Analysis of Primer- Derived, Nonspecific Amplification Products in RAPD-PCR

Transcription

1 Analysis of Primer- Derived, Nonspecific Amplification Products in RAPD-PCR BioTechniques 22: (June 1997) Recently, we applied the randomamplified polymorphic DNA polymerase chain reaction (RAPD-PCR) (17), also known as arbitrarily primed PCR (AP-PCR) (16), procedure to fingerprint genomes of various varieties and wild relatives of sugarcane (Saccharum spp.) (4). Unlike restriction fragment length polymorphism (RFLP) (2), RAPD-PCR is a simpler, faster, less laborious and less expensive procedure. However, it has a few disadvantages. Most polymorphic RAPD markers are dominant, and therefore, heterozygotes are not accurately distinguished (17). RAPD markers may not be reproducible between experiments or laboratories (1,6,10,15). Researchers must standardize RAPD conditions (7,10) or use RAPD markers that are reproducible at least in a set environment (5,15). Another problem that, to our knowledge, has largely been ignored in RAPD-PCR literature is the existence of primer-derived, nonspecific amplification products in negative control reactions containing all the reaction components except for a DNA template. Such primer-derived artifacts were observed by Williams et al. in the original RAPD paper (17) and later by Tingey and del Tufo (13), Tingey et al. (14) and Lanham et al. (9). Nonspecific products were also observed in negative control reactions with different pairs of universal rdna primers (3) and with human immunodeficiency virus (HIV)- specific primers SK145/SK431 (7). These artifacts presumably are absent if a genomic DNA template is included in the reaction (7,11,13,14,17). We show that 19 of 20 Operon A Kit primers and 4 of 20 Operon C Kit primers (Operon Technologies, Alameda, CA, USA) produced artifact DNA bands in negative control reactions. We further demonstrate that some of these primerderived DNA products were amplified even in the presence of a DNA template. RAPD-PCR was performed in a PTC-100 Programmable Thermal Controller (Model 60; MJ Research, Watertown, MA, USA) in a 25-µL volume. Unless mentioned otherwise, the reaction mixture contained 10 mm Tris-HCl, ph 8.3, 10 mm KCl, 1.5 mm MgCl 2, 0.2 mm each of datp, dttp, dgtp and dctp, 0.4 µm primer, 2 U of Stoffel fragment (Perkin-Elmer, Norwalk, CT, USA), 25 ng of genomic DNA and was overlaid with two drops of mineral oil. The following thermal cycling program was used: 95 C for 5 min; 40 cycles at 93 C for 40 s; 40 C for 30 s and 72 C for 1 min; with a final extension at 72 C for 5 min; with a ramp speed of 2.36 s per 1 C from 93 C to 40 C, 1.73 s per 1 C from 40 C to 72 C, and 2.31 s per 1 C from 72 C to 93 C. Amplified PCR products were resolved by electrophoresis at 5.7 V/cm in 0.7% agarose/0.4% Synergel (Diversified Biotech, Boston, MA, USA) binary gels containing ethidium bromide (EtdBr; 0.5 µg/ml) in 0.5 TBE buffer for 1.5 h and were visualized on a UV transilluminator (Spectronics, Westburg, NY, USA). A total of 40 primers from two 10- mer kits (A and C) were tested. All primers have a 60% or 70% G + C content. Without a DNA template, all but OPA-20 of the A kit and four (OPC-02, -05, -08 and -11) of 20 C-kit primers produced polymorphic DNA bands (Figure 1A, OPA-01, -02 and -11 not shown). The sizes of these DNA products ranged from approximately 100 to greater than 2000 bp. The experiment was repeated twice, and polymorphic DNA bands were produced each time. Amplified polymorphic DNA bands were also produced when the annealing temperature was 34 C or when three other brands of DNA polymerase were used: Taq DNA Polymerase from Perkin-Elmer or Promega (Madison, WI, USA) and AmpliTaq DNA Polymerase from Perkin-Elmer (Figure 1B). No amplified DNA products were observed when both DNA template and primer were omitted from the reaction or when the primer was omitted from the RAPD- PCR mixture (Figure 1A). Contrary to our finding, OPA-17 did not produce any DNA product in a DNA-minus control in another RAPD study (8). Reagents prepared in our laboratory Vol. 22, No. 6 (1997)

2 were checked routinely to assure that PCRs were free of contamination. No amplified DNA bands were observed in negative control reactions involving a pair of either specific (12) or universal primers (unpublished data). We suspected that these nonspecific polymorphic DNA products were amplified from DNA contamination of the primer kits or from the polymerase (3). To determine whether the particular batch of primer kits was problematic, we ordered nine of these primers (OPA-01, -02, -04, -10, -11, -16, -17, -18 and -19; Integrated DNA Technologies [IDT], Coralville, IA, USA). Without exception, the nine primers produced polymorphic patterns in the absence of a DNA template. These results indicated that the Operon 10-mer Kits A and C were free of DNA contamination. Another experiment was conducted in which the Stoffel enzyme solution (10 U/µL) was pretreated with DNase I (Life Technologies, Gaithersburg, MD, USA) in a 50-µL volume containing 200 mm Tris-HCl, 20 mm MgCl 2, 500 mm KCl, 5 µl Stoffel enzyme solution and 0.29 U of DNase I. The mixture was incubated at 37 C for 30 min, followed by addition of 1 µl 100 mm EDTA and heating at 95 C for 10 min. Two units of this DNase I-treated Stoffel enzyme were used in negative control reactions with primer OPA-10, -17, -18 or -19. Again, discrete DNA bands were produced. This result and the fact that no artifact DNA bands were observed in negative control reactions involving universal rdna primers (unpublished data) provided strong evidence that there were no residual DNA molecules in polymerase enzyme solutions and that the polymorphic DNA bands shown in Figure 1 were indeed amplified from the primers themselves. We do not know the cause of primerderived amplification. It seems likely that the presence of these products is not due to random annealing. Three lines of evidence support this hypothesis. First, these primer-derived, nonspecific DNA products are fairly large, ranging from approximately 100 to greater than 2000 bp (Figure 1). Second, most of these products were heatresistant and resumed their doublestranded structure when cooled down gradually to room temperature after heating in boiling water for 10 min (data not shown). The third line of evidence was from a Southern blot experiment. Without a DNA template, primer OPA-17 produced two major (band a = 480 bp and band b = 400 bp) and one minor (band c = 670 bp) DNA products (Figure 2A). However, a different, cultivar-specific banding pattern was observed when 25 ng genomic DNA of any of five sugarcane varieties were added to the reaction (lanes HoCP , LCP 82-89, CP , LCP and CP ). No amplified products were observed with DNA of a sixth variety (lane CP ). There were bright DNA bands in lanes HoCP and LCP that co-migrated with band b. There were also faint bands from other sugarcane variety lanes that co-migrated with band a or b. To clarify whether these DNA products shared any homologous sequences, the RAPD-PCR products were resolved in duplicate gels (Figure 2A) and blotted to Zeta-Probe Membranes (Bio-Rad, Hercules, CA, USA). Bands a and b were excised from a separate gel and purified using QIAquick Gel Extraction Kit (Qiagen, Chatsworth, CA, Figure 1. Ten-mer, primer-derived amplification products in 0.7% agarose/0.4% Synergel binary gels stained with EtdBr. (A) Products amplified by Stoffel fragment from RAPD negative control reactions containing either: no DNA, no primer; DNA, no primer; or primer, no DNA. Primer name is shown at the top of each lane. Sizes of PCR markers (Sigma Chemical, St. Louis, MO, USA) are indicated at left. (B) Amplified products from negative control RAPD reactions by four different brands of DNA polymerase: Taq (Promega), Taq (Perkin-Elmer), AmpliTaq and Stoffel. Primers OPA-10 or OPA-17 were ordered from two companies (Operon and IDT). The (-) lanes had no primer BioTechniques Vol. 22, No. 6 (1997)

3 USA) and labeled with [ 32 P]dCTP using a Multiprime DNA Labeling System (Amersham, Arlington Heights, IL, USA). The blot on the left was hybridized with band a and the one on the right with band b according to Pan et al. (Reference 12; Figure 2B). As also shown in Figure 2B, we found that: (i)bands a and b did not cross-hybridize; (ii) the intensely stained DNA bands in lanes HoCP and LCP did not hybridize to band b, and therefore they were true sugarcane DNA-templated amplification products; (iii)bands a and b were amplified, though the signals were weak, when genomic DNAs of sugarcane varieties HoCP , LCP 82-89, CP and LCP were included in RAPD-PCRs; and (iv)both bands were absent when genomic DNA of CP was present. Since the same amount of sugarcane genomic DNA was included in each reaction, we can not explain why adding genomic template DNA prevented primer-derived, nonspecific amplifications in some but not all RAPD-PCRs. In conclusion, this report describes a common, yet rarely reported, phenomenon in RAPD-PCR: the existence of primer-derived, nonspecific amplification products. Three lines of evidence suggest that it is the intrinsic property of some 10-mer primers that leads to the nonspecific amplification. Such non- Figure 2. Analysis of RAPD products by gel electrophoresis and Southern blot hybridization. (A) EtdBr-stained RAPD products in duplicate in a 0.7% agarose/0.4%synergel binary gel. Primed with OPA- 17, the RAPD reactions contain either no DNA or 25 ng each of genomic DNA of sugarcane varieties HoCP , LCP 82-89, CP , LCP , CP and CP Sizes of PCR markers are indicated at left. (B) Autoradiograph of Southern blot hybridization of the gel in A using the 32 P-labeled band a (left half) and band b (right half) (see text). Both bands were cloned into the PCR 2.1 Vector (Invitrogen, San Diego, CA, USA) and sequenced. No matching sequence was found in the database using the GCG program. Vol. 22, No. 6 (1997)

4 specific amplification events are likely to persist, though at an apparently reduced rate, even in the presence of a DNA template. It is recommended that proper controls (e.g., a primer-only negative control) be included in every RAPD-PCR experiment and that only intensely stained RAPD products that do not co-migrate with a nonspecific band from the negative control be used in further analysis. If one wishes to clone an intensely stained RAPD marker that co-migrates with a primer-derived, nonspecific product (for example, band a or b in Figure 2), one should expect to obtain two kinds of clones: those that contain the desired RAPD marker and those that contain a primer-derived, nonspecific amplification product. Disclaimer: Product names and trademarks are mentioned to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA does not imply the approval of the product to the exclusion of others that may also be suitable. REFERENCES 1.Annamalai, P., H. Ishii, D. Lalithakumari and R. Revathi Polymerase chain reaction and its applications in fungal disease diagnosis. J. Plant Dis. Protect. 102: Botstein, D., R.L. White, M. Skolnick and R.W. Davis Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am. J. Hum. Genet. 32: Böttger, E.C Frequent contamination of Taq polymerase with DNA. Clin. Chem. 36: Burner, D.M., Y.-B. Pan and R.D. Webster. Genetic diversity of North American and Old World Saccharum assessed by RAPD analysis. Genet. Res. Crop Evol. Vol. 44 (In press). 5.Fritsch, P., M.A. Hanson, C.D. Spore, P.E. Pack and L.H. Rieseberg Constancy of RAPD primer amplification strength among distantly related taxa of flowering plants. Plant Mol. Biol. Rep. 11: Hadidi, A., L. Levy and E.V. Podleckis Polymerase chain reaction technology in plant pathology, p In R.P. Singh and U.S. Singh (Eds.), Molecular Methods in Plant Pathology. CRC/Lewis Publishers, Boca Raton. 7.Higuchi, R., C. Fockler, G. Dollinger and R. Watson Kinetic PCR analysis: realtime monitoring of DNA amplification reactions. Bio/Technology 11: Joshi, C.P. and H.T. Nguyen Application of the random amplified polymorphic DNA technique for the detection of polymorphism among wild and cultivated tetraploid wheat. Genome 36: Lanham, P.G., S. Fennell, J.P. Moss and W. Powell Detection of polymorphic loci in Arachisgermplasm using random amplified polymorphic DNAs. Genome 35: Li, W.L Standardization of RAPD (random amplified polymorphic DNA) using Opti- Taq system. biosolutions Newsl. 1: Operon Technologies. 10-mer Kits Product Information. Alameda, CA. 12.Pan, Y.-B., M.P. Grisham and D.M. Burner A PCR protocol for the detection of Xanthomonas albilineans, the causal agent of sugarcane leaf scald disease. Plant Dis. 81: BioTechniques Vol. 22, No. 6 (1997)

5 13.Tingey, S.V. and J.P. del Tufo Genetic analysis with random amplified polymorphic DNA markers. Plant Physiol. 101: Tingey, S.V., J.A. Rafalski and J.G.K. Williams.Genetic analysis with RAPD markers, p Proceedings of the Symposium: Applications of RAPD Technology to Plant Breeding, 1992 November 1; Minneapolis, MN. 15.Weeden, N.F., G.M. Timmerman, M. Hemmat, B.E. Kneen and M.A. Lodhi. Inheritance and reliability of RAPD markers, p Proceedings of the Symposium: Applications of RAPD Technology to Plant Breeding, 1992 November 1; Minneapolis, MN. 16.Welsh, J. and M. McClelland Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res. 18: Williams, J.G.K., A.R. Kubelik, K.J. Livak, J.A. Rafalski and S.V. Tingey DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acid Res. 18: UT, USA) sequenced the clones and conducted database search. Address correspondence to Yong-Bao Pan, USDA/ARS, Southern Regional Research Center, Sugarcane Research Unit, P.O. Box 470, Houma, LA 70361, USA. Internet: usda.gov Received 8 July 1996; accepted 18 November Y.-B. Pan, D.M. Burner, K.C. Ehrlich 1, M.P. Grisham and Q. Wei USDA/ARS, SRRC SRU, Houma 1 FFS, New Orleans, LA, USA This research was partially funded by a grant from the American Sugar Cane League. Dr. R. Wang (USDA/ARS, Logan, Vol. 22, No. 6 (1997) BioTechniques 1077