PCR 龙平, 崔占虎, 李虔全, 徐建平, 张春红, 周立社 * *, 李旻辉 ( 包头医学院, 内蒙古包头 014060) DNA 30 28 DNA trnl-trnf PCR PCR 136 bp 323 bp 10% PCR PCR Astragalus membranaceus Fisch. Bge. var. mongho- licus Bge. Hsiao A. membranaceus Fisch. Bge 1 1 DNA 2-3 DNA 9 ITS 10 Hedysarum polybotrys Hand. -Mazz. PCR PCR 11-13 PCR 4-6 4 7 1 2013-03-07 2012BA128B02 201107009 2011X1002 * Tel 010 64014411-2956 E-mail li _ minhui @ yahoo. cn * Tel 13604726512 E-mail zhoulishe@ sohu. com 4-5 8 2 CTAB 1 TAE Promega Fluka DNA Taq Takara 2 000 bp DNA Markar Takara PCR Eppendorf 5332 DYY-12 Eppendorf 5810R 2581
Syngene GBOXHR 18 5 5 Retsch MM400 KQ- 28 2 500DE Eppendorf 58 1 1 Table 1 The sources of Astragali Radix and Hedysarum Radix No. 1 AMM2011_NMGTY Astragalus membranaceus var. mongholicus 2 2 AMM2011_NMGGY A. membranaceus var. mongholicus 2 3 AMM2011_NMGCF A. membranaceus var. mongholicus 2 4 AMM2011_NMG A. membranaceus var. mongholicus 10 5 AM2011_SXHY A. membranaceus 5 6 AM2011_GSLX A. membranaceus 5 7 AM2011_NMGCF A. membranaceus 2 8 AM2012_AH A. membranaceus 2 9 HP2011_GSWD Hedysarum polybotrys 2 10 HP2011_GSTC H. polybotrys 21 11 HP2011_NMGTY H. polybotrys 5 2 1 2 2. 1 trnl-trnf 14 BioEdit Primer Premier 5 2 1 PCR 2 Fig. 1 The specific primer design schematic diagram of Astragali Radix and Hedysari 136 323 bp Radix 2 Table 2 PCR Primer and PCR reaction conditions 5'-3' PCR trnl GGAAATCGGTAGACGCTACG 94 5 min 35 94 30 s 56 30s 72 1 min 30 s 72 7 min trnf ATTTGAACTGGTGACACGAG AM-F GTTCAAATCAGTCACTCCGA AM-R CGACGGACTTTCCTCTTAAT 95 5 min 32 95 30 s 52 30 s 72 1 min 40 s 72 7 min HP-F AATCAGTCACTCCACCCT HP-R TAAGTAAGTTTCAGTAGTAGTAATG 2. 2 DNA PCR DNA PCR 100 mg CTAB PCR PCR DNA DNA 10 mg 95 5 min 95 30 s L - 1 trnl trnf PCR 52 30 s 72 1 min 40 s DNA 20 μl 35 72 7 min 3 μl 10 buffer 2 μl dntp1. 6 μl 1% 100 ~ 150 V 0. 25 μl 5 pmol L - 1 EXTaq 0. 2 μl 5 U 15 min μl - 1 DNA 0. 5 μl 14. 7 μl10 ng PCR 2582
PCR dntp PCR 3. 1 trnl- 2. 3 PCR trnf PCR PCR Clastul W 2 PCR 20 μl PCR 10 buffer 2 μl dntp1. 6 μl PCR 0. 25 μl 5 pmol L - 1 EXTaq 0. 2 μl 5 U μl - 1 DNA 0. 5 μl 14. 7 μl 1 95 5 min 95 PCR 1 30 s 52 30 s 72 1 min 40 s 72 7 min 35 4 3. 2 PCR 2. 4 PCR 52 DNA PCR PCR 10 buffer 2 μl dntp1. 6 μl AM-F AM-R 0. 3 μl 5 pmol L - 1 HP-F HP- R 0. 2 μl EXTaq 0. 2 μl 5 U μl - 1 DNA 0. 5 μl 14. 7 μl PCR dntp 0. 8 μl 2. PCR 5 μl 6 loading AM-F /AM-R 1. 2 buffer EB 1. 5% 0. 3 μl HP-F /HP-R SYNGENE 0. 8 0. 2 μl PCR 1% 5% 10% 20% 40% dntp PCR 60% 80% DNA PCR dntp 3 PCR PCR dntp 4 32 35 dntp 1 /2 3 3 Table 3 PCR Optimization of PCR amplification AM1 AM2 AM3 AM4 AM5 AM6 HP1 HP2 HP3 32 33 34 + + + + + + + + + 35 + + + + + + + + + T m / 50 52 + + + + + + + + + 54 + + + 56 / pmol 0. 2 /0. 3 + + + AM-F /R /HP-F /R 0. 25 /0. 25 + + + 0. 3 /0. 2 + + + + + + + + + dntp / nmol 0. 8 + + + + + + + + + 1. 6 + + + + + + + + + 2. 0 AM1. AM2. AM3. AM4. AM5. AM6. HP1. HP2. HP3. +.. 2583
3. 3 PCR trnl-trnf 1 000 bp PCR 136 bp PCR 323 bp 2 PCR 1% 5% 250 100 bp 1 ~ 7. 1% 10% 8. M. DNA 2 000 1 000 750 500 5% 10% 20% 40% 60% 80% 20% 40% 60% 80% Fig. 3 Agarose gel electrophoresis of specific PCR amplification 3 products amplified from different percentage mixture of Astragali 10% Radix and Hedysari Radix 3 - PCR A. trnl-trnf B. C. M. DNA 4 PCR PCR PCR 10% PCR 1. S. 2010 283. 2. J. 2012 37 21 3203. 3. J. 2 000 1 000 750 500 250 100 bp 1. 2. 3. 4. 5. 2010 29 844. 4. J. 2002 12 6 26. 6. 7. 8. 5. J. 2007 25 9. 10. 5 1053. 11. 12. 6. 2 PCR J. 2011 20 5 435. Fig. 2 Agarose gel electrophoresis of specific PCR amplification 7. products amplified from Astragali Radix and Hedysari Radix J. 2005 11 1 46. 2584
8. J. 2001 13 3 37. 9. DNA 13 Wang H Kim M K Kim Y J et al. Molecular authentication of J. 2006 8 3 33. 10. ITS J. 2012 37 24 3773. 11. PCR J. 2012 37 24 3752. 12 Wang H Kim M K Kwon W S et al. Molecular authentication of Panax ginseng and ginseng products using robust SNP markers in ribosomal external transcribed spacer region J. J Pharm Biomed Anal 2011 55 5 972. the Oriental medicines Pericarpium citri reticulatae and Citri unshius pericarpium using SNP markers J. Gene 2012 494 1 92. 14 Kress W J Wurdack K J Zimmer E A et al. Use of DNA barcodes to identify flowering plants J. Proc Natl Acad Sci USA 2005 102 8369. Study on identification of Astragali Radix and Hedysari Radix by PCR amplification of specific alleles LONG Ping CUI Zhan-hu LI Qian-quan Xu Jian-ping ZHANG Chun-hong ZHOU Li-she * LI Min-hui * Baotou Medical College Inner Mongolia Baotou 014060 China Abstract To explore the new method of discriminating Astragali Radix and Hedysari Radix by using PCR amplification of specific alleles 30 samples of the different Astragali Radix materials and 28 samples of Hedysari Radix were collected. The total DNA of all samples were extracted trnl-trnf sequence from Astragali Radix and Hedysari Radix was amplified by PCR and sequenced unidirectionally. These sequences were aligned by using Clustul W. Primer was designed and the PCR reaction systems including annealing temperature dntp etc were optimized. All samples were amplified by PCR with specific primer DNA from Astragali Radix would be amplified 136 bp whereas PCR products from all of Hedysari Radix were 323 bp. This method can detect 10% of intentional Hedysari Radix DNA into Astragali Radix. PCR amplification of alleles can be used to identify Astragali Radix and Hedysari Radix successfully and is an efficient molecular marker for authentication of Astragali Radix and Hedysari Radix. Key words Astragali Radix PCR amplification of specific alleles molecular identification doi 10. 4268 /cjcmm20131607 2585