化学发光Southern blot法检测HBV体外复制及其应用于药物对HBV的抑制分析

目的：建立化学发光Southern blot检测细胞内HBV DNA的方法,同时检测3种不同靶点抗乙肝药物的体外作用。方法：用地高辛标记HBV探针,优化杂交条件,检测来自HepG2及HepG2.2.15 HBV DNA复制中间体;利用建立的化学发光Southern blot检测HBV DNA的方法检测经拉米夫定、Bay41-4109、α-Galcer以不同药物浓度处理的HepG2.2.15HBV DNA复制中间体的水平。结果：（1）标记的HBV探针的检测灵敏度为0.1pg,杂交系统的检测灵敏度为1pg,可检测到HepG2.2.15细胞内的HBV DNA特异性信号;（2）以该法检测胞内HBV DNA可见3种药物都有明显的抑制作用,其半数有效量（IC50）分别为1.53μmol/L、0.41μmol/L、0.01μmol/L。结论：胞质HBV DNA的水平能准确地反映不同靶点抗HBV药物的抗病毒效果,建议在观察药物特别是中药抗病毒研究中采用。     

转基因甘蔗植株Southern杂交体系的优化北大核心CSCD

对甘蔗Southern杂交体系的优化,旨为转基因甘蔗Southern杂交鉴定分析提供参考。以转基因甘蔗为材料,就探针不同标记方法的比较、甘蔗基因组DNA的提取、基因组DNA酶切量、酶切时间及杂交过程等方面,对地高辛标记的Southern杂交技术进行了优化研究。结果表明,改良的CTAB法提取的甘蔗DNA能满足后期实验的要求;PCR法标记探针的效率较随机引物法标记探针的效率高,更适合用于Southern杂交;40μg的DNA在400μL酶切体系中,酶切10 h可获得良好的酶切效果;杂交温度40℃,杂交18 h,可获得清晰的杂交条带。     

转基因甘蔗植株Southern杂交体系的优化

对甘蔗Southern杂交体系的优化,旨为转基因甘蔗Southern杂交鉴定分析提供参考。以转基因甘蔗为材料,就探针不同标记方法的比较、甘蔗基因组DNA的提取、基因组DNA酶切量、酶切时间及杂交过程等方面,对地高辛标记的Southern杂交技术进行了优化研究。结果表明,改良的CTAB法提取的甘蔗DNA能满足后期实验的要求;PCR法标记探针的效率较随机引物法标记探针的效率高,更适合用于Southern杂交;40μg的DNA在400μL酶切体系中,酶切10 h可获得良好的酶切效果;杂交温度40℃,杂交18 h,可获得清晰的杂交条带。     

橡胶树转基因植株Southern杂交体系的优化

以地高辛标记的Southern杂交技术已广泛应用于多个物种中。选择橡胶树为材料,就基因组DNA的提取、探针标记方法的选择以及具体的杂交操作过程等方面对该体系进行了优化。结果表明,至少40μg高质量的DNA在300μL的大酶切体系中,酶切12 h可获得良好效果;PCR法标记的探针杂交条带清晰、背景浅,其效率明显强于随机引物法标记的探针。本研究优化的体系信号强、背景浅和灵敏度高,为橡胶树Southern杂交鉴定分析提供参考。     

液相杂交快速检测特异DNA

检测特异DNA片段的方法中,传统Southern blot技术由于其高度可重复性及能够显示条带大小的特性,一直是DNA检测的“黄金标准”.但是杂交时间长,步骤复杂,放射性污染等问题亟待解决.为了简化Southern blot,研究使用了一种液相杂交快速检测DNA的方法,即使用异硫氰酸荧光素(FITC)标记的dUTP掺入探针后,在溶液中与待检测DNA样本42℃下杂交,然后琼脂糖凝胶电泳检测荧光杂交信号.利用质粒为模板,优化了探针制作、杂交液组成、杂交时间和温度等参数.在FITC-dUTP∶ dTTP比例为1∶3、模板质粒浓度为50μg、1×杂交缓冲液(25 mmol/LTris,10mmol/L EDTA,8mmol/L Nacl,PH =8.0)中95℃变性5～9 min和42℃杂交3h的实验条件下,可检出1.2μg的质粒,探针灵敏度为7.3 ng/μl.这种方法不需要转膜,曝光,大大节约了时间,简化了操作,荧光检测也为该方法同时检测多色样本提供了可能,可广泛应用于核酸检测.     

基于地高辛标记对小麦进行Southern杂交分析主要影响因素的优化和验证

以转基因小麦和野生型小麦DNA为材料,对利用地高辛标记对小麦基因组DNA进行Southern杂交分析的影响因素进行了优化研究,包括探针制备与纯化、样品DNA量、酶切体系、真空转印条件、杂交条件、免疫检测方法等。结果表明,对随机引物标记的模板和标记后的探针进行纯化可明显提高探针的标记效率,10μg高质量的DNA样品在80μl的体系中,酶切8～12h可获得良好的效果;真空转膜时使用碱性液比中性液获得的转膜效果更干净;试剂纯度、杂交温度及杂交炉转速等均对杂交效果产生重要影响;配合改进的CSPD涂布方法,使用化学发光检测系统比单纯使用X光片显像更易操作,背景更干净;本研究所优化的地高辛标记的小麦Southern杂交分析显示出较高的灵敏度和信噪比,结果稳定,可克服同位素标记对实验条件、设备及实验人员身体状况等限制,在普通实验室推广应用。     

化学发光法检测体外HBV复制

目的:建立化学发光法检测体外HBV复制水平的方法,研究其灵敏度和稳定性.方法:用HBV DNA重组质粒pCH9转染到人肝癌细胞株HepG2和Huh7中,5d后收集细胞并抽提其HBV复制中问体DNA,转印后以地高辛标记HBV DNA为探针进行杂交,用化学发光法检测杂交结果,同时进行探针灵敏度的检测.结果:转染后HepG2和Huh7细胞提取HBV DNA中检测出较强的复制中间体的信号,分别为松散环状DNA(rcDNA),双链线性DNA(dslDNA),单链DNA(ssDNA),探针检测的灵敏度可达到lpg,接近同位素法检测的灵敏度.整个实验重复3次获得同样结果.结论:成功建立了稳定的化学发光法检测体外HBV复制水平的方法.     

地高辛标记探针Southern印迹杂交技术要点及改进

Southern印迹杂交技术是检测特定DNA片段的常规方法之一,其操作程序繁琐,对基因组DNA的提取、纯化、酶切等均有较高要求,要获得理想杂交结果需要反复摸索.总结地高辛标记探针Southern印迹杂交的技术要点,并结合具体操作提出改良方案.     

新课程中一笔带过的3个技术——Southern印迹、Northern印迹和Western印迹

“基因工程”是人教版高中生物学新教材中的重要内容,也是高考的热点和难点之一。该内容涉及到分子生物学中常用的3种印迹技术：Southern blot、Northern blot和Western blot。结合高考试题对这3种技术的具体操作过程和用途进行了介绍,以期师生对该部分内容有更加深入的理解。     

利用柱花草为受体表达口蹄疫病毒外壳蛋白VP1的研究

将口蹄疫病毒外壳蛋白VP1基因克隆到植物表达载体pBI121,并转化到根癌农杆菌(Agrobacteriumtumefaciens)菌株LBA4404中,采用叶盘转化法转化柱花草(Stylosanthesspp.)栽培品种热研二号柱花草(S.guianensiscv.ReyanⅡ),获得了转基因植株,经PCR、PCR-Southern blot和Southern blot分析表明VP1基因已整合到转基因柱花草植株的核基因组中。经RT-PCR、Northern blot分析表明VP1基因已在转基因柱花草中获得转录。     

Development of Prevotella intermedia-specific PCR primers based on the nucleotide sequences of a DNA probe Pig27

The aim of this study was to develop Prevotella intermedia-specific PCR primers based on the P. intermedia-specific DNA probe. The P. intermedia-specific DNA probe was screened by inverted dot blot hybridization and confirmed by Southern blot hybridization. The nucleotide sequences of the species-specific DNA probes were determined using a chain termination method. Southern blot analysis showed that the DNA probe, Pig27, detected only the genomic DNA of P. intermedia strains. PCR showed that the PCR primers, Pin-F1/Pin-R1, had species-specificity for P. intermedia. The detection limits of the PCR primer sets were 0.4 pg of the purified genomic DNA of P. intermedia ATCC 49046. These results suggest that the PCR primers, Pin-F1/Pin-R1, could be useful in the detection of P. intermedia as well as in the development of a PCR kit in epidemiological studies related to periodontal diseases.     

Direct molecular analysis of the fragile X syndrome in a sample of Egyptian and German patients using non-radioactive PCR and Southern blot followed by chemiluminescent detection

Molecular genetic analysis of individuals from 6 Egyptian and 33 German families with fragile X syndrome and 240 further patients with mental retardation was performed applying a completely non-radioactive system. The aim of our study was the development of a non-radioactive detection method and its implementation in molecular diagnosis of the fragile X syndrome. Furthermore, we wanted to assess differences in the mutation sizes between Egyptian and German patients and between Egyptian and German carriers of a premutation. Using non-radioactive polymerase chain reaction (PCR), agarose gel electrophoresis and blotting of the PCR products, followed by hybridisation with a digoxigenin-labelled oligonucleotide probe (CGG)₅ and chemiluminescent detection, we identified the fragile X full mutation (amplification of a CGG repeat in the FMR-1 gene ranging from several hundred to several thousand repeat units) in all patients. We observed no differences in the length of the CGG repeat between the Egyptian and German patients and carriers, respectively. However, in one prenatal diagnosis, we detected only one normal sized allele in a female fetus using the PCR-agarose assay, whereas Southern blot analysis with the digoxigenin labelled probe StB 12.3 revealed presence of a full mutation. Our newly established nonradioactive genomic blotting method is based on the conventional radioactive Southern blot analysis. Labelling of the probe StB 12.3 with digoxigenin via PCR allowed the detection of normal, premutated and fully mutated alleles. For exact sizing of small premutated or large normal alleles, we separated digoxigenin labelled PCR products through denaturing poly-acrylamide gelelectrophoresis (PAGE) and transfered them to a nylon membrane using a gel dryer. The blotted PCR-fragments can easily be detected with alkaline phosphate-labelled anti-digoxigenin antibody. The number of trinucleotide repeat units can be determined by scoring the detected bands against a digoxigenated M13 sequencing ladder. Our newly developed digoxigenin/chemiluminescence approach using PCR and Southern blot analysis provides reliable results for routine detection of full fragile X mutations and premutations.     

Blame it on Southern,but it's a western blot

Edwin M. Southern is a professor emeritus at the University of Oxford. He is perhaps best known for development of the “Southern blot” (Dr. Southern was at the University of Edinburgh when he wrote his landmark paper). The Southern blot provided a scientific breakthrough by allowing scientists to detect a particular DNA sequence without first purifying it from the rest of the genome; the basic method involves the transfer of the DNA to a membrane, followed by detection with a specific probe. Although few people perform Southern blots as originally carried out by Southern, due in part to the more recent technique of the polymerase chain reaction, the basic concept continues to play an important role in molecular biology.     

Sequential chemiluminescent detection of target DNAs without stripping and reprobing.

We present a simple method for sequential chemiluminescent detections of two different DNA loci on a single Southern blot. First, an enzyme-linked DNA probe for a unique sequence is detected with a horse-radish peroxidase (HRP) substrate followed by the detection of another enzyme-linked DNA probe for a different unique sequence with an alkaline phosphatase (AP) substrate that simultaneously inhibits the chemiluminescence generated by HRP. Such sequential detection steps eliminate the need to strip and reprobe blots and can be performed with no intervening steps.     

Molecular evidence of<Emphasis Type="Italic">Bartonella</Emphasis> DNA in ixodid ticks in Czechia

We report the development of a novel method for detection of Bartonella DNA in ixodid ticks. The assay is based on a specific amplification of a part of 16S rRNA gene and 16S-23S rRNA intergenic spacer region of Bartonella sp. by nested PCR and Southern blot hybridization with specific DNA probe; the method is highly sensitive and specific. The screening of 327 unfed ticks collected in different urban and suburban areas of Czechia in 2003-2005 revealed the presence of Bartonella DNA in four Ixodes ricinus individuals (1.2%), two males, one female and one nymph.     

Using non-radioactive probes on plants: a few examples

Due to costs in using and disposing of radiochemicals and to health considerations, we have been developing applications which include non-isotopic detection of DNA and proteins using chemiluminescence. Our major interests are in the detection of viral nucleic acids and in the analysis of transgenic plants. Generally, probes were labelled with digoxigenin, either by the random priming method or by PCR, and then detected with CSPD or CDP-Star. We routinely use a tissue blotting protocol for diagnosing TYLCV, a plant virus becoming a pest in the Mediterranean region. Test results were comparable with those using the same radiolabelled probe. When total nucleic acids are extracted from the plant samples and used in dot-blot or Southern blot assays, viral DNAs are promptly detected by chemiluminescence. In transgenic plants, chemiluminescence was used to detect the transgene on genomic Southern blots, the transgenic mRNAs on Northern blots, and the transgenic protein on Western blots. In Southern and Northern blots, the quality of the results obtained was usually satisfactory, but not as good as with a radiolabelled probe, the main problem being the signal-to-background ratio. Our goal is now to improve the quality of results in demanding applications such as genomic Southern blots, by reducing the background on membranes. © 1998 John Wiley & Sons, Ltd.     

Genomic Southern analysis with alkaline-phosphatase-conjugated oligonucleotide probes and the chemiluminescent substrate AMPPD

We have used a chemiluminescent detection method to improve both the sensitivity and the speed of detection of human genes with oligonucleotide probes. A direct chemiluminescent substrate (AMPPD) was used in combination with an alkaline-phosphatase-labeled oligonucleotide probe to detect the human tissue of plasminogen activator gene by Southern blot analysis. X-ray exposures obtained after 4 h were comparable to those obtained after 7 days with a 32P-labeled oligomer. After 16 h, the signal was 12 times greater than the 32P signal. The detection of the single-copy tissue plasminogen activator gene in 0.25 micrograms of human genomic DNA (76,000 molecules) was achieved. The improved sensitivity obtained by chemiluminescent detection should increase the usefulness of oligonucleotide probes in the direct Southern analysis of human genetic disorders.     

Rapid and simple detection of PCR product DNA: a comparison between Southern hybridization and fluorescence polarization analysis

The essential aim of this study was to compare two different methods, Southern hybridization and fluorescence polarization (FP) assay. They both detect specific hybridization and were examined using common asymmetric PCR products and probes. FP assay clearly showed the hybridization of probe DNAs with the asymmetric PCR products of their target genes. Southern blot patterns presented excellent consistency with the results of FP assay. In both methods, two types of Shiga toxin (vero toxin) genes held in enterohaemorrhagic Escherichia coli (EHEC) were used as target genes. For detection of the two genes, stx1 and stx2, two respective DNA probes were synthesized. Both in FP assay and in Southern hybridization, the probe for stx1 hybridized only with the product of stx1 and vice versa. The results of the DNA detection using different methods were completely in agreement. Moreover, FP assay makes it possible to detect the hybridization rapidly. In our high NaCl concentration condition, hybridization between the probes and the asymmetric PCR products could be monitored within about 15min.     

Southern杂交方法用于近交系小鼠H-2基因检测

目的建立一种简便、快捷、准确的检测方法用于近交系小鼠的遗传检测。方法根据近交系小鼠的H-2基因序列设计相应的探针,并标记生物素,利用微孔板Southern杂交技术,使探针与模板DNA杂交,再加入亲和素标记的辣根过氧化物酶进行酶显色反应,通过酶标仪检测杂交结果,以确定近交系小鼠的基因型。结果57BL／6和C57B：／10为H-2^b型;DBA／2和Scid为H-2^d型;615和C3H为H-2^k型;NCPC／2、TA1、TA2和T739均为H-2^b型。结论通过Southern杂交检测可以确定近交系小鼠的基因型。该检测方法简便、易行,检测结果客观,可以应用于近交系小鼠的遗传检测。