An efficient method for purification of PCR products for sequencing

A high-throughput DNA sequencing method that generated high quality data was developed. A frame fashioned from a standard agarose gel combined with 0.1%-0.2% low-melting point (LMP) agarose gel was used to isolate the PCR product of interest. Collected PCR products were centrifuged without any reagents and the supernatants were directly used for a sequencing reaction. This method is simple and labor efficient, provides high quality sequences at a low cost, and bypasses problems with impure PCR products. This technique has been used for single nucleotide polymorphism (SNP) discovery in Populus angustifolia trees.     

Extraction of nucleic acids from agarose gel--a quantitative and qualitative comparison of four different methods

Agarose gel electrophoresis is commonly used to separate different species of nucleic acids. We compare four different methods of extraction which are commonly used. These methods include buffer extraction, electroelution, glass bead extraction and extraction of DNA from low-melting agarose. The results show that DNA extracted by these four methods is comparable in their ligability to the PMT 21 vectors and the plasmids with insert can be used for subsequent transfections of competent bacteria. There is a higher yield for buffer extraction and electroelution when compared with glass bead extraction and low melting agarose (p less than 0.05). To conclude, the four commonly used methods for DNA isolation are comparable qualitatively. But the simplest method, namely buffer extraction, has the highest yield.     

酿酒酵母染色体DNA样品制备和CHEF分析

等高锁状均匀电场（contolllsclampedhomogeneouselectncfield,CHED电泳技术与分子杂交、酵母人工染色体（yeastartificialchromosome,YAC）等分子生物学技术的有机结合,推动了真菌电泳核型、染色体作图和染色体DNA长度多型性（chromosomalDNAlellgthpolymorphism,CLP...     

An automated method for DNA preparation from thousands of YAC clones.

We describe an automated method for the preparation of yeast genomic DNA capable of preparing thousands of DNAs in parallel from a YAC library. Briefly, the protocol involves four steps: (1) Yeast clones are grown in the wells of 96-well microtiter plates with filter (rather than plastic) well-bottoms, which are embedded in solid growth media; (2) These yeast cultures are resuspended and their concentrations determined by optical density measurement; (3) Equal numbers of cells from each well are embedded in low-melting temperature agarose blocks in fresh 96-well plates, again with filter bottoms; and (4) DNA is prepared in the agarose blocks by a protocol similar to that used for preparing DNA for pulsed-field gels, with the reagents being dialyzed through the (filter) bottoms of the microtiter plate. The DNA produced by this method is suitable for pulsed-field gel electrophoresis, for restriction enzyme digestion, and for the polymerase chain reaction (PCR). Using this protocol, we produced 3000 YAC strain DNAs in three weeks. This automated procedure should be extremely useful in many genomic mapping projects.     

Rapid separation of DNA molecules by agarose gel electrophoresis: use of a new agarose matrix and a survey of running buffer effects.

This report describes the use of a new type of agarose (FastLane agarose) for faster separation of DNA by agarose gel electrophoresis. DNA molecules separated in this agarose exhibited electrophoretic mobilities up to 30% higher than similar separations in standard analytical grade agarose. DNA molecules of all sizes examined showed higher mobilities in FastLane agarose. The mobility increase was predominantly due to the low electroendosmosis of FastLane agarose and was most pronounced in pulsed field gel electrophoresis separations. The magnitude of mobility increase varied depending on the conditions used for electrophoresis.     

The Role of Duplex Ends in the Spontaneous Interaction of Homologous Linear DNA Fragments

The spontaneous interaction of homologous linear DNA fragments was studied with a model of purified PCR products by agarose gel electrophoresis. To interact, duplexes required not only homology of internal regions, but also complementary ends. Fragments differing in terminal sequences did not interact. The yield of Holliday junctions (HJ), the simplest product of DNA–DNA interaction, depended on dissociation of fragment ends. Compared with genomic fragments, those with low-melting AT ends interacted with each other more efficiently and those with high-melting GC ends, less efficiently. Incubation temperature affected the equilibrium HJ concentration in solution of homologous fragments. A conclusion was made that HJ formation is initiated by nucleation of dissociated duplex ends.     

Purification and cloning of DNA fragments fractionated on agarose gels

Purification of DNA fragments from acrylamide or agarose gels is a commonly used technique in the molecular biology laboratory. This article describes a rapid, efficient, and inexpensive method of purifying DNA fractions from an agarose gel. The purified DNA is suitable for use in a wide range of applications including ligation using DNA ligase. The procedure uses standard high-melting-temperature agarose and normal TBE electrophoresis buffer. In addition, the protocol does not involve the use of highly toxic organic solvents such as phenol.     

Localization of low-melting regions in phage T7 DNA

Specific fragmentation of T7 DNA at glyoxal-fixed denatured regions by the S1 endonuclease followed by restriction analysis made it possible to localize four low-melting regions in phage T7 DNA. These regions have the following coordinates:0.5-1.2;14.8+/-0.3;46.3+/-0.5; 98.4+/-0.3 (in T7 DNA length units). The location of the low-melting regions was refined by means of electron-microscopic denaturation mapping and gel electrophoresis of partially denatured DNA. The obtained localization of the low-melting regions is consistent with the available data on the sequence of T7 DNA. The map of low-melting regions was compared with the genetic map of T7 DNA.Images     

Development of a method based on alkaline gel electrophoresis for estimation of oxidative damage to DNA in Escherichia coli

A method for estimating DNA strand breakage and subsequent repair based on alkaline gel electrophoresis was developed and tested with isogenic strains of Escherichia coli deficient in DNA repair enzymes. Samples from a cell suspension were removed at 2 min intervals following a 15 min exposure to 20 mmol l^-1 H₂O₂. Catalase was added and the cells were embedded in blocks of low-melting point agarose and lysed. After alkaline gel electrophoresis, photographs of the gels were taken and the relative lengths of the distributions of DNA fragments were measured with a scanner and computer. The lengths were correlated with survival of the cells exposed to H₂O₂ and with the importance of particular DNA repair enzymes. Alkaline gel electrophoresis appears to be a relatively simple method for analysing the level of H₂O₂-caused DNA damage and repair in E. coli.     

The role of duplex ends in the spontaneous interaction of homologous linear DNA fragments

The spontaneous interaction of homologous linear DNA fragments was studied with a model of purified PCR products by agarose gel electrophoresis. To interact, duplexes required not only homology of internal regions, but also complementary ends. Fragments differing in terminal sequences did not interact. The yield of Holliday junctions (HJ), the simplest product of DNA-DNA interaction, depended on dissociation of fragment ends. Compared with genomic fragments, those with low-melting AT ends interacted with each other more efficiently and those with high-melting GC ends, less efficiently. Incubation temperature affected the equilibrium HJ concentration in solution of homologous fragments. A conclusion was made that HJ formation is initiated by nucleation of dissociated duplex ends.     

Rapid purification of DNA from agarose gels by centrifugation through a disposable plastic column

A simple and rapid method for purifying DNA from agarose gels is described. Agarose slices containing DNA are placed in a disposable plastic column and the DNA is separated from the agarose by centrifugation in a microfuge. Recoveries averaging 25% are obtained for DNA of 14 kb or less. The recovered DNA can be labeled to high specific activity, cleaved with restriction endonucleases, and ligated efficiently using standard cloning vectors.     

Rapid re-amplification of PCR products purified in low melting point agarose gels

A rapid, simple method is described for performing sequential amplifications of purified products produced by the PCR. After the initial amplification, an aliquot of the reaction is run on a low melting point agarose gel. A Pasteur pipet is used to punch out a gel plug from the amplified band. The DNA in this plug is then used directly as the template for a second round of amplification. Relatively large amounts of agarose can be tolerated without noticeable effects on amplification. Use of a composite gel made from agarose and linear polyacrylamide increases the ease and utility of this technique. These gels are simple to cast, easier to handle and permit several replicate plugs to be obtained from a single band. This method is well suited to experiments which use "nested" primers to increase the sensitivity and specificity of amplification or any method in which PCR amplification follows DNA purification by electrophoresis in LMP agarose gels.     

中国安徽庐江鸭乙型肝炎病毒全基因组克隆酶谱分析和部分病毒基因正负单链探针的构建

用鸭乙型肝炎病毒(DHBV)阳性的安徽庐江鸭血清感染DHBV阴性的北京雏鸭,扩增病毒,将提取的DHBV-DNA插入pUC18质粒,转化E.coli JM 105。酶切重组质粒及South-ern转膜杂交结果证实,质粒pLJ76的插入片段为DHBV全基因组。用EcoR Ⅰ等11种限制性内切酶对pLJ76进行酶谱分析,并与美国,西德的已知DHBV基因组比较。定向克隆该株病毒不同基因编码区片段,构建正负单链探针,将斑点杂交和单链电泳检出的M13阳性重组子与已知序列的DHBV基因组作比较,提示获得了该株病毒基因组的S、Pre-S、P和X/C等蛋白编码区的正、负单链克隆株。     

胚胎脑片免疫荧光组织化学双重漂染技术在神经元发生研究中的应用

目的为更客观地观察胚胎脑神经祖细胞的增殖、分化和迁移,建立胚胎脑片免疫荧光组织化学双重漂染技术。方法灌流固定,取胚胎14天(E14)大鼠脑,低熔点琼脂糖包埋,振动切片机连续冠状切片,免疫荧光组织化学双重漂染,激光扫描共聚焦显微镜下观察。结果波形蛋白(Vimentin)和乙酰胆碱转移酶(ChAT)双阳性细胞呈黄色荧光,胞体位于脑室区,长突起呈放射状。ChAT阳性细胞呈红色荧光,除胞体位于脑室区,长突起呈放射状伸展外,可见皮层板区也有阳性细胞集聚。结论此项技术可直接观察到胚胎脑神经祖细胞和成神经细胞的完整形态,并通过免疫荧光组织化学方法鉴定其表型,从而阐明相互之间的关系。     

Convenient and versatile DNA extraction using agarose plugs for ribotyping of problematic bacterial species.

We describe a convenient, versatile and safe method for preparing bacterial DNA for ribotyping analysis. In this method, extraction of bacterial DNA from Salmnonella typhi and Burkholderia pseudomallei. and subsequent restriction endonuclease digestion, was performed in agarose blocks/plugs thus minimizing shearing and loss of DNA, problems commonly associated with liquid phase phenol extraction. Digested DNA in the plugs was then electrophoresed directly, transferred to nylon membranes and hybridized with labeled rDNA probes in the usual manner to provide reproducible restriction patterns. This method is particularly useful for bacterial species where standard DNA extraction in the liquid phase using phenol has been problematic (e.g. B. pseudomallei) but can be used for any bacterial species. The DNA extracted within the agarose plugs can be stored for long periods and can be used in other, widely-used typing methods such as pulsed-field gel electrophoresis (PFGE) and PCR-based techniques. Embedding live cells directly in agarose plugs also minimizes the risk of exposure to these virulent human pathogens among laboratory workers.     

Production of hybrid calluses via donor-recipient fusion between Microcitrus papuana and Citrus sinensis

X-ray irradiated embryogenic protoplasts of Microcitrus papuana Swing. were electrically fused with iodoacetic acid-treated embryogenic protoplasts of Newhall navel orange [Citrus sinensis (L.) Osb.]. Seven cell lines were established by low-melting agarose embedding culture of fusion-treated protoplasts. Cytological examination of 4 cell lines showed that each cell line consisted of many aneuploid (45.10%, 38.98%, 32.69% and 34.85%, respectively) and diploid cells (52.94%, 59.33%, 63.46% and 62.12%. respectively), whereas only a few tetraploid cells (1.96%, 1.69%, 3.85% and 3.03%, respectively) were detected. Analyses of random amplified polymorphic DNA with four 10-mer primers confirmed the hybrid characteristics of the cell lines, which in combination with chromosome counting proved that the cell lines were asymmetric hybrids. This revised version was published online in June 2006 with corrections to the Cover Date.     

Marked improvement of PAC and BAC cloning is achieved using electroelution of pulsed-field gel-separated partial digests of genomic DNA.

We describe a simple electroelution method for purifying large, gel-fractionated DNA molecules that alleviates the need for melting of the agarose and subsequent enzymatic agarose digestion. The method yields DNA that is visibly more intact than that purified from a standard agarose-digestion protocol and is more amenable to large-fragment cloning with PAC and BAC vectors. These findings are notable in that PAC and BAC library construction is a very labor-intensive and costly procedure, such that any net improvement in cloning efficiency is highly advantageous. This method also should prove useful towards other applications which require purification of very large DNA molecules, such as YAC cloning.     

Short protocol for pulsed field gel electrophoresis of a variety of Clostridia species

While pulsed field gel electrophoresis has become an important tool for genotyping of bacteria, one of its drawbacks is that standard methods are rather time-consuming. In order to overcome this problem, shortened procedures for DNA preparation have been developed for some bacterial species. The aim of this study was to examine if a short procedure used for pulsed field gel electrophoresis of Clostridium botulinum could be applied to other Clostridia species. For this, the protocol was modified and used to prepare the DNA of 34 strains of 25 different Clostridia species. In contrast to a standard procedure, which takes at least 5 days from DNA extraction to completion of the electrophoresis, this protocol yielded results within 2 days. In order to directly compare the results of the short protocol with those of the standard, long procedure, parallel DNA preparations were performed using both methods and the two DNA samples thus obtained per strain were then run on the same gel. Briefly, the procedure was as follows. After embedding the bacterial cells in agarose, the agarose blocks were incubated for 1 h in lysis solution containing lysozyme, mutanolysin, lysostaphin and RNase. This was followed by a 1-h proteinase K treatment. Then, slices were cut from the agarose blocks and washed for 15 min in TE buffer, these washes were repeated four times with fresh TE. After a 2-h restriction with SmaI, electrophoresis was carried out overnight.     

Chemical and viral transformation of cultured skin fibroblasts from patients with familial polyposis coli

Treatment of skin fibroblasts from an FPC patient with 4NQO or MNNG followed by sequential passaging caused morphological changes of the cells, which showed characteristics of transformed cells such as a high frequency of colony formation in agarose, increased growth ability, and chromosomal abnormalities. This and other fibroblast lines from 5 of 12 FPC patients had an increased susceptibility to 4NQO cytotoxicity, which was caused by enhanced 4NQO-reductase activity rather than by reduced DNA repair. However, the susceptibility to cytotoxicity of MNNG and repair of MNNG-damaged DNA were normal in FPC cells. The tumor promoters TPA and DHTB enhanced the frequency of chemical transformation of the FPC fibroblasts, and protease inhibitors suppressed the promoter-enhanced transformation. The skin fibroblasts from many FPC patients exhibited increased susceptibility to transformation by murine sarcoma viruses. Analysis of the viral DNA and RNA after infection revealed that the increased susceptibility is determined at an early stage of transformation. Two out of 5 MNNG-transformed clones of FPC fibroblasts, isolated from agarose, had increased expression of c-Ki-ras or c-Ha-ras, and 4 of 4 MSV-transformed clones showed high expression of viral Ki-ras. These clones grew further after isolation from agarose, but were mortal and did not form tumors in nude mice. The present results suggest that additional changes in morphologically transformed FPC fibroblasts are required for malignant transformation.     

Characterization of an inexpensive, nontoxic, and highly sensitive microarray substrate

An agarose film has been proposed as an efficient substrate for producing microarrays. The original film preparation procedure was simplified significantly by grafting the agarose layer directly onto unmodified microscope glass slides instead of aminated glass slides, and the blocking procedure was replaced with a wash in 0.1x standard saline citrate (SSC) and 0.5% sodium dodecyl sulfate (SDS) without decreasing the performance of the produced microarrays. Characterization of the grafted agarose film using atomic force microscopy (AFM) and scanning electron microscopy (SEM) showed that the agarose film had a 10-fold increase in surface roughness compared to glass and that the interior of the agarose film was porous, with pore sizes between 100-500 nm. A comparison of hybridization on aldehyde-activated agarose-coated microarray slides and commercial amino-reactive microarray slides showed that aldehyde-activated agarose-coated slides had the highest signal-to-noise ratio of 850, suggesting that the aldehyde-activated agarose microarray slides are suitable in applications where analytes have a wide concentration range. By immobilizing the DNA probes using ultraviolet (UV) light, the signal-to-noise ratio was further increased to 3000 on the agarose microarray slides. The specificity of the UV cross-linked DNA probes was demonstrated using 21 and 25 bp long capture probes, enabling discrimination of target molecules differing in only one base.