiluminescence detec-tion kit (Boehringer, Mannheim) according toinstructions of the supplier. Southern hybridiza-tion experiments were carried out using 6 µL ofthe amplification products obtained with eachprimer after transfer onto nylon filters(Hybond-N, Amersham). Hybridization andmembrane washes were carried out under thestringency conditions previously described [6].

2.7 Sequencing of RAPD markers

Sequencing of RAPD markers was performedusing the standard technique described by [20].

2.8. Analysis of duplexes

The predictable secondary structures and in-ternal stability of primer-template duplexeswere obtained by using the DNASIS pro-gram [21].

3. Results

3.1. Generation of RAPD profiles

Genomic DNA from H. influenzae Rd wasamplified as described in Materials and meth-ods using eight 10-nt primers (table I). Two ofthem, RF2 and AP12, differed only for thenucleotide at the 3’ end. RAPD products wereseparated by agarose gel electrophoresis. Asshown in figure 2, RAPD patterns showed 1–4bands (24 bands total) whose dimensionsranged in size from 350 to 3800 bp (table I).

Amplification reactions were performed inthree independent experiments and no differ-ence in RAPD patterns was found in the stan-dard conditions used (not shown).

3.2. RAPD band cloning

The amplification products were used with-out any previous purification for direct cloning

Figure 2. RAPD profiles obtained from amplification of H. in-fluenzae Rd genomic DNA using eight different decamers (AP12,RF2, CD1, 1247, 1253, 1281, 1283, and 1290) as primers. Ld:ladder 123 bp (dimension of the first fifteen bands: 123, 246, 369,492, 615, 738, 861, 984, 1107, 1230, 1353, 1476, 1599, 1722,1845, 1968).

86 Mori et al.

into the plasmid vector pCRTMII, employing thecloning strategy reported in figure 1. Recombi-nant plasmid DNA was extracted from a total ofabout 250 transformants and analyzed by agar-ose gel electrophoresis. A few bands werecloned by previous purification from agarosegel and subsequent insertion into the pCRTMIIvector. In this way, we obtained a total of 29different RAPD bands from the eight amplifica-tion patterns. To verify which of the 29 clonedfragments corresponded to the 24 RAPD ampli-fication bands visible by agarose gel electro-phoresis, each of them was labeled withdigoxigenin-11-dUTP and used in Southern hy-bridization experiments, using as targets theeight RAPD mixtures. Results showed thattwenty-three bands actually corresponded tobands visible on agarose gel (data not shown).The other six RAPD fragments corresponded tononvisible amplified fragments. Only one of thevisible bands (corresponding to the 1100-bpmarker obtained with primer RF2) was notcloned, even when it was purified from agarosegel. This might be due to possible lethal effectsof this sequence when cloned in E. coli onto ahigh copy number plasmid.

3.3. RAPD band sequencing

The nucleotide sequence of at least 200 nucle-otides downstream from both ends of each ofthe 23 RAPD markers was determined as re-ported in Materials and methods. We deter-mined the nucleotide sequence of multipleclones for each RAPD marker because of thepossible comigration of RAPD bands not onlybetween the different isolates of single species,but also within a single strain, as has recentlybeen reported [16]. Results showed the absenceof comigrating bands and indicated that all therecombinant plasmids contained inserts show-ing at their termini the nucleotide sequence ofthe primer used in the relative RAPD experi-ments, suggesting that no rearrangement oc-curred during the cloning step.

Furthermore, each of the 46 nucleotide se-quences obtained was compared to the H. influ-enzae Rd whole genome sequence [7]. We didnot find any difference between cloned frag-

ment sequences and the DNA sequence re-ported by Fleischmann et al. [7] except for theprimer annealing sites (see below). The com-parison also enabled determination of the exactlength of the amplified fragment; in 22 cases, itcorresponded to the expected dimension result-ing from gel electrophoresis analysis. In onlyone case (the 570-bp marker 71 obtained withprimer 1283) were the two values different, withthe length of the amplified fragment beingsmaller than the actual distance between thetwo primer-template annealing sites (see be-low).

3.4. Analysis of the primer annealing sites

The nucleotide sequence of primer-templateannealing sites was determined through align-ment of the 46 sequences obtained with the H.influenzae Rd whole genome sequence [7]. Re-sults of this analysis revealed the following: i) atotal of 88 mismatches were found, with anaverage value of 1.9 mismatches per annealingsite (table I); ii) only one primer-template an-nealing site perfectly matched the primer se-quence (marker 113); 14 primer annealing sitesshowed one mispaired base, 20 had two, 10 hadthree, and 1 had four mismatches (table I); iii)most of the primer-template annealing sitescontaining 3 or 4 mismatches showed mispairedbases clustered in the 5’ moiety of the primermolecule (table I); iv) only one event of primer-template mismatch at the 3’ terminal base of theprimer was observed (see marker 118 obtainedwith primer 1253), but this mismatch site wasstabilized by five adjacent G or C matches. Thissequence allowed the 3’ end of the primer to bebound to the target site and the polymerase toextend the primer sequence; v) as shown infigure 3, the number of mismatches declinedfrom the 5’ towards the 3’ end of the primermolecule. In particular, most of the mismatches(80.7%) fell into the first four bases from the 5’end of the primer molecule, which might there-fore be subdivided into two different moieties:the 5’ domain (containing the first four bases)and the 3’ domain (containing the remaining sixbases); and vi) one RAPD band resulted fromthe amplification of a DNA sequence one hun-

Molecular nature of H. influenzae RAPD markers 87

dred base pairs downstream from the primer-template annealing site. In this case, the primerbound two separate sequences in the templateDNA, resulting in a hairpin loop formation.More precisely, eight bases at the 5’ end ofprimer 1283 matched a sequence on the targetDNA located 106 bp away from the dinucle-otide that matched the two 3’ terminal bases ofthe primer.

Further, the thermodynamic properties of allprimer-template interactions were analyzed us-ing DNASIS software in order to predict thesecondary structure and stability of the variousprimer-template combinations, some of whichare shown in figure 4. It was observed that oftenthe primer did not yield the predicted matchaccording to the nucleotide sequence of targetsites, but it shared a different base pairing inwhich possible matches were not formed. Thus,a primer sequence might form a loop of one ortwo nucleotides that led to a more stable sec-ondary structure and probably to more efficientamplification. ∆G of all primer-template du-plexes obtained was calculated as indicative oftheir relative internal stability. Primer-templateannealing sites within the same RAPD bandgenerally (19 out of 23 amplified bands) sharedtwo high ∆G values, ranging from -10.0 to -24.9Kcal/mol. No relevant relationships betweenpredicted stability of primer-template interac-tions and relative intensity of RAPD bands wereobserved.

3.5. Analysis of target sequences

The determination of RAPD band nucleotidesequences also led us to investigate which chro-mosomal regions were amplified and to verifywhether amplified sequences were really ran-dom or if there were particular regions of thechromosome preferentially amplified because ofphysical or functional features of the DNAtarget sequence. Results of this analysis (fig-ure 5) revealed that amplified fragments con-tained both coding and noncoding regions,whereas primer-template annealing sites fellmostly within coding sequences, representingthe largest portion of the H. influenzae genome.Further, genes involved in RAPD amplificationsshared different functions, suggesting that therewas no kind of selection of a particular func-tional repertoire of genes.

An analysis of the localization of the twenty-three RAPD markers on the 1830137-bp H.influenzae Rd genome was carried out (figure 6).We found that amplified sequences were notclustered but rather, appeared to be scatteredalong the genome.

4. Discussion

Results reported in the present paper showthat, in the standard conditions of RAPD ampli-fication, a low degree of sequence homologybetween the primer and the template DNAyields amplification patterns which seem todepend on local sequence annealing conditions.Generally, we observed a number of amplifiedfragments much higher than that expected onthe basis of the Caetano-Anollés equation, aspreviously reported [2, 4]. For instance, wewould expect 0.04 amplified fragments and 0.1fragments with primer 1290 and 1283, respec-tively. Conversely, the number of observed frag-ments is four in both cases. Similar results havebeen obtained with the other six primers (notshown). This inconsistency could be explainedby data reported in this work. We have ob-served that all but one primer-template anneal-ing sites show at least one mismatched base(generally located in the 5’ moiety of the primer

Figure 3. Number of mismatches per nucleotide position (from5 position 1, to 3 position 10) of the primer.

88 Mori et al.

molecule) but even duplexes harboring up tofour mismatches exhibited high internal stabil-ity to allow RAPD amplification. In the lattercases, the duplex arises through the formationof secondary structures and is stabilized by thepresence of more stable GC pairs adjacent tomismatch(es). In such cases, the primer mayform a small loop possibly linked to a morestable secondary structure. Unusual primer-template interactions, in which the annealingsite is formed by the junction of two separateDNA sequences, joined to each other by ahairpin loop, are also possible. Our results also

suggested that even a short homologous region(3–4 bases at the 3’) could yield successfulamplification. These experimental data are indisagreement with the estimation by Cateano-Anollés [2], who suggested that a perfect ho-mology to the first 5–6 nt from the 3’ terminus ofthe primer is required for successful amplifica-tion. However, our data are in accordance witha previous conclusion by Hilton et al. [9] basedon the analysis of a single RAPD band obtainedfrom the amplification of Salmonella DNA,showing that a four base homology at the 3’ endis sufficient to permit amplification.

Figure 4. Hypothetical secondary structures of 16 primer-template duplexes and relative ∆G values determined by program DNASIS.

Molecular nature of H. influenzae RAPD markers 89

Moreover, in most cases (44 out of 46), aperfect match of at least the first two nucle-otides at the 3’ terminus of the primer is re-quired. This is also underlined by the different

patterns obtained with primers RF2 and AP12,differing only in the tenth base of the molecule.

The results also showed that the region con-taining the first 6 nucleotides of the primer-

Figure 5. Schematic representation of chromosomal regions from H. influenzae Rd amplified using eight different decamers as primers.Dimension and map position of each RAPD band and the primer used for its amplification are indicated. Arrows indicate ORFs and theirtranscription direction. Lines indicate intergenic sequences. DNA sequences corresponding to amplified regions are represented bysolid lines, whereas flanking regions are indicated by dashed lines.

90 Mori et al.

template annealing sites did not contain morethan 3–4 mispaired bases. This finding sug-gested that a higher number of mismatches in

this region could strongly inhibit RAPD ampli-fication. In addition, the local sequence contextof the primer-template annealing site may playan important role. Accordingly, the analysis ofthermodynamic properties of primer annealingsites revealed that the internal stability ofprimer-template duplexes ranged from -4.5 to-24.9 Kcal/mol, and that often the most stableprimer-template interaction did not result fromthe simple pairing between the primer and thetarget DNA. A different secondary structuremay be preferred in which some homologousmatches are shifted along the nucleotide se-quence of the target site.

Differences in DNA conformation, such as thepresence of secondary structures depending onnucleotide sequence and GC content, wouldaffect priming events and amplification yield.Therefore, both the number of homologousmatches in the primer annealing site and thelocal sequence context of RAPD fragments in-fluence its amplification.

Our findings support the use of mini-hairpinprimers, characterized by a short binding se-quence, to increase the sensitivity and reproduc-ibility of the RAPD technique [2].

The analysis of the localization of the 23RAPD markers on the H. influenzae chromo-some (figure 6), revealed that they do not accu-mulate in genomic regions with a particulargenomic organization, i.e. the origin and termi-nus sites of DNA replication, or repeats.

Recent studies have revealed that functionalorganization of bacterial genomes is associatedwith specific patterns of base composition varia-tion. The GC skew, i.e. the quantity(G-C)/(G+C), averaged over a sliding windowof arbitrary length 10 kb, and the purine skew,i.e. the quantity (G–A)/(G+A) are strongly cor-related with the direction of replication of thebacterial chromosome. For all the genomes ana-lyzed by several authors [1, 8, 10, 12-14](see also www.sciencemag.org/cgi/content/full/279/5358/1827a and references therein),the minimum of the purine skew corresponds tothe origin of replication. Since G+C content andGC skew can influence the kinetics of formationof temporary secondary structures during the

Figure 6. Schematic representation of the circular map of H.influenzae Rd chromosome showing the position of the putativereplication origin (ori) and the total or single distribution ofRAPD bands.

Molecular nature of H. influenzae RAPD markers 91

PCR cycles, we have analyzed the positions ofthe amplified fragments with respect to thecurves of purine skew, GC skew and GC contentalong the H. influenzae genome, by considering asliding window of 10 kb as in the above citedworks. We have found no correlation betweenthe patterns of RAPD amplification and thevalues of these functions (data not shown).These findings can be interpreted as an addi-tional clue that RAPD amplification depends ona combination of DNA local features affectingthe interaction between the primer moleculeand target sequences and it is unrelated toglobal or semi-local (10 kb) base compositionproperties.

Thus, the complex nature of RAPD amplifi-cation indicates that we must be cautious whenassuming that the estimated genetic distancebased on RAPD polymorphisms is simply cor-related with processes of nucleotide diver-gence [5] based on single base mutations. Westress that the understanding of the molecularmechanism of RAPD amplification will prob-ably reveal new practical aspects of this meth-odology and different means of gaining insightinto certain genomic properties.

Résumé — Nature métabolique des marqueursRAPD du génome de Haemophilus influenzae Rd.Malgré la généralisation de la technique RAPD (ran-dom amplified polymorphic DNA) il n’y a pas de preuveexpérimentale du mécanisme moléculaire de l’am-plification aléatoire à partir d’un modèle complexe.Pour explorer ce mécanisme, nous avons cloné etséquencé 23 bandes RAPD sélectionnées amplifiées àpartir de l’ADN génomique de Haemophilus influen-zae Rd, en utilisant huit décamères amorces différantpar leur contenu en GC et/ou par leur séquencenucléotidique. Étant donné que la séquence du gé-nome de H. influenzae est connue dans sa totalité, laséquence nucléotidique de chaque site d’annelageamorce-modèle a été identifiée. Les résultats ontmontré qu’en moyenne une homologie de huit pai-res de bases est impliquée dans les événementsd’amorçage, et que le nombre de bases non homolo-gues s’appariant décline exponentiellement de laterminaison 5’ de l’amorce à la terminaison 3’. L’in-teraction entre l’amorce et le modèle ADN peut êtrestabilisée par la formation de structures secondaires,

et l’appariement de la région terminale 3’ n’est pasnécessaire pour la réussite de l’amplification. Lacomplexité du processus d’annelage suggère que,dans les conditions de la réaction étudiée, de nom-breux sites d’annelage amorce-modèle sont étendusdans les premiers cycles, et ces différences dansl’efficacité d’amorçage et le processus de réplicationconduisent à l’amplification de fragments RAPD. Enoutre, la distribution des régions amplifiées du chro-mosome de H. influenzae a été analysée. © Elsevier,Paris

amplification RAPD / séquence RAPD / site d’an-nelage amorce-modèle / situation cartographique

Acknowledgments

We are grateful to Prof. Giorgio Mastromei forhelpful discussion and for critical revision of themanuscript. We are deeply indebted to twoanonymous reviewers for their many usefulsuggestions in improving the manuscript.

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