Genomic Fingerprinting by Application of rep-PCR

Abstract

Several families of short repetitive DNA sequences, widely distributed in the genome, have been identified in bacteria (1). They have an intercistronic location, are not translated, and their function is unclear, although they may be involved in transcription termination, mRNA stability or chromosomal organization. Repetitive extragenic palindrome (REP) elements (2), also known as palindromic units (PU) (3), and enterobacterial repetitive intergeinc consensus (ERIC) sequences (4) are the best characterized of these elements and were mitially identified in Salmonella typhimurium and Eschenchla colt, respectively. The REP consensus sequence was formulated through DNA sequence comparisons of intercistronic regions of different operons (3,5) and comprises a 38 nucleotide palindromic sequence that can form a stable stem-loop structure with a 5-bp variable loop m the central region (2). There may be 50—1000 copies of the REP sequence in the genome, frequently present in complex clusters (2), with each cluster comprising as many as 10 copies (6). REP sequences have been located between genes within an operon or at the end of an operon, in different orientations and in tandem arrays, and in operons distributed throughout the genome (2,3). The REP sequence has been identified in intergenic regions within operons from different bacterial species (7,8). REP like sequences have been shown to exist throughout the eubacterial kingdom, although the consensus sequences may differ among different bacteria 9 11).

1 Introduction

Several families of short repetitive DNA sequences, widely distributed in the genome, have been identified in bacteria (1). They have an intercistronic location, are not translated, and their function is unclear, although they may be involved in transcription termination, mRNA stability or chromosomal organization. Repetitive extragenic palindrome (REP) elements (2), also known as palindromic units (PU) (3), and enterobacterial repetitive intergeinc consensus (ERIC) sequences (4) are the best characterized of these elements and were mitially identified in Salmonella typhimurium and Eschenchla colt, respectively. The REP consensus sequence was formulated through DNA sequence comparisons of intercistronic regions of different operons (3,5) and comprises a 38 nucleotide palindromic sequence that can form a stable stem-loop structure with a 5-bp variable loop m the central region (2). There may be 50—1000 copies of the REP sequence in the genome, frequently present in complex clusters (2), with each cluster comprising as many as 10 copies (6). REP sequences have been located between genes within an operon or at the end of an operon, in different orientations and in tandem arrays, and in operons distributed throughout the genome (2,3). The REP sequence has been identified in intergenic regions within operons from different bacterial species (7,8). REP like sequences have been shown to exist throughout the eubacterial kingdom, although the consensus sequences may differ among different bacteria 9 11). The precise function of these elements has not been determined, but several functions have been postulated, including roles m gene regulation and retroregulation (2,12,13). These functions may be a consequence of a stem loop structure m a specific chromosomal locatton (1). Like their REP counterparts, the larger 126-bp ERIC elements have an extragenic location and contain a highly conserved central inverted repeat (4,14). However, despite similarities in structure, the ERIC elements have no sequence homology to the REP consensus (4).

The term rep-PCR refers to a general method that utilizes oligonucleotide primers matching interspersed repetitive DNA sequences to yield DNA fingerprints of individual bacterial isolates (11). The use of degenerate, repetitive sequence oligonucleotides as primers in PCR-based amplification was described by Versalovic et al. (11). It was hypothesized that repetitive DNA sequences were dispersed in the E.coli chromosome in different orientations and separated by various distances. As such, these dispersed repetitive sequences could be used as primer binding sites ( Fig. 1 ). As the rep primers are targeted at endogenous interspersed repetitive sequences in the genomes of numerous bacterial species, the rep-PCR method enables a rational approach to primer design, such that the same primer set can generate genomic fingerprints for bacteria that contain these sequences. Outwardly directed primer sets, based on the REP or ERIC consensus sequences and designed to match inverted repeat sequences, can be used to generate bands clearly resolvable by agarose gel electrophoresis after PCR amplification using template genomic DNA from species that contain these sequences (11) The bands represent amplrficatlon of DNA between adjacent repetitive elements( Fig. 1 ) within the approximate 5-kb limitation of the Taq poiymerase extension. Band patterns provide DNA fingerprints that discriminate bacterial strains (11,15–18). Differences in band sizes represent polymorphisms m the distances between repetitive sequence elements in different genomes. The method has been used to type bacterial pathogens, investigate infectious disease outbreaks, and identify clonal or sporadic outbreak isolates (11,18–23). Time-consummg genomic DNA extraction, purification, and quantitation may be avoided by the use of cell lysates as the template for PCR amplification (19,24). Although this increases the attractlveness of the method for investlgations that require high-throughput strain typmg, it is not suitable for all strains.

Fig. 1.

The rep-PCR method. Outwardly directed primers are designed to be complementary to the rep consensus sequences. For REP-PCR this is a 38-bp sequence, however, for ERIC-PCR the primers are based on the central inverted repeat of the 126 bp consensus. Under the conditions of PCR, the primers amplify between adjacent repetitive elements within the limits of Taq polymerase extension. The PCR products are electrophoresed and the resultant bands generate a fingerprint. Differences in band sizes represent polymorphisms in the distance between adjacent repetitive elements.

This chapter describes a PCR-based method for typing bacterial strains using outwardly directed primer sets based on REP and ERIC consensus sequences. Use of cell lysates is preferable for rapidity and simplicity. However, DNA template preparation methods have been included for both Gram-positive (25) and Gram-negative (26) bacteria

2 Materials

2.1 Preparation of Crude Cell Lysates for PCR

• 1. Sterile distilled water

• 2 200 mM NaC1 wash buffer

• 3 TE buffer. 10 mM Tns-HCl, 1 mM EDTA, pH 8 0

2.2 Preparation of DNA Template for PCR

2.2.1 Gram-Positwe Bacteria

• 1 Lysozyme: 10 mg/mL in sterile distilled water (freshly prepared).

• 2 Lysis buffer 5 M guanidine lsothlocyanate, 0 1 M EDTA, pH 7 0, store at room temperature.

• 3 Ammonium acetate 7 5 M,

• 4 Chloroform.lsoamyl alcohol (24. 1). store at 4°C

• 5 Isopropanol: store at 4°C

• 6 70% ethanol: store at 4°C

• 7. TE buffer. 10 mMTns-HCl, 1 mMEDTA, pH 8 0 (autoclave at 120°C for 20 mm and store at room temperature)

2.2.2 Gram-Negative Bacteria

• 1. TE buffer: 10 rnMTris-HCl, 1 rnMEDTA, pH 8 0 (autoclave at 12O°C for 20 min and store at room temperature).

• 2. Sodium dodecyl sulfate (SDS): 10% (w/v), store at room temperature.

• 3. Proteinase K: 20 mg/mL, store in small single-use aliquots at −2O°C

• 4. Sodium chloride 5 M

• 5 Hexadecyl trimethyl ammonium bromide (CTAB)/NaCl solution 10% (w/v) CTABm07MNaCl

• 6 Chloroform lsoamyl alcohol (24 1) store at 4°C

• 7 Phenol:chloroform lsoamyl alcohol (25 24. 1). store at 4°C

• 8 Isopropanol store at 4°C

• 9 70%ethanol store at 4°C

2.3 Oligonucleotide Primers

The REP primers are based on the 38-bp REP consensus (11); Fig. 1 ) (see Note 1) and the ERIC primers are based on the central Inverted repeat (11) of the published ERIC consensus sequence (4). These primers may be obtained commercially (see Notes 2 and 3).

• 1 REPlR-I: 5’-IIIICGICGICATCIGGC-3’ (I denotes the nucleotlde mosme)

• 2 REP2-I. 5’-ICGICTTATCIGGCCTAC-3’.

• 3 ERIC1R S-ATGTAAGCTCCTGGGGATTCAC-3’

• 4 ERIC2: 5’-AAGTAAGTGACTGGGGTGAGCG-3’

2.4 Buffers and Stock Solutions for PCR

All components of PCR reactions should be prepared in sterile deionized water dedicated for PCR This can be obtained commercially (e.g., Sigma Chemical Co., St. Louis, MO).

• 1 PCR buffer (1 0X stock). 200 mMTns-HCl, pH 8 4, 500 mM KCl Commercially available from Taq polymerase manufacturer (see Note 4)

• 2 MgCl₂ 50 mM stock diluted for use at a concentration of 2 mM

• 3 Deionized distilled water

• 4 dNTPs a mixture of all four dNTPs at 10 mM each m sterile, deionized water Dilute from commercially supplied, buffered stock solutions (100 mM) Dispense into 20–50 μL ahquots (sufficient for 40–100 reactions) and store frozen Mmlmlze freeze thawing(see Notes 5 and 6 )

• 5 Primers. 100 μM stock solutions of each primer m sterile deionized water Store frozen m 100 μL ahquots (see Note 4 )

• 6 Taq DNA polymerase. 5 U/μL, usually obtained commercially (see Note 6)

• 7. Mineral oil. commercially available.

• 8 Agarose: use a Molecular Biology quality grade suitable for the preparation of high concentration gels on which to separate fragment range of 100 bp to 5 5 kb (e g, Ultrapure from Life Technologies Ltd [Glbco-BRL, Galthersburg, MD])

• 9 TBE electrophoresis buffer (1OX concentrate)’ 0 9 MTrls base, 0 9Mboric acid, 0.02M EDTA

• 10 DNA dilution buffer (TE) 10 mM Tns-HCl, 1 mM EDTA, pH 8 0

• 11 Sample loading buffer (5X concentrate) 60% sucrose, 0 025% (w/v) bromopheno1 blue m TE buffer

• 12 1-kb and/or 100-bp lambda DNA markers available commercially

• 13 Ethtdium bromide (EB) 5 mg/mL m water. Caution: EB is a powerful mutagen and potential carcinogen Always wear gloves when handling the solid or liquids containing the chemical, and dispose of appropriately EB solutions can be purchased from commercial sources; this represents the safest way of preparing the stock solution

3 Methods

3.1 Preparation of Cell Lysates for PCR

• 1 To prepare a crude cell lysate from a pure overnight broth culture, transfer 200 μL of the culture to a clean microcentrifuge tube and pellet by centrifugation at 1O,OOOgmaxfor 4 min.

• 2 Discard the supernatant, taking care not to disturb the pellet, and resuspend m 500 μL of 200 mMNaC1 to wash the cells

• 3 Pellet the cells by centrifugation, ensuring that as much as possible of the 200 mM NaCl is removed and resuspend in 100–200 μL of water or TE buffer to an approximate cell density equal to an A600 value of 0 5(see Note 7)

• 4. Heat to 95°C on a heating block or block of thermal cycler for 10 mm to encourage cell drsruption

• 5. Centrifuge briefly (∼15 s) before using 5 μL as template for PCR

3.2 Preparation of DNA Template for PCR

3.2.1 Gram-Positive Bacteria

• 1. Harvest bacterial cells into a 1 5-mL microcentrifuge tube and pellet by centrifugation at 10,000g_max for 3 min The recommended pellet volume is 50–100 μL Volumes of reagents, however, may be proportionately scaled up or down as required

• 2 Resuspend the cells by vortexing m 100 μL of lysozyme solutton and incubate at room temperature for 30 min

• 3 Lyse the cells by addition of 200 μL of 5 M guanidine isothrocyanate lysis buffer and mix by inversion. After the solution has cleared add 150,μL of ammomum acetate and mix gently

• 4. Add 450 μL of chloroform isoamyl alcohol and emulsify the lysate by vigorous shaking or vortexing.

• 5. Separate the phases by centrifugation at l0,000g_max for 2 min and transfer the upper aqueous phase to a fresh tube

• 6 Add 0.54 vol of isopropanol to the aqueous phase and mix by inversion to precipitate the high molecular weight DNA

• 7. Immediately separate the precipitated DNA, which forms a dense stringy precipitate within a few seconds of mixing, from the liquid phase with a pipet tip (see Note 8).

• 8 Wash the DNA twice in 1 mL of ethanol and dry under vacuum

• 9. Dissolve the dried pellet m TE buffer (see Note 9).

3.2.2 Gram-Negative Bacteria

• 1 Harvest bacterial cells by centrifugation as for Gram-positive bacteria.

• 2 Resuspend the bacterial pellet m 567 μL of TE buffer by repeated pipeting

• 3 Add 30 μL of 10% SDS and 3 μL of 20 mg/mL proteinase K to give a final concentration of 100 μg/mL protemase K in 0 5% SDS Mix thoroughly and incubate for 1 h at 37°C (see Note 10)

• 4 Add 100 μL of 6 M NaCl and mix well (see Note 11).

• 5 Add 80 μL of CTAB/NaCl solution. Mix and Incubate at 65°C for 10 min.

• 6 Add 800 μL of chloroform. lsoamyl alcohol Mix and centrifuge 5 min at 10,000g_max to remove the CTAB-protein! polysaccharide complexes, which appear as a white interface after centrifugation.

• 7 Remove the supernatant to a fresh microcentrifuge tube, taking care to avoid disrupting the Interface.

• 8 Add 750 μL of phenol chlorofonn lsoamyl alcohol, mix well, and separate phases by centrifugation

• 9. Transfer the upper aqueous phase to a fresh microcentrifuge tube and repeat phenol:chloroform isoamyl alcohol extractions until the interface is clear

• 10. Add 0.6 vol (∼450 μL) of lsopropanol to precipitate the nucleic acids and invert the tube until a stringy clump becomes visible (see Note 8)

• 11 Transfer the pellet to a fresh tube containing 70% ethanol Wash the pellet twice in ethanol to remove residual CTAB

• 12. Dry under vacuum and resuspend pellet m an appropriate volume of TE buffer (see Note 9)

3.3 Polymerase Chain Reaction

3.3.1 Preparation of Reaction Mixes

• 1 Use 25 μL reaction volumes m small microfuge tubes of a size and shape compatible with the thermal cycler m use

• 2. Prepare a master mix for the PCR reaction. The volumes of each component are dependent on the number of mdividual reactions to be prepared (see Note 12) The following components are added in order, to a separate tube (volumes given are per reaction)14 75 μL of sterile distilled water, 2 5 μL of 10X PCR buffer, 1.25 μL of 50 mM M4MgCl₂ 0.5 pL of dNTP mix, 0 5 μL of each primer (REPlR-I and REP2-I or ERICIR, and ERIC2). Replace cap and store on ice

• 3 Aliquot each template in a total volume of 5 μL to a fresh labeled microfuge tube (see Note 13).

• 4. Add Taq polymerase to a concentration of l–2 U per reaction to the reactlon mix Mix well by inversion.

• 5 Immediately aliquot the reaction mix to each tube of template taking great care to avoid cross-contamination. The simplest way is to use a separate pipet tip for each transfer (see Note 5).

• 6 Overlay the reactlon mixture with mineral 0i1 and pulse to ensure no reaction mix is left on the walls of the tube

3.3.2 Thermal Cycling

• 1 When using REP primers, program an Initial denaturing step of 95°C for 7 min, followed by 30 cycles of 90°C for 30 s; 42°C for 1 min, and 65°C for 8 min with a final cycle of 90°C for 30 s, 40°C for 1 min and 65°C for 16 min. Program the machine to maintain the samples at 4°C until the PCR products can be analyzed by gel electrophoresis. When using ERIC primers, the annealing temperature is increased to 52°C but all times and the temperatures of denaturing and extension steps are identical to those used for the REP primers.

• 2. For reactions mvolving the use of cell lysates as template, it may be necessary to add EDTA to a concentration of 50 mM as soon as possible after completion of thermal cycling (see Note 14).

3.3.3 Analysis of PCR Products

• 1 Prepare a 1.5% agarose gel containing 1X TBE electrophorests buffer

• 2. Add 5 μL of loading dye to the finished PCR. There is no need to remove the mineral oil; add the dye by pipeting it onto the inside wall of the tube and centrifuge briefly to mix it into the aqueous phase.

• 3. Load an appropriate volume of the sample into the well of the gel (e.g., 10 μL for a 2 min well). Load DNA markers to at least one well, flanking the sample wells

• 4. Run the gel in 1X TBE at 10 V/cm until the bromophenol blue dye has migrated approx 415 the length of the gel

• 5. Stain the gel in 600 ng/mL EB in water. Caution Wear gloves whenever handling the gel

• 6 Photograph the gel using a UV transilluminator to visualize the DNA bands

3.4 Evaluation of Results

The band pattern following agarose gel electrophoresis of products of rep-PCR amplification is specific for the test bacterium and primer combination utilized (see Note 15). A typical gel photograph is shown in Fig. 2 . Amplification product sizes resulting from rep-PCR generally range from < 100 bp-5 kp (the latter is the limit of Taq polymerase extension). Fingerprints should be visually inspected. Profiles should be considered highly similar when all visible bands from the test isolates have the same apparent migratron distance. Variations in intensity or shape of bands should not be used to justify designation of a separate pattern type, parttcularly when cell lysates are used as templates, as this observation often results from dirfferences in the amount of DNA in the reaction available for amplification. The profile obtained for a sample constitutes a fingerprint for the test micro-organism, and can be compared with those obtained for other bacteria under the same reaction conditions.

Fig. 2.

REP-PCR profiles of Listeria monocytogenes, including six epidemiologitally related pairs, generated using primers REPlR-I/REP2-I. Lane 1, 1 kb h ladder molecular weight marker; lane 2, serovar 1/2a; lane 3, serovar 1/2a; lane 4, serovar 1/2c; lane 5, serovar 1/2c; lane 6, serovar 1/2a; lane 7, serovar 4bX; lane 8, serovar U2a; lane 9, serovar 1/2a; lane 10, serovar 1/2a; lane 11, serovar 1/2b; lane 12, serovar 4bX; lane 13, serovar 1/2a; lane 14, serovar4b; lane 15, serovar 1/2b; lane 16, serovar4b; lane 17, serovar 1/2b; lane 18, serovar 1/2a; lane 19, serovar 1/2a; lane 20, serovar 1/2a.

Failure to generate a rep-PCR profile may be caused by several factors. These Include

• 1. Sequences homologous to the REP or ERIC consensus sequences may not be present in the target organism,

• 2. Incomplete cell lysis (see Note 7) or concentration of target DNA is too low;

• 3. Inhibition of PCR by a factor(s) introduced with the template (see Note 5); and

• 4. A component(s) in the reaction is limiting (see Notes 16 and 17 ).

3.4.1 Computer-Assisted Analysis of Fingerprints to Evaluate Similarity Among Amplification Profiles

The patterns may be recorded by a scanner or densitometer linked to a computer. The digitized, optimized density values are transfered to a computer and are stored as densitograms of patterns or as a bitmap image file. Using a software package such as Gel Compar (Applied Maths, Belgium) or Dendron (Solltech, Oakdale, CA) the tracks are further processed. This involves normalization of tracks, generation of databases, and grouping or identification of tracks by quantification of their resemblance. However, this is only feasible if optimization of the PCR reactions, ensuring reproducibillty between runs, has been undertaken.

4 Notes

• 1. Primer pairs REPIR-I plus REP2-I (11) are based on the 38-bp REP consensus and contain the nucleotide mosine at ambiguous positions Inosine contains the base hypoxanthine and can form Watson-Crick base pairs with any of the four nucleotide bases (A,C,G, and T) Weaker than AaT base pairs, however, they form the least destabilizing, but most discriminatory pairs

• 2. Primers should be COP-cartridge or even HPLC purified. This appears to enhance reproducibilty between batches of primers (author’s unpublished observation)

• 3. A fluorophore-enhanced rep-PCR (FERP) technique has been recently developed (27) that combines DNA amplificatton and fluoresence detection for the analysis of bacterial pathogens If this system is desired, a covalently linked fluoresent dye should be added to the 5′ end of each of the primers described in Subheading 2 . This is useful for the laser detection of specific PCR products when the amplicons are resolved by polyacrylamide gel electrophoresis (PAGE)

• 4 Commercially available Taq polymerases (e.g, Life Technologies [Gtbco BRL, Gaithersburg, MD] or Ampli Taq [Perkm Elmer, Norwalk, CT]) are supplied with 10X PCR buffer This may contain 500 mM MgC1₂, or this component may be supplied separately, allowing for easy optimization Some manufacturers supply a 1% solution of the detergent W-1, which should be added at a final concentration of 0 05% (v/v), and is thought to improve the thermostability of the enzyme The experience of the author is that this component does not enhance the appearance of the amplification products. It is advisable to use cloned Tuq polymerase, licensed for PCR, as negative control lanes were clear of contamination, in contrast to correspondmg control reactions generated using uncloned Tag polymerase

• 5. Contammation of PCR must be minimized. Reaction products must not contaminate stock reaction components or they will act as templates m any reactions set up using the contaminated component solutions yielding misleading results This can be minimized using clean, sterile pipet tips for each reaction component, establishing separate areas for handling of reaction components and products, using designated pipets or positive-displacement pipets and tips to prevent aerosols, using commercially obtained sterile distilled water, and by using aliquotted batches of dNTPs, primers, and PCR reaction buffers to appropriate volumes

• 6. For long term storage of stocks of primers, dNTPs, or DNA templates (includmg crude lysates), it is advisable to store concentrated solutions in small aliquots at -20°C. Solutions of DNA are susceptible to degradation by nucleases at higher temperatures.

• 7. Differences in amplification profiles obtained using cell lysates compared with purified DNA templates have been observed by the author. These mainly consist of greater numbers and intensities of small (C200 bp) amplicons when generated from cell lysates, whereas larger fragments tend to predominate when DNA is used as a template Inhibition of the PCR may result from the use of a cell lysate containing too many cells It is easier to standardize cell concentration when liquid cultures are used Although an OD reading provides a good approximation of cell concentration, it is not appropriate for a large number of samples In this case, an OD readmg could be obtained for a single sample, and the remainder diluted to match The volumes of cells used can be adjusted accordingly For this reason it is advisable to optimize conditions of cell lysis, taking mto account the buffers and detergents (if any) used, cell concentration, and whether centifugation of the lysate is required

• 8 If no stringy DNA precipitate forms with the addition if isopropanol usmg either of the DNA extraction methods outlined, it is likely that cell lysls was incomplete, or that the DNA has sheared into relatively low molecular weight pieces DNA should not be collected by centrifugation because this results in the formatlon of a compacted pellet, which is diffcult to resuspend. In addition, a cloudy precipitate, probably consisting mainly of RNA and protein, forms In some preparations and this is collected with the DNA if centrifugation is used

• 9 The nucleic acid concentration should be spectrophotometrlcally determined A 1 mg/mL solution of double-stranded DNA has an absorbance of 20 OD U at 260 nm (1 e., an OD_Z6, of 1 is equivalent to 50 μg/mL of dsDNA) Alternatively, a small aliquot (0.2–l μL) may be run on 0 6% agarose gel with a sample of known concentration run in adjacent well to give an estimate of the DNA concentration in samples. The DNA should be stored frozen m allquots at−20°C at 5s 100 ng/μL and diluted m TE before use

• 10 As the detergent lyses the bacterial cell wall, the solution should become viscous If cell lysis is poor, It may be necessary to predigest the bacterial cell wall with lysozyme

• 11 To ensure that nucleic acids are retained in solution, thereby allowing CTAB to form polysaccharide complexes, it is necessary to add concentrated salt solution The protein/polysaccharide CTAB complexes are removed by precipitation

• 12 For multiple PCR reactions encompassing a number of different bacterial lysates and/or DNA templates, a master PCR reaction mix should be prepared, allowmg for control reactions and pipettmg inaccuracies

• 13 When the volume of cell lysate or DNA to be added to each PCR reaction is greater than 1 μL, the volume of water added to the reactlon mix should be adjusted accordingly.

• 14 Some bacteria contains nucleases that will degrade DNA products amplified from cell lysates; this is especially noticable for Salmonella spp. For this reason, it is advisable to add EDTA to a final concentration of 50 mMto the reaction lmmedlately after completion of the PCR

• 15 Optimization of reaction conditions should be undertaken for different templates. The factors most likely to cause variation of rep-PCR profiles include magnesium ion concentration and annealing temperature. Many workers use the annealing temperature (40°C) reported by Versalovic et al (11) for use with these primers, although temperatures as high as 45°C have been reported to generate reproducible patterns with a sensible number of amplicons for the purpose of typing (24) Magnesium ion concentration has a significant effect on the complexity of the profiles, as has the addition of dimethylsulfoxide (24) PCR-amplified fragments of a size close to the limit of Taq polymerase extension (4—5 kb) are not always reproducible, and should not be considered when interstrain comparisons are made

• 16. In general, more dlscriminatory DNA fingerprints are provided by rep-PCR when the REP primers (REPlR-I/REP2-I). rather than the ERIC primers (ERICIIU ERIC2) are employed The level of discrimination afforded using the REP primers has been observed to be at least as high as ribotyping (24) (author’s unpublished observation), but the method has the advantage of speed and simplicity

• 17. Some strains may be observed to generate profiles consisting of smears, with or without few visible bands These strains may contains large amounts of substances that can inhibit PCR, such as lipopolysaccharides, or may release nucleases that degrade DNA released from lysed cells. In this case, it is preferable to use a purified DNA template However, it should be noted that small differences occur between profiles generated from DNA, and cell lysates and analyses should be performed with the same template preparation method