[methods in molecular biology] plant dna fingerprinting and barcoding volume 862 || development of...

123

Nikolaus J. Sucher et al. (eds.), Plant DNA Fingerprinting and Barcoding: Methods and Protocols, Methods in Molecular Biology, vol. 862,DOI 10.1007/978-1-61779-609-8_10, © Springer Science+Business Media, LLC 2012

Chapter 10

Development of Sequence Characterized Amplifi ed Region from Random Amplifi ed Polymorphic DNA Amplicons

Kalpana Joshi and Preeti Chavan

Abstract

Among the PCR-based markers that are most widely used in molecular genetic studies, SCARs (sequence characterized amplifi ed regions) have the advantage of being less sensitive to the conditions of a standard PCR due to its primer size when compared to RAPD (random amplifi ed polymorphic DNA) and hence are more specifi c and reproducible. Moreover, SCARs require no radioactive isotopes and detect only a single locus. Here, we describe the development of SCAR from RAPD amplicons.

Key words: SCAR , Polymorphisms , DNA marker , Genotyping

RAPD (random amplifi ed polymorphic DNA) analysis has been widely used for population genetic studies, genetic mapping, geno-typing, and marker-assisted selection of desirable traits. However, RAPD analysis is sensitive to reaction conditions, sometimes lead-ing to nonreproducible results. Further, nonstringent annealing temperatures can result in nonspecifi c amplifi cation which may not be reproducible thereby undermining their direct use as markers. Therefore, RAPD amplicons are converted to SCAR (sequence characterized amplifi ed region) markers. In this technique, the RAPD marker termini are sequenced and longer primers (22–24 nucleotide bases) are designed for specifi c amplifi cation of a par-ticular locus ( 1 ) . SCAR primers being longer in length (usually 18–22 nucleotides) have higher annealing temperatures and thus are specifi c and reproducible. SCARs are useful in genetic mapping studies (codominant SCARs), map-based cloning, comparative mapping, or homology studies among related species, genotyping, and marker-assisted selection of desired traits. SCAR markers have been applied for species identifi cation in various organisms such as

1. Introduction

124 K. Joshi and P. Chavan

plants ( 2– 4 ) , insects ( 5, 6 ) , microbes ( 7 ) , and animals ( 8 ) . Here, we describe the development of SCAR markers from RAPD ampli-cons. Following DNA isolation and RAPD analysis, the amplicon of interest is purifi ed, cloned in a suitable vector, and sequenced to develop SCAR markers as described below.

1. Operon primer Kits A, B, C, D, E, F, G, and H (Primer con-centration: 5 pmol/ μ l).

2. Taq polymerase, 3 U/ μ l (Fermentas). 3. 10 mM dNTP mix: Mix 2.5 mM concentration each of dATP/

dCTP/dGTP/dTTP. 4. Taq Buffer: 100 mM Tris–HCl (pH 9), 15 mM MgCl 2 ,

500 mM KCl, and 0.1% gelatin. 5. Store the above biochemicals at −20°C.

1. Low melting agarose (Sigma). 2. Wizard SV kit for purifi cation of PCR fragment from gel

(Promega).

1. Purifi ed RAPD product. 2. 100 mM dATP. 3. Taq polymerase Buffer. 4. Taq polymerase (3 U/ μ l) (Fermentas).

1. A-tailed product. 2. 2× T4 ligase buffer. 3. T4 ligase. 4. 50 ng/ μ l PGEM ® -T Easy vector (Promega). 5. Sterile deionized water.

1. Escherichia coli XL1-Blue. 2. Luria broth. 3. Glycerol. 4. TSB solution: 10 ml TSB contains 86 μ l of 5 M NaCl, 1 g

polyethylene glycol (mol. wt 8,000), 500 μ l DMSO, 100 μ l of 1 M MgCl 2 , 100 μ l of 1 M MgSO 4 , 1 ml glycerol, 5 ml Luria broth, and sterile deionized water to make up volume to 10 ml.

5. Liquid nitrogen.

2. Materials

2.1. RAPD

2.2. Purifi cation of RAPD Product

2.3. A-Tailing of RAPD Product

2.4. Ligation Reaction

2.5. Preparation of Competent Cells

12510 Development of Sequence Characterized Amplifi ed Region…

1. Competent cells ( E. coli XL1Blue). 2. 5× KCM Buffer: 0.5 M KCl, 0.15 M CaCl 2 , 0.25 M MgCl 2 . 3. Ligation mixture.

1. Luria broth powder (HiMedia). 2. X-gal (2% w/v): Dissolve 20 mg X-gal in 1 ml of dimethyl

formamide. 3. IPTG (20% w/v): Dissolve 0.2 g IPTG in 1 ml sterile deion-

ized water. 4. 10 mg/ml Ampicillin (US biologicals).

NotI from Nocardia otidiscaviarum (recognition sequence: 5 ¢ -GC/GGCCGC-3 ¢ ).

Nuclease-free bovine serum albumin (BSA). 10× Assay buffer B: 100 mM Tris–HCl (pH 8), 1 M NaCl,

100 mM MgCl 2 , 1 mM DTT. SP6 promoter primer GAT TTA GGT GAC ACT ATA. T7 promoter primer TAA TAC GAC TCA CTA TAG GG.

1. 96-Well reaction plate. 2. Big Dye Terminator sequencing buffer (5X). 3. Sequencing Master mix (ABI). 4. Primers. 5. Purifi ed DNA template (insert).

To optimize the PCR conditions for RAPD, set up the PCR using 10–100 ng DNA, 50–300 μ M dNTP, 1–2.5 mM MgCl 2 , 0.5–2 U Taq polymerase, and 30–45 cycles and 35–40°C of annealing tem-perature. The RAPD reaction conditions optimized by us are as given in Table 1 (see Note 1 ).

1. After setting up the reactions, amplifi cation is performed in a thermocycler as described in Table 2 . Operon primers are short oligonucleotides of 10 bp length, hence low annealing tem-peratures are used for amplifi cation.

2. After completion of amplifi cation place the tubes on ice, add 3 μ l gel loading buffer to each tube, mix well by centrifuga-tion for 20 s at 7155 × g and load on a 1.5% agarose gel con-taining ethidium bromide. Electrophorese at 65 V for 1.5 h and visualize the gel on a UV transilluminator (UVP) (see Note 2 ).

2.6. Transformation

2.7. Selection of Clones

2.8. Screening for Recombinants

2.9. Sequencing

3. Methods

3.1. RAPD


Select a species specifi c RAPD fragment for conversion to SCAR (Fig. 1 ).

1. Set up ten PCR reactions for the selected RAPD fragment. 2. Thermal cycling parameters are as described earlier except for

a prolonged fi nal extension step of 15 min at 72°C so as to add an adenine nucleotide overhang at the 3 ¢ end of the insert.

3. Load the PCR products on a 1.2% low melting agarose gel containing ethidium bromide. After electrophoresis in TBE buffer, visualize the gel under a long-wavelength UV lamp and slice out the fragment of interest in a minimal volume of aga-rose using a clean sterile scalpel.

4. Purify the RAPD fragment from the agarose gel using the commercial kit as per the instructions of the manufacturer.

3.2. Purifi cation of RAPD Product

Table 1 RAPD reaction

Reaction components Volume (effective concentration)

DNA template 4–5 μ l (25 ng)

10× Taq polymerase buffer A 2.5 μ l (1×)

1 mM dNTP 2.5 μ l (100 μ M)

3 U/ μ l Taq polymerase 0.3 μ l (0.9 U)

5 pmol/ μ l RAPD Primer 2 μ l (10 pmol)

Sterile deionized water q.s.

Table 2 Thermal cycling parameters for RAPD reaction

Step Temperature (°C) Time (min)

1 Initial denaturation 94 5

2 Denaturation 94 1

3 Annealing 36 1

4 Extension 72 2

Go to step 1 and repeat 45 times

5 Final extension 72 5

Hold at 4°C


5. Dissolve the purifi ed DNA in minimum volume of nuclease-free deionized water (18–20 μ l) and store at −20°C until further use.

6. Ensure purifi cation of only the desired RAPD fragment by loading on an agarose gel (Fig. 2 ).

The purifi ed RAPD product is then A-tailed to increase cloning effi ciency.

1. The A-tailing reaction is set up in a PCR tube as described in Table 3 .

2. Incubate the reaction at 72°C for 20 min in a thermal cycler. 3. After incubation remove the tube from the thermal cycler and

store at 4°C. 4. Precipitate the A-tailed RAPD product with two volumes of

ethanol and store at −20°C for 30 min. 5. Centrifuged at 21913 × g for 10 min at 4°C to obtain a pellet. 6. Allow the pellet to dry so as to remove ethanol traces and dis-

solve in 8 μ l of sterile deionized water.

3.3. A-Tailing of RAPD Product

Fig. 1. RAPD analysis . Lane 1: Low range DNA ruler (0.1, 0.2, 0.3, 0.6, 1, 1.5, 2, 2.5, and 3 kb). Lane 2: Zingiber offi cinale DNA amplifi ed by primer OPC-9. Lane 3: Zingiber zerum-bet DNA amplifi ed by primer OPC-9. Lane 4: Zingiber cassumunar DNA amplifi ed by primer OPC-9. Lane 5: Z. offi cinale DNA amplifi ed by primer OPG-6. Lane 6: Z. zerumbet DNA amplifi ed by primer OPG-6. Lane 7: Z. cassumunar DNA amplifi ed by primer OPG-6. Arrows indicate putative species-specifi c RAPD fragments selected for conversion to SCAR.


7. Load 3 μ l of the purifi ed product on a 1.5% agarose gel and visualize on a UV transilluminator.

The purifi ed A-tailed product is ligated with PGEM ® -T Easy vector.

1. The ligation reaction is set up as shown in Table 4 . 2. Incubate the reaction at 4°C for 12–16 h overnight.

1. Prepare a 3-ml starter culture of E. coli XL1Blue in Luria broth and allow to grow at 37°C for 14–15 h overnight.

2. Add 1% of fresh starter culture to Luria broth, incubate at 37°C shaking at 120 rpm until the O.D.560 = 0.3–0.5 is obtained.

3.4. Ligation Reaction

3.5. Preparation of Competent Cells

Fig. 2. Purifi ed RAPD fragment. M : Low range ruler (0.1, 0.2, 0.3, 0.6, 1, 1.5, 2, 2.5, and 3 kb). Lane 1: Purifi ed 1,368-bp RAPD fragment amplifi ed by primer OPC-9.

Table 3 A-tailing reaction

Reaction components Volume ( m l)

Purifi ed RAPD product 87

100 mM dATP 2

Taq polymerase Buffer 10

Taq polymerase (3 U/ μ l) 1


3. Centrifuge at 2795 × g for 5 min at 10°C to pellet down the cells. Discard the supernatant completely.

4. Add 10% glycerol and resuspended the pellet. 5. Centrifuge at 5,000 rpm for 5 min at 10°C. Discard the super-

natant and add 5 ml TSB to the pellet. Resuspend the pellet and store on ice for 15 min.

6. Prepare aliquots in precooled microcentrifuge tubes. Snap freeze the tubes immediately by dipping in liquid nitrogen and store at −70°C.

1. Thaw the competent cells on ice. 2. Set up the transformation reaction as shown in Table 5 . 3. After mixing the reaction components, store the tube on ice

for 30 min and then at room temperature for 10 min. 4. Add 500 μ l Luria broth to the tube and shake at 37°C for 2 h.

3.6. Transformation

Table 4 Ligation reaction


A-tailed product 2–3

T4 ligase 1

2× T4 ligase buffer 5

50 ng/ μ l PGEM ® -T Easy vector 1

Sterile deionized water q.s.

Table 5 Transformation reaction


Competent cells 50

5× KCM 20

Ligation mixture 10

Sterile DI 20


Luria Agar/ampicillin/IPTG/X-gal plates are prepared as follows:

1. Dissolve 2 g Luria broth powder in 80-ml deionized water and make up the volume to 100 ml.

2. Add 2 g of agar powder and autoclave the medium. Cool the medium to 55°C and add ampicillin to a fi nal concentration of 50 μ g/ml. Mix gently and pour 30–35 ml in a sterile plate.

3. After complete solidifi cation of the medium, spread 40 μ l of X-gal and 7 μ l IPTG stock on the plate. Wrap the plates in aluminum foil and incubate at 37°C for 1–2 h.

4. Innoculate the plates with the transformation reaction mixture and incubate at 37°C for 12–15 h overnight.

5. Identify the recombinant clones by blue/white selection, since the vector is lacZ genetically marked.

6. Pick up and inoculate a single white colony in 4-ml Luria broth containing ampicillin (80 μ g/ml) and incubate at 37°C shak-ing at 120 rpm for 12–15 h overnight. Isolate recombinant plasmids by the miniprep method as described by Sambrook and Russel ( 9 ) .

7. Dissolve the isolated recombinant plasmids in 100 μ l of Tris–EDTA buffer.

8. Remove RNA by RNase A treatment as described previously. Finally, dissolve the DNA in 20 μ l of Tris–EDTA buffer.

9. Prepare glycerol stocks of the recombinant cells by adding 50 μ l of sterile glycerol to 450 μ l of culture, mix by vortexing, and freeze immediately by dipping the tubes in liquid nitro-gen. Store at −70°C.

Screening by restriction endonuclease analysis: Set up restriction digestion reaction (Table 6 ) with enzymes having sites in the mul-tiple cloning site fl anking the insert to check for the presence of desired insert in the purifi ed recombinant plasmids. Incubate the reaction for 4 h at 37°C. Check the size of the restriction digestion product by electrophoresis in 1% agarose gel containing ethidium bromide.

Screening by PCR: Confi rm the presence of desired insert by amplifi cation with vector-specifi c SP6 and T7 promoter primers. The sequences of the primers are given in Table 7 . Set up the PCR reactions in a 25 μ l volume with reaction conditions same as that for RAPD with the exception of annealing temperature of 55°C and 35 thermal cycles. Analyze the amplifi cation products by elec-trophoresis in 1.2% agarose gel containing ethidium bromide.

3.7. Selection of Clones

3.8. Screening for Recombinants


Sequence the insert from three clones for each RAPD fragment on an ABI 373 automated sequencer (Applied Biosystems, Inc) using ABI BigDye Terminator cycle sequencing kit as per manufacturer’s instructions.

1. PCR reaction for sequencing: Set up separate reactions with SP6 and T7 promoter primer as described in Table 8 . The thermal cycling conditions for PCR were as described in Table 9 .

3.9. Sequencing

Table 6 Restriction digestion of recombinant plasmids


Purifi ed Plasmid DNA 5

10× Buffer B 2

2 U/ μ l RE 1

1 mg/ml nuclease-free BSA 2

Table 7 Primer sequences

Primer Sequence 5 ¢ –3 ¢

SP6 promoter primer GAT TTA GGT GAC ACT ATA

T7 promoter primer TAA TAC GAC TCA CTA TAG GG

Table 8 PCR reaction for sequencing


Purifi ed RAPD insert 2

BigDye Terminator Sequencing buffer (5×) 3

Ready Reaction Mix 2

T7/SP6 promoter primer (5 pmol/ μ l) 1

Deionized water 2


2. After completion of PCR amplifi cation, prepare the samples for loading into the automated sequencer.

3. Add 80 μ l of 80% isopropanol to each reaction and incubate in the dark for 15 min.

4. Centrifuge the plate at 1789 × g for 45 min. 5. After centrifugation add isopropanol and decant by placing the

plate in inverted position on a tissue paper. 6. Centrifuge the plate in this position at 14,000 rpm for 1 min. 7. Dry the plate completely and then add 10 μ l formamide, incu-

bate at 95°C for 3–5 min followed by incubation on ice for 3–5 min.

8. Load the plates into the automated sequencer. Align the out-put sequences generated (see Note 3).

9. Analysis of sequence data: Perform homology searches within GenBank’s nr database (All GenBank+RefSeq Nucleotides+EMBL+DDBJ+PDB sequences but no EST, STS, GSS, or phase 0, 1, or 2 HTGS sequences) for all sequenced RAPD fragments using NCBI-BLAST algorithm with the BLASTN 2.2.17 program. Select the unique sequences for use as SCAR markers.

Design the SCAR marker sequences by identifying the original 10-bp sequence of the RAPD primer and adding the next approxi-mately 6–10 bp in the DNA sequence. Alternatively, approximately 20-bp sequences can be picked from the sequence using Primer 3 or FastPCR software program. The species-specifi c SCAR primers are custom synthesized (Integrated DNA Technologies, Inc) (see Note 4 ).

3.10. SCAR Marker Design

Table 9 Thermal cycling parameters for sequencing PCR

Step Temperature Time

1 96°C 10 s

2 50°C 5 s

3 60°C 4 min

4 Go to step 1 and repeat 25 times

5 Hold at 4°C


Test each of the designed SCAR primer pairs (one forward and one reverse) on DNA from the species of interest. A gradient of tem-peratures in the range of T m ± 5°C of the SCAR primers can be used for PCR to determine the optimal annealing temperature. Ensure amplifi cation of the band with exact molecular weight as expected from the sequence data (Fig. 3 ). Once the optimal annealing tem-perature for each set of SCAR primer pairs is determined, test them in closely related species to rule out nonspecifi c amplifi cation.

1. The RAPD reactions are assembled as follows: When amplifying several DNA samples with a large number of primers, a master mix containing water, buffer, magnesium chloride, dNTPs, Taq polymerase, and DNA can be prepared and dispensed in PCR tubes. Primers are added to each tube separately. Alternatively, when many DNA samples are to be amplifi ed using one primer, the DNA samples are fi rst added to the PCR tubes followed by a master mix of all other ingredi-ents including the common primer. All reactions are to be set up on ice.

2. Repeat each PCR at least three times and choose only sharp, reproducible bands that are specifi c for the species of interest for conversion to SCARs.

3. Best sequencing results are obtained approximately 200 bp downstream of the sequencing primers. Internal primers may be designed based on the initial sequence read-outs obtained using SP6 and T7 promoter primers and can be used to sequence the ends of each RAPD fragment. Also for large

3.11. Testing SCAR Primers in Zingiber Species

4. Notes

Fig. 3. Species-specifi c SCAR marker for Zingiber offi cinale Roscoe. M : Low range ruler (0.1, 0.2, 0.3, 0.6, 1, 1.5, 2, 2.5, and 3 kb). Lane 1–13: Z. offi cinale accessions; Lane 14–19: Zingiber zerumbet accessions; Lane 20: Z. cassumunar accession.


fragments that are not sequenced completely using the SP6 and T7 promoter primers, internal nested primers may have to be designed based on the initial sequence read outs.

4. Five to six different primer pairs may be designed for each RAPD fragment that amplify different smaller sized amplicons from the cloned RAPD fragment. Among these the primer pair that amplifi es a species-specifi c and reproducible fragment is chosen. This is especially useful if the RAPD fragment is large and the developed SCAR marker is to be amplifi ed from DNA isolated from a botanical formulation or dried powder where the DNA is likely to be fragmented. In such cases designing primers that amplify smaller amplicons (<500 bp) is useful.

References

1. Paran I, Michelmore RW (1993) Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce. Theor Appl Genet 85: 985–993

2. Chavan P, Warude D, Joshi K, et al (2008). Development of SCAR marker as a comple-mentary tool for identifi cation of Zingiber offi -cinale Roscoe from crude drug and multi-component formulation. Biotech Appl Biochem 50:61–69

3. Lee MY, Doh EJ, Park CH, et al (2006) Development of SCAR marker for discrimina-tion of Artemisia princeps and A. argyi from other Artemisia herbs. Biol Pharm Bull 29:629–633

4. Warude D, Chavan P, Joshi K, et al (2006) Development and application of RAPD-SCAR marker for identifi cation of Phyllanthus emblica LINN. Biol Pharm Bull 29: 2313–2316

5. Kethidi DR, Roden DB, Ladd TR, et al (2003) Development of SCAR markers for the DNA-based detection of the Asian long-horned

beetle, Anoplophora glabripennis (Motschulsky).Arch Insect Biochem Physiol 52:193–204

6. Manguin S, Kengne P, Sonnier L, et al (2002) SCAR markers and multiplex PCR-based iden-tifi cation of isomorphic species in the Anopheles dirus complex in Southeast Asia. Med Vet Entomol 16:46–54

7. Trebaol G, Manceau C, Tirilly Y, et al (2001) Assessment of the genetic diversity among strains of Xanthomonas cynarae by randomly amplifi ed polymorphic DNA analysis and devel-opment of specifi c characterized amplifi ed regions for the rapid identifi cation of X. cynarae . Appl Environ Microbiol 67:3379–3384

8. Yau FC, Wong KL, Wang J, et al. Generation of a sequence characterized amplifi ed region probe for authentication of crocodilian species. J Exp Zool. 2002; 294:382–386

9. Sambrook J, Russell DW(2001) Molecular Cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, New York.

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