선택한 단어 수는 입니다.
최소 단어 이상 선택하여야 합니다.
최소 단어 이상 선택하여야 합니다.
선택한 단어 수는 30입니다.
최대 10 단어까지만 선택 가능합니다.
최대 10 단어까지만 선택 가능합니다.
문제 정의
- 1). Therefore, we are confident in our results and used these polymorphic RAPD amplicons in further investigations.
- This study was designed to produce a rapid genetic test for identification of ‘Hashuoh’ and ‘Baekshuoh’ and their potential contaminants.
제안 방법
- In the development of SCAR markers to differentiate between C. wilfordii and C. auriculatum, we prepared SCAR primer sets from nucleotide sequences of 12 RAPD polymorphic amplicons, and primer specificity was verified using multiple samples for each species. As shown in Fig.
- multiflorum. In this study, we investigated reliable SCAR markers for differentiating P. multiflorum from C. wilfordii and C. auriculatum, and developed two RAPD-derived SCAR markers from polymorphic amplicons that were specific for P. multiflorum (Figs. 1 and 2c). Furthermore, we established a multiplex-PCR method for rapid molecular discrimination of these three species, as previously described (Henegariu et al.
- To control for individual variations, we examined two or three samples per species. In this study, we used 16 Operon 10-mer random primers to analyze polymorphic patterns, and identified 45 distinct species-specific DNA fragments from 12 of the primers. These polymorphic fragments, designated 1–7 DNA fragments, varied in size, ranging from 200 to 2,100 bp (Fig.
- Sixteen Operon 10-mer RAPD primers, RAPD Kit A (OPA 13–20) and Kit C (OPC 13–20), were used to screen the eight samples of the three medicinal plant species to determine the potential of clear polymorphisms and reproducibility (Operon Technologies, Germany).
- Species-specific amplification and multiplex-PCR were carried out using single species-specific primer pairs, and six combinations of the three forward and three reverse primer pairs. Reactions were carried out as previously reported (Choo et al.
- The PCRs were carried out in 30-ll reaction mixtures containing 10 mM Tris–HCl (pH 9.0), 2.5 mM MgCl2, 200 lM of each dNTP, 10 mM (NH4)2SO4, 0.5 U Taq DNA polymerase (Solgent, Korea), 30 pmole each primer, and 10–20 ng template DNA.
- To develop the SCAR markers for C. wilfrodii and C. auriculatum, five sets of primers were prepared based on the corresponding nucleotide sequences of four speciesspecific RAPD amplicons, HA13-5, HC15-3, HC15-4, and HC20-4.
- 1; and data not shown). To identify SCAR markers, we analyzed the nucleotide sequences of 23 species-specific RAPD amplicons, consisting of 6 DNA fragments for C. wilfordii, 5 for C. auriculatum, and 12 for P. multiflorum (Fig. 1). From the resulting nucleotide sequences, two unique RAPD amplicons were registered in the NCBI GenBank dbGSS, and used to develop RAPDderived SCAR markers (Table 2; Fig.
- 1993). To overcome these causal factors while developing SCAR markers, we tested multiple samples for each species, and used only those primers that performed similarly with all of the samples from each species (Fig. 1). Therefore, we are confident in our results and used these polymorphic RAPD amplicons in further investigations.
대상 데이터
- AY548208). All plant materials were given accession numbers and preserved in the Herbarium of the Korea Institute of Oriental Medicine (Table 1).
- The nucleotide sequences of the DNA amplicons were determined from both strands by dideoxynucleotide chain termination using an automatic DNA sequencer (ABI 3730; Applied Biosystems, USA). Six amplicons were used to develop SCAR markers and were registered with the NCBI GenBank dbGSS (accession nos. HC14-2; FI857165, HC18-2; FI857166, HC15-3; FI857167, HC20-4; FI857168, HA13-5; FI857169, HC15-4; FI857170, http://www.ncbi. nlm.nih.gov/dbGSS).
- multiflorum, collected from native habitats and Chung-buk Agricultural Research and Extension Service Center, were used in this study (Table 1). The identification of species was performed by The Classification and Identification Committee of the Korea Institute of Oriental Medicine, composed of nine experts in the fields of plant taxonomy, botany, pharmacognosy, and herbology. The identification was further confirmed by the comparison of ITS sequences against those in the NCBI GenBank: P.
- Three samples each of C. wilfordii and C. auriculatum, and two samples of P. multiflorum, collected from native habitats and Chung-buk Agricultural Research and Extension Service Center, were used in this study (Table 1). The identification of species was performed by The Classification and Identification Committee of the Korea Institute of Oriental Medicine, composed of nine experts in the fields of plant taxonomy, botany, pharmacognosy, and herbology.
- auriculatum, which can be used to differentiate Polygoni Multiflori Radix and Cynanchi Wilfordii Radix. Three sets of primers were designed based on the corresponding sequences of two RAPD amplicons, HC14-2 and HC18-2 (Table 2; Fig. 2). As a result, we obtained two SCAR markers specific to P.
이론/모형
- The primers were prepared from the sequences of corresponding species-specific RAPD amplicons, excluding the 10-mer RAPD primer sequences. The melting temperature, GC contents and secondary structures of each primer were verified using CyberGene AB primer design tools (http://www.cybergene.se, Stockholm, Sweden).
성능/효과
- auriculatum have highly homologous genomic DNA sequences, and that the selection of primers is very important for developing specific SCAR markers. Even though we could not obtain a SCAR marker for C. wilfordii, the merged results of these SCAR markers are enough to differentiate between C. auriculatum and C. wilfordii.
- In this study, we analyzed RAPD patterns to develop several reliable SCAR markers that can be used for discrimination of C. wilfordii, C. auriculatum, and P. multiflorum. Furthermore, we established two rapid molecular authentication markers by combining the primers from each SCAR marker that can be used simultaneously in multiplex-PCR.
- The identification of species was performed by The Classification and Identification Committee of the Korea Institute of Oriental Medicine, composed of nine experts in the fields of plant taxonomy, botany, pharmacognosy, and herbology. The identification was further confirmed by the comparison of ITS sequences against those in the NCBI GenBank: P. multiflorum (accession no. EF016293), C. wilfordii (accession no. AY548207), and C. auriculatum (accession no. AY548208). All plant materials were given accession numbers and preserved in the Herbarium of the Korea Institute of Oriental Medicine (Table 1).
- 2c, d). These results indicate that theHC15-3 and HC20-4 SCAR markers are not enough to differentiate between C. wilfordii and C. auriculatum. However, these markers are suitable for distinguishing Cynanchi Wilfordii Radix and Polygoni Multiflori Radix.
- 2c, d). These results suggest that C. wilfordii and C. auriculatum have highly homologous genomic DNA sequences, and that the selection of primers is very important for developing specific SCAR markers. Even though we could not obtain a SCAR marker for C.
- multiflorum. This result came up to our expectation because P. multiflorum is a member of the Polygonaceae family, and C. wilfordii and C. auriculatum belong to the Asclepiadaceae family. Even though some primers did not generate specific PCR products, we obtained polymorphic amplicons to distinguish between the three species.
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