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Ready-To-Go RAPD Analysis Beads are designed as pre-mixed,pre-dispensed reactions for performing random amplified

polymorphic DNA (RAPD) analysis. With RAPD analysis,genomic polymorphisms can be detected at multiple loci usingonly nanogram quantities of DNA. The RAPD reactions areprovided as room-temperature-stable dried beads that contain allthe necessary reagents, except primer, for performing RAPD

analysis. Each kit contains 100 individual RAPD reactions,control RAPD Analysis Primer and two controlE. coli DNAs.

XY-065-00-11

ReadyToGo

RAPD Analysis Beads

Rev. 7

Note: After opening, store unused reactions in the resealable pouch with

desiccant provided and /or store in a desiccator.

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CONTENTS

Patent and License Information 3Components 4

Overview 5

Recommendations for Storage 7

Requirements for Template DNA 7Requirements for RAPD Primers 8

Protocol: Introduction 11

Procedure A: RAPD Reaction 14

Procedure B: Gel Analysis 15Appendix 1: Additional Reagents Required 16

Troubleshooting Guide 17

Function Testing, Storage, References 22

Ordering Information 23

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PATENT AND LICENSE INFORMATION

Before using this product, carefully read the Patent and License Informationbelow. By using this product, you indicate your acceptance of the conditionsdescribed below.

This product is designed for use in the Random Amplified Polymorphic(RAPD) process covered by patents and pending applications including

U.S. 5,126,239 owned by E. I. du Pont de Nemours and Company ofWilmington, DE. Amersham Biosciences has a limited licensefrom du Pont de Nemours and Company to produce and sell this product tothe pur-chaser. The purchase of this product includes a fully paid-up, limitednonexclusive license under the RAPD patent to use this product to performthe RAPD process for research purposes, provided that the RAPD process is

conducted using nucleic acid polymerase which has been licensed byHoffmann-La Roche Inc. for use in the polymerase chain reaction (PCR)process and provided the RAPD is conducted in conjunction with a thermo-cycler licensed by the Perkin-Elmer Corporation for use in the PCR process.

Purchase of this product conveys to the user the right to utilize the RAPD

process for research purposes only. No right or license is conveyed to thepurchaser to develop a commercial product, to perform any diagnostictesting or to offer any commercial service using the RAPD process.

Further information about acquiring rights under the RAPD process toperform commercial testing or for commercial product development can be

obtained by contacting E. I. du Pont de Nemours and Company, NewBusiness Development at facsimile number 302-892-1581.

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COMPONENTS

RAPD Analysis Room-temperature-stable bead containingBeads: buffer, dATP, dCTP, dGTP, dTTP, BSA,AmpliTaq and Stoffel fragment.

Control E. coli 1 g ofE. coli BL21(DE3) DNA;

BL21(DE3) DN A : lyophilized.

Control E. coli 1 g ofE. coli C1a DNA; lyophilized.C1a DN A:

RAPD Analysis 2.5 nmol of lyophilized primer

Primer 2: (5'-d[GTTTCGCTCC]-3').

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OVERVIEW

Random amplified polymorphic DN A (RAPD) analysis is atechnique for rapidly detecting genomic polymorphisms (1),utilizing a single short oligonucleotide primer of arbitrarysequence in a polymerase chain reaction (PCR)*. The PCRreaction is carried out under low stringency conditions to generate

a reproducible array of strain-specific products that are sub-sequently analyzed by gel electrophoresis.

RAPD analysis has been used in numerous applications, includinggene mapping, detection of strain diversity, population analysis,

epidemiology, and the demonstration of phylogenetic and tax-onomic relationships (reviewed in reference 2). RAPD analysis hasbecome a widely used technique since it enables the quick detec-tion of polymorphisms at a number of different loci using onlynanogram quantities of genomic DNA. Furthermore, RAPD

analysis can be carried out on organisms for which there is little orno information concerning genomic sequences or organization,thus making it possible to analyze polymorphisms for virtuallyany organism from which relatively pure genomic DNA can beisolated.

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*The PCR process is covered by U. S. Patents 4,683,195 and 4,683,202 owned byHoffmann-La Roche Inc. Use of the PCR process requires a license.

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Ready-To-Go RAPD Analysis Beads provide the reagents forRAPD reactions in a convenient ambient-temperature-stable bead.

The beads are manufactured using a proprietary technologylicensed to Amersham Biosciences. Ready-To-Go RAPDAnalysis Beads have been optimized for RAPD reactions andcontain thermostable polymerases (AmpliTaq DNA polymeraseand Stoffel fragment), dNTPs (0.4 mM each dNTP in a 25 lreaction volume), BSA (2.5 g) and buffer [3 mM MgCl2, 30 mMKCl and 10 mM Tris, (pH 8.3) in a 25 l reaction volume]. Thetwo different thermostable polymerases, combined in aproprietary ratio, produces a more complex RAPD fingerprintingpattern than either of the polymerases alone. The only reagentswhich must be added to the reaction are an arbitrary primer andtemplate DNA. The Ready-To-Go bead format significantlyreduces the number of pipetting steps, thereby increasing thereproducibility of the RAPD technique and minimizing the risk of

contamination.In addition to the RAPD Analysis Primer included in the kit, aprimer set (27-9501-01) consisting of six primers (10-mers) ofarbitrary sequence is also available. This primer set has beensuccessfully used in conjunction with the Ready-To-Go RAPDAnalysis Beads for the study of a variety of different organisms,including bacteria, fungi, insects, plants, algae and humans.

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Recommendations for StorageAfter opening the kit, store unused reactions at ambienttemperature in the resealable pouch with desiccant provided.

It is important to seal the pouch completely so that the beads donot absorb moisture. In regions with high relative humidity, werecommend that the resealed pouch be stored in a desiccator.

Reconstituted Control DNA and RAPD Analysis Primer shouldbe stored at -20C.

Requirements for Template DNARAPD analysis can be performed on genomic DNA from virtually

any organism. The use of pure, high quality genomic DNA iscritical for good RAPD analysis. Since the most frequent cause ofpoor quality RAPDs is poor quality DNA, it is important topurify genomic DNA using methods that will yield pure,undegraded DNA. To ensure high quality DNA, we recommend

using GenomicPrep DNA Isolation Kits from AmershamPharmacia Biotech (see page 23, Ordering Information). Tomaximize the reproducibility of the RAPD technique, werecommend that all comparisons of RAPD reactions be madeusing DN A purified by the same procedure.

The amount of genomic DNA used in a RAPD reaction can varyfrom as little as 1 ng to as much as 100 ng. As a general rule,5-50 ng is sufficient, but templates may differ in their optimal

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amounts. Sometimes a poor quality RAPD can be improved bydecreasing or increasing the amount of template. Therefore, it is

recommended that a range of template concentrations be tested todetermine the amount needed to give the desired banding pattern.

E. coli cells can be added directly to a RAPD reaction withoutprior extraction of the DNA. However, the reproducibility of the

RAPD technique is maximized if purified genomic DNA is used.

Requirements for RAPD PrimersPrimers used for RAPD analysis should consist of a single oligo-nucleotide of arbitrary sequence. The size of the primer is typically

7-15 bases in length. Primers with different arbitrary sequencesand different sizes will give different banding patterns with thesame DNA. We have obtained the best results with a variety ofdifferent templates using primers that are 10 bases long.

RAPD primers should at a minimum be purified using aNAP-10 column (17-0854-01, -02). Their GC content shouldbe at least 60% , and they should contain no hairpin structures.

Different amounts of primer, relative to template, give differentsubsets of bands in a RAPD reaction. High concentrations of

primer result in primarily low molecular weight bands, while lowconcentrations of primer result in a banding pattern comprisedprimarily of high molecular weight bands. We have found that formost templates, 25 pmol of primer and 10 ng of template is

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optimal for generating the most complex banding pattern.

RAPD Primers from AmershamBiosciencesAmersham Biosciences offers a set of six RAPD primers.Each primer is a 10-mer of arbitrary sequence that is specificallydesigned and tested for use in RAPD analysis. The RAPDAnalysis Primer Set (27-9501-01) contains 2.5 nmol of each ofthe following primers:

RAPD Analysis Primer 1 - (5'-d[GGTGCGGGAA]-3')

RAPD Analysis Primer 2 - (5' -d[GTTTCGCTCC]-3' )

RAPD Analysis Primer 3 - (5'-d[GTAGACCCGT]-3')

RAPD Analysis Primer 4 - (5'-d[AAGAGCCCGT]-3')*

RAPD Analysis Primer 5 - (5'-d[AACGCGCAAC]-3' )*

RAPD Analysis Primer 6 - (5'-d[CCCGTCAGCA]-3')*

When used with the controlE. coli DNAs in a RAPD reaction,

each of the RAPD primers will generate a unique bandingpattern. Figure 1, page 10 shows the approximate bandingpattern that can be expected when the control DN A RAPDreactions are electrophoresed on a 2% agarose gel. Note,however, that slight variations in control RAPD patterns may

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Primer 2 is also packaged as a component of Ready-To-Go RAPDAnalysis Beads (27-9500-01).

* see reference 3

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10

BL21 BL21 BL21 BL21 BL21 BL21( D E 3 )C 1 a (DE3) C1a ( D E 3 )C 1 a (DE3) C1a (DE3) C1a ( D E 3 )C 1 a

RAPDPrimer 1

RAPDPrimer 2

RAPDPrimer 3

RAPDPrimer 4

RAPDPrimer 5

RAPDPrimer 6

Figure 1. Analysis of control DN A RAPD reactions on a 2% agarose gel.Each reaction was performed with 10 ng of control DNA and 25 pmol ofprimer using the reaction conditions described in Procedure A.

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result from differences in thermocycling and/or electrophoresisconditions.

PROTOCOL

IntroductionThe reaction conditions described in Procedure A have beenoptimized for genomic DNA from a wide variety oforganisms. Any variations to the suggested conditions inrelation to reaction volume, primer concentration, DNA

template concentration or cycling profile can result in poorRAPD results (see the Troubleshooting Guide on page 17). It isstrongly recommended that all RAPD reactions be performedinitially using the conditions stated in Procedure A. Subsequentreactions may then be further optimized to produce the desired

banding pattern for a particular model system.

Control DNA and PrimerThe twoE. coli strain DNAs and the RAPD analysis primerincluded in the kit are provided as controls to assay the ability of

the RAPD beads to amplify DNA and identify polymorphisms.Each tube ofE. coli DNA contains 1.0 g of lyophilized genomicDNA which should be reconstituted with 200 l of sterile waterto give a final concentration of 5 ng/l. Reconstituted control

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DNA should be stored at -20C.

The tube of RAPD Analysis Primer 2 contains 2.5 nmol of primer.The primer should be reconstituted with 500 l of sterile distilledwater to give a final concentration of 5 pmol/l. Reconstitutedcontrol primer should be stored at -20C.

When 10 ng (2 l) ofE. coli DNA is used in a RAPD reactionwith 25 pmol (5 l) of RAPD Analysis Primer 2, a unique subsetof PCR products is produced. When the two control DNAs areanalyzed on a gel, they will display a differential banding pattern.Figure 2, page 13 demonstrates the polymorphisms that are

observed with the two control DNAs and RAPD Primer 2 usingthe RAPD reaction conditions described in Procedure A.

In the procedures which follow, components provided with the kitare highlighted by the use ofboldface type. Additional reagentsrequired for the procedures are listed in Appendix 1 (page 16).Other Amersham Biosciences products for use withReady-To-Go RAPD Analysis Beads can be found in the OrderingInformation on page 23.

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13

100Base-

PairLadder

800 bp

BL21( D E 3 )C 1 a

RAPDPrimer 2

Figure 2. RAPD analysis ofE. coli BL21 (DE3) and C1aDNA (10 ng each) using RAPD Analysis Primer 2(25 pmol) and the reaction conditions described inProcedure A.

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Procedure A: RAPD Reaction

When performing RAPD analysis, exercise extreme care toprevent DNA contamination. Always use sterile, filter pipette tipsand avoid carry-over contamination of stock solutions.

Check that the bead is visible in the bottom of the tube ofRAPD

Analysis Beads. If necessary, tap the tube against a hard surface to

force the bead to the bottom of the tube.

Add the following to a tube containing the RAPD AnalysisBead:

25 pmol of a single RAPD primer X l

5-50 ng template DNA Y lDistilled water to total of 25 l

Mix the contents of the tube by gently vortexing, or byrepeatedly pipetting the mixture up and down. Centrifuge

briefly to collect the contents at the bottom of the tube.

Overlay the reaction with 50 l of mineral oil.

Place the samples in a thermocycler and cycle using the follow-ing cycle profile: 1 cycle at 95C for 5 minutes followed by 45

cycles at 95C for 1 minute, 36C for 1 minute and 72C for 2minute.

Continue with Procedure B, Gel Analysis.

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Procedure B: Gel AnalysisAfter amplification, the banding pattern of the randomly amp-lified DNA must be visualized and analyzed. RAPD analysis canbe done on either agarose or polyacrylamide gels. For agarosegels:

Pour a long (e.g. GNA 200) 2% agarose gel using 1X TAE or TBE

buffer (see Appendix 1) containing 0.5 g/ml of ethidiumbromide.

Add 1 l of 6X tracking buffer (see Appendix 1) to 5 l of the

amplified sample and load onto the gel.

Electrophorese the sample until good separation of RAPD bands

is observed and the bromophenol blue from the tracking dye is

2.5 cm or less from the bottom of the gel (e.g. 150 volts for

3 hours).

For better resolution of low molecular weight bands, polyacryla-mide gel systems may be used. The loading level for apolyacrylamide gel should be approximately 0.5 l of the 25 lRAPD reaction. For visualization on acrylamide gels, werecommend using the DNA Silver Staining Kit from Amersham

Pharmacia Biotech.

Note:Bands and/or smears in a no template control are normalin RAPD Analysis. See page 21 for further information.

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TROUBLESHOOTING GUIDE

This Guide is organized in the following fashion: A problem isstated, followed by its possible cause(s) and solution(s). The gelphotographs are provided to give visual examples of commonproblems associated with RAPD analysis.

No bands are visible and/or there is excessivesmearing on the gel.

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The quality of the DN Atemplate was poor. Poorquality DNA is the most

common reason for sub-optimal RAPDs. Assay thetemplate DNA on a gel toensure that it is not degraded.

The wrong thermocyclingprofile was used. Altering therecommended thermocyclingconditions, especially thedenaturation temperature,can affect the bandingpattern (Figure 3). Use thethermocycling conditionsdescribed in Procedure A.

Figure 3. RAPDresults ofE. coliBL21(DE3) DNA(10 ng) using

various denatura-tion temperaturesduring thermo-cycling. All otherreaction condi-tions were identi-

cal to those de-scribed in Pro-

cedure A.M = 100 Base-Pair Ladder

(27-4001-01).

M

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Insufficient DN A was used in the RAPD reaction. Dependingon the purification method used, the amount of DNA needed

to give a good banding pattern can vary (Figure 4). Titrate thegenomic DNA in the reaction until the smearing is eliminated.

The thermocycler was not functioning properly. Impropercycling conditions can result in banding pattern variations andsmearing. Check that your thermocycler is functioningproperly and switch to a different thermocycler if necessary.

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Figure 4 . RAPD results using25 pmol of primer with variousamounts of template DNA purified

as follows:E. coli DNA wasisolated using RapidPrepMicroGenomic DNA Isolation Kit forCells and Tissue (27-5225-01);

lettuce DN A was isolated by theCTAB method (4); trout DNA was

isolated by the Proteinase Kmethod of Grimberg et al. (5). Allother reaction conditions were

identical to those described inProcedure A. M = 100 Base-PairLadder (27-4001-01).

E. coli trout

M M

lettuce

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The wrong reaction volumewas used. The RAPD

Analysis Beads should onlybe used in a final reactionvolume of 25 l (Figure 5).

No primer was added to thereaction. The RAPDAnalysis Beads contain noprimer. Therefore, primermust be added by thecustomer.

Inadequate primer amountswere used in the RAPDreaction. Too little primercan result in smearing whileexcessive primer to template

ratios can cause an abun-dance of low molecularweight bands and smearing(Figure 6). Titrate theprimer in the reaction until

the smearing is eliminated.

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Figure 5. RAPDresults using various

reaction volumeswith 10 ng ofspinach DN A. Allother reactionconditions wereidentical to thosedescribed inProcedure A.

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A reproducible banding pattern is not achieved whenusing the same template and primer.

The amount of template used was different. Different primerto template ratios in a RAPD reaction can result in differentbanding patterns on a gel (Figure 4). It is critical that theexact same amount of template is used each time. Usingmaster mixes (i.e. pre-mixed solutions of template andprimer) will increase reproducibility.

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The amount of primerused was different.Different primer to

template ratios in aRAPD reaction canresult in different band-ing patterns on a gel(Figure 6). It is critical

that the exact sameamount of primer isused each time. Usingmaster mixes (i.e.pre-mixed solutions of

template and primer)will increase reproduci-bility.

Figure 6. RAPDresults using variousamounts of primerwith 10 ng of porcine

DN A. All otherreaction conditionswere identical tothose described inProcedure A.M = 100 Base-Pair

Ladder (27-4001-01).

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The quality of the DN A template was poor. Degraded DNAgives a poor banding pattern. Assay the template DNA on a

gel to ensure that it is not degraded. The thermocycler was not functioning properly or a differ-

ent thermocycler was used. Differences in thermocyclerdesign and ramping speeds can result in significantdifferences in banding patterns. Whenever possible, use thesame thermocycler to avoid these variations. Impropercycling conditions can also result in banding variations.Check that your thermocycler is functioning properly andswitch to a different thermocycler if necessary. Variations

within a single thermocycler can result due to temperaturevariations across the block. This problem can be avoided byswitching to another, less variable thermocycler.

There are visible bands and/or smears in a notemplate control sample.

A no template control usually gives some bands and/orsmears in a RAPD reaction. Bands and/or smears in a notemplate control are normal in RAPD analysis (1, 6, 7).The bands and/or smears probably arise from small

amounts of DNA contamination in the polymerases. Thesebands are not observed when 10 ng of template is present ina reaction. At these levels, the template DNA will out-compete any contaminant DNA that may be present.

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FUNCTION TESTINGEach batch of Ready-To-Go RAPD Analysis Beads is tested toensure its ability to generate a differential banding patternbetween the two controlE. coli strains using the control primer.

STORAGE

Store ambient. After opening the kit, store unused reactions in theresealable pouch with desiccant provided. Reconstituted ControlE. coli DNA and RAPD Primer should be stored at -20C.

REFERENCES

1. Williams, J. G. et al.,Nucl. Acids Res. 18, 6531 (1990).

2. Welsh, J. et al., PCR 2: A Practical Approach, McPherson, M.J., Hames, B. D., Taylor, G.R. ed., chapter 11, IRL Press(1995).

3. Akopyanz, N. et al., N ucl. Acids Res. 20, 5137 (1992).

4. Doyle, J. et al., Phytochem. Bull. 19, 11 (1987).

5. Grimberg, J. et al., N ucl. Acids Res. 17, 8390 (1989).

6. Klein-Lankhorst, R. M. et al., Theor. Appl. Genet. 83, 108(1991).

7. Hadrys, H. et al.,Molecular Ecology 1, 55 (1992).

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Ready-To-Go, NAP and GenomicPrep are registered trademarks ofAmersham BiosciencesLimited or its subsidiaries. AmershamBiosciences is a trademark of Amersham plc.

AmpliTaq is a trademark of Hoffman-La Roche

All goods and services are sold subject to the terms and conditions ofsale of the company within the Amersham Biosciences groupthat supplies them. A copy of these terms and conditions of sale isavailable on request.

Amersham Biosciences 1999All rights reservedPrinted in the United States for Amersham BiosciencesAmersham Biosciences UK Limited. Amersham Place, LittleChalfont, Buckinghamshire, England HP7 9NA.Amersham Biosciences. SE-751 84, Uppsala, Sweden.

Amersham Biosciences. 800 Centennial Avenue, PO Box1327 Piscataway, NJ 08855, USA.Amersham Biosciences Europe GmbH . Munzinger Strasse 9,D-79111, Freiburg, Germany.

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