2 Southern 183 method was not only easier to operate but also cleaner in background comparing X-ray imaging optimized. The improved digoxigenin-labele

Transcription

1 Journal of Plant Genetic Resources Southern / / / DNA DNA Southern DNA 10μg DNA 80μl 8 ~ 12h CSPD X Southern PCR Southern Optimizing and Confirming of Digoxigenin Based Southern Blotting for Wheat Genome Analysis LIU Lu 1 2 NIU Yan-yan 2 3 LEI Hao 4 LIN Zhi-shan 2 ZHAO Xiang-yu 1 YE Xing-guo 2 ZHANG Xian- sheng 1 1 State Key Laboratory Shandong Agricultural University Development of molecular biology laboratory Shandong Taian National Key Facility for Crop Gene Resources and Genetic Improvement / Key Laboratory of Crop Genetics and Breeding of ministry of Agriculture /Institute of Crop Sciences Chinese Academy of Agricultural Sciences Beijing University of Science and Technology of China Hefei Beijing City University Beijing Abstract The Southern blot analysis based on isotope labeling technique has some shortages such as facility limitation environmental pollution and poor stability. Therefore establishing a safe stable and efficient Southern hybridization protocol is very important to detect the integration of exogenous genes in transgenic wheat. In this study by using the DNAs from wild-type wheat and transgenic wheat asmaterial the DIG-labeled Southern blot analysis was improved through modifying several key steps including probe preparation and purification usage of DNA samples amount digestion system vacuum transfer conditions stringent hybridization conditions and immunoassay detect. The results showed that the purifications of DNA template and probe-labeled could significantly improve the efficiency of the probe labeling when random prime labeling method was used. Desirable digestion result could be a- chieved when 10μg DNA samples with high quality was enzymed in 80μl reaction system for 8 ~ 12 hours. In the vacuum transfer step alkaline liquid was better than neutral liquid to obtain clean transmembrane effect. Reagent purity the temperature and the rotation speed in hybridization inside the hybridization oven impacted on the hybridization results greatly. Application of chemiluminescence detection system combining with improved coating CSPD ZX liu1985lu@ 126. com linzs@ caas. net. cn

2 2 Southern 183 method was not only easier to operate but also cleaner in background comparing X-ray imaging optimized. The improved digoxigenin-labeled Southern blot technique showed good sensitivity and high ratio of signal to noise for wheat DNA analysis can be used stably and widely in ordinary laboratory overcoming restrictions of isotope labeling to experimental conditions equipments and physical status of researcher. Key words Wheat Digoxigenin Random primer PCR DNA labeling Immunological detection Southern blotting Southern PCR 827 bp pta 1 Sma1PtaF 5' TCC CCC CGG GGC TCC TTC CTC ATG GCC T 3' Xba1PtaR 5' GCT CTA GAG CTC ATT TCG CTT ATT TCA CC 3' DNA Southern 221bp μl 10min Southern 4μl Dig-High prime vial 1 20μl h Southern 2μl 0. 2M EDTA -20 PCR PCR DIG Probe Synthesis Kit 17000Mb 5 Cat. No Roche 50μl 140 MgCl 2 PCR buffer 10 conc GC vial 3 5μl PCR DIG Probe Synthesismix vial 3 2 5μl forward PCR primer 10 conc 5μl re- PCR primer10 conc 5μlEnzymemix vial Southern verse μl DNA DNA1 ~ 50ng Pinellia ternate pta DNA10 ~ 100pg PCR DIG Probe Synthesismix vial 2 dntp stock solu- 4 PCR Southern tion vial 424bp 827bp High pure PCR Product pta T 3 Purification kit REF Version μl AMRESCO DIG High Prime DNA Labeling and Detection Starter kit Ⅱ REF ~ 500ng DNA Roche 20μl 100μl g 30 ~ 60s 500μl 0. 45μm g 60s 200μl g 60s 30μl g 60s - 20 Roche pta PRTL μm

3 DNA CTAB ~ 42 1h 60 ~ 65 CTAB 10ml Robbins scientific mmol /LTris-Cl ph mmol /L EDTA 1. 5 mol / L NaCl 2% m /v PVP40 2% m /v CTAB 2% v /v β- 20ml 4g 30 ~ 60min rpm 20min 10000r /min 10min 65 ~ min DNA DNA70% 100% 9 DNA 1ml ddh 2 O 20μl RNaseA 10mg /ml 37 1h RNA / DNA A260 /A280 A260 /A230 OD Buffer ddh 2 O Thermo SCIENTI- Detection Buffer CSPD FICMANODROP 2000 Spectrophotometer μl 10μg DNA 10pg 9 80U Promega 8μl 10 buffer 1μl BSA 10mg /ml ddh 2 O 80μl 8 ~ 12 h 5μl DNA DNA 5μl ~ 60min loading buffer DNA 1ng DNA 0. 7% 1 X Kodark 40 V 12 h 60V 7 ~ 1 h 8h 1h 15s 1min s 1min MHCL 15 ~ 20min UVP 37 30min 1h mol /L 30min 1h NaOH 1mol /L NaCl Hybond2N+ Amersham Pharmacia biotech ddh 2 O 10min 1. 8 MAX Robbins scientific MODEL 1000 JAPAN 30min 2h 100μl PCR 94 5min 5min 5 ~ 7ml ~ min Washing Buffer Blocking Solution Anti-Dig-AP Washing CSPD CSPD CSPD CSPD 5min 37 Chemi DOC-IT Imaging system min % SDS 0. 2mol /L NaOH 5min SSC Bio-Rad 9 5min 4

4 2 Southern Southern 10pg 3pg 1pg 0. 3pg 0. 1pg 0. 03pg 0. 01pg 0 1B 0. 1pg A B 1 6ml 12 2μl pta 1 Table 1 Fig. 1 1 Detection of probe labeling efficiency by random primed labeling method A 1 221bp DNA 3 827bp DNA B 6 221bp DNA 7 827bp DNA A probe before purification upper row one spot the 221bp digoxigenin-labeled probe middle row three spots pointed with horizontal arrow digoxigenin-labeled DNA. lower row six spots digoxin labeled 827bp DNA B probes after purification upper row digoxin labeled 221bp probe middle row pointed with horizontal arrow digoxin labeled control DNA lower row seven spots digoxin labeled 827bp DNA 20μl The newly synthesized DNA amount in 20 μl labeling reaction system under optimal conditions DNA ng Amount of purified template DNA ng Amount of newly synthesized 1h DNA 20h PCR optimized condition 4 dig-labeled probes 5 non-dig-labeled positive control 2 PCR DNA 2μl PCR 0. 5μl /ml μl /ml PCR 3 A 3B Fig. 2 2 PCR Detection of the probe labeling efficiency by PCR labeling method A PCR 827bp B PCR 424bp 4 5 A Using asymmetric PCR -labeled 827bp probe 1 Non-diglabeled positive control 2 downstream primers diluted 10 times 3 downstream primers diluted 20 times B PCR-labeled 424bp probe in

5 Southern PCR DNA PCR DNA DNA μg DNA 5 DNA 3 DNA 10μg Fig. 3 Comparison of hybridized results before and after probe purification A before purification B after purification 2. 2 DNA Southern DNA Southern DNA DNA DNA 4A 4B OD OD260 / ~ DNA Southern OD260 / Fig. 5 Southern hybridization patterns with DNA different amount of DNA EcoRⅠ HindIII pta 6 2 Fig. 4 Table 2 4 DNA Electrophoresis patterns of wheat genomic DNA A some samples with degradation B DNA samples with better integrity DNA OD DNA concentration and OD values of some wheat samples ng /μl Material number Concentration 0D260 /0D280 0D260 /0D230 11Fa Fa CL CL CL CL CL CL CL Kn Fig. 6 6 Results of enzyme digestion A incomplete digestion B completed digestion 10μg DNA 80μl 8 ~ 12h > 200μl DNA DNA DNA DNA

6 2 Southern 187 DNA 2. 5 Southern 2. 3 Southern 2 10 SSC pta Southern 0. 4mol /L NaOH 1mol /L NaCl 7 10ug DNA EcoRⅠ HindⅢ 9A B PCR Fig. 7 7 Comparison of two methods of membrane transferring A fully transfer alkaline condition B and C incompleted transfer neutral condition 2. 4 Southern cm 2 10ml 1 ~ 2h 5 ~ 7ml 4r /min DNA CSPD 8B D pare 12marker lkb ladder 13 positive sample 14 negative sample A 3 C X DNA DNA SDS 8 CSPD A C B D A B X C D Southern Fig. 8 Southern hybridization patterns with CSPD uniform A C or uneven B D coating with different imaging methods A B X-ray C D chemi luminescence 9 Fig. 9 Hybridization patterns of different probe labeling methods 1 marker λhindiii EcoRⅠ HindIII Marker 1kb ladder marker 1kb ladder marker λhindiii digestion with EcoRⅠ 6 plasmid not digested digestion with HindIII 2 and 7 5 and 10 the same sample pair 11 marker 1kb ladder 3 and 8 4 and 9 the same negative sample CTAB CTAB DNA 2 DNA 1 24h 2 2h Southern

7 h 24h 2 3 ~ 4 10ug DNA DNA 300 ~ 1500bp 800bp ~ 0. 03pgDNA 10μg DNA 10pg 9 DNA PCR ~ 0. 03pgDNA DNA 10pg DNA 10ng PCR B or anthocyanin synthetic genesj mol /L Tris-Cl ph7. 5 1mol /L J 6 Cui C J Song F Tan Y NaCl 2 SSC 2 DNA L. J DNA 9 DNA DNA 0. 25M 15 ~ 20min 10 DNA 7 J DNA J. Tm = % G 13McCabe M S Power J B + C /l l = length of hybrid in base pairs Topt = Tm - 20 to 25 G + C % = 61% l = 827 Tm = /827 Topt 49 ~ 54 C 50 C 1 Southern E M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. JMol Biol Southern J CL UGPase J Saika H Sakamoto W Maekawa M et al. Highly efficient visual selection of transgenic rice plants using green fluorescent protein. Plant Biotechnol DNA et al. Stable chloroplast transformation of immature scutella and inflorescences in wheat Triticum aestivum. Acta Biochim Biophys Sin Shanghai CTAB DNA J PCR DNA J J D W. 7 M Southern Kruchen B Rueger B. The DIG system nonradioactive and highly sensitive detection of nucleic acids J. Biochemica de Laat A M M et al. Detection of single-copy genes in DNA from transgenic plants by nonradioactive Southern blot analysis J. Mol Biotechnol Hêltke H J Ankenbauer W Muhlegger K et al. The digoxigenin DIG system for nonradioactive labeling and detection of nucleic acids an overview J. Cell Mol Biol Southern J