Direct sequencing of bacteriophage T4 DNA with a thermostable DNA polymerase

We present a simple and convenient protocol for the direct sequencing of bacteriophage T4 genomic DNA. The method utilizes the thermostable DNA polymerase from Thermus aquaticus (Taq) and 32P-end-labeled oligodeoxyribonucleotide primers to produce extension products that allow the analysis of at least 200 nucleotides (nt) on a single sequencing gel. Single-nt changes in the template were easily detectable following an overnight exposure of the autoradiograms. Comparison of sequences from fully modified T4 DNA containing glucosylated hydroxymethyldeoxycytosine or from templates containing cytosine showed little difference in sequence clarity. These techniques considerably simplify the molecular analysis of T-even bacteriophages and should be compatible with automated sequencing methods which employ 5'-end-labeled primers.     

Sequence analysis and editing for bisulphite genomic sequencing projects

Bisulphite genomic sequencing is a widely used technique for detailed analysis of the methylation status of a region of DNA. It relies upon the selective deamination of unmethylated cytosine to uracil after treatment with sodium bisulphite, usually followed by PCR amplification of the chosen target region. Since this two-step procedure replaces all unmethylated cytosine bases with thymine, PCR products derived from unmethylated templates contain only three types of nucleotide, in unequal proportions. This can create a number of technical difficulties (e.g. for some base-calling methods) and impedes manual analysis of sequencing results (since the long runs of T or A residues are difficult to align visually with the parent sequence). To facilitate the detailed analysis of bisulphite PCR products (particularly using multiple cloned templates), we have developed a visually intuitive program that identifies the methylation status of CpG dinucleotides by analysis of raw sequence data files produced by MegaBace or ABI sequencers as well as Staden SCF trace files and plain text files. The program then also collates and presents data derived from independent templates (e.g. separate clones). This results in a considerable reduction in the time required for completion of a detailed genomic methylation project.     

Primer design for large scale sequencing.

We have developed PRIDE, a primer design program that automatically designs primers in single contigs or whole sequencing projects to extend the already known sequence and to double strand single-stranded regions. The program is fully integrated into the Staden package (GAP4) and accessible with a graphical user interface. PRIDE uses a fuzzy logic-based system to calculate primer qualities. The computational performance of PRIDE is enhanced by using suffix trees to store the huge amount of data being produced. A test set of 110 sequencing primers and 11 PCR primer pairs has been designed on genomic templates, cDNAs and sequences containing repetitive elements to analyze PRIDE's success rate. The high performance of PRIDE, combined with its minimal requirement of user interaction and its fast algorithm, make this program useful for the large scale design of primers, especially in large sequencing projects.     

用一个简单快速的RT-PCR技术筛选白细胞介素-6作用相关基因

为了研究白细胞介素-6(IL-6)作用相关基因以及一些可能受IL-6调控的基因,利用一个简单快速的以PCR为基础的方案,检测了IL-6处理和未处理的Sko007细胞中基因表达的差异,克隆并鉴定了差异表达基因的cDNA片段.首先用6-mer寡核苷酸引物进行反转录从而最大限度地将mRNA编码区序列生成cDNA;然后用2或3个较长的随机引物进行PCR扩增,并以不同引物组合重复PCR增扩;扩增产物在2%琼脂糖凝胶上电泳分离,回收差异片段并直接用于克隆、测序及进一步分析.在此研究中,获得了3个表达序列标签(EST),其中一个为新的基因片段,反向RNA杂交有力证实了它们与IL-6作用的相关性.进一步的生物信息学分析表明,新基因片段STRF17在多种组织中表达.     

Microsatellite loci in the phytoparasitic nematode Globodera.

A Globodera pallida genomic library, population Guiclan (Pa2/3), was screened for TG and TC microsatellite motifs. Screening of 50,000 clones revealed 48 positive matches. After sequencing, primers were designed to amplify 14 microsatellite loci. The specificity of the loci was tested with DNA templates of other populations of G. pallida, and also on other species of Globodera. Appearance of amplification products on several of these DNA templates showed that the microsatellite flanking regions are relatively conserved between G. pallida populations as well as between Globodera species. Evidence for allele polymorphism between individuals was demonstrated by using nine loci primers, in G. pallida population Guiclan and from a population of a closely related species G. "mexicana". Some alleles appeared to be species specific.     

The phylogenetic potential of entire 26S rDNA sequences in plants

18S ribosomal RNA genes are the most widely used nuclear sequences for phylogeny reconstruction at higher taxonomic levels in plants. However, due to a conservative rate of evolution, 18S rDNA alone sometimes provides too few phylogenetically informative characters to resolve relationships adequately. Previous studies using partial sequences have suggested the potential of 26S or large-subunit (LSU) rDNA for phylogeny retrieval at taxonomic levels comparable to those investigated with 18S rDNA. Here we explore the patterns of molecular evolution of entire 26S rDNA sequences and their impact on phylogeny retrieval. We present a protocol for PCR amplification and sequencing of entire (approximately 3.4 kb) 26S rDNA sequences as single amplicons, as well as primers that can be used for amplification and sequencing. These primers proved useful in angiosperms and Gnetales and likely have broader applicability. With these protocols and primers, entire 26S rDNA sequences were generated for a diverse array of 15 seed plants, including basal eudicots, monocots, and higher eudicots, plus two representatives of Gnetales. Comparisons of sequence dissimilarity indicate that expansion segments (or divergence domains) evolve 6.4 to 10.2 times as fast as conserved core regions of 26S rDNA sequences in plants. Additional comparisons indicate that 26S rDNA evolves 1.6 to 2.2 times as fast as and provides 3.3 times as many phylogenetically informative characters as 18S rDNA; compared to the chloroplast gene rbcL, 26S rDNA evolves at 0.44 to 1.0 times its rate and provides 2.0 times as many phylogenetically informative characters. Expansion segment sequences analyzed here evolve 1.2 to 3.0 times faster than rbcL, providing 1.5 times the number of informative characters. Plant expansion segments have a pattern of evolution distinct from that found in animals, exhibiting less cryptic sequence simplicity, a lower frequency of insertion and deletion, and greater phylogenetic potential.      

Internal transcribed spacer repeat-specific primers and the analysis of hybridization in the Glycine tomentella (Leguminosae) polyploid complex

Polyploid and diploid hybridization is a ubiquitous and evolutionarily important phenomenon in the plant world. Determining the parental species of a hybrid, however, is difficult. Molecular markers such as the nuclear ribosomal DNA gene complex, particularly its internal transcribed spacer (ITS) region, have proved powerful in determining hybrid parentage. In some cases, population and genomic phenomena, such as genetic drift and concerted evolution, result in the loss of all or many of the tandemly repeated copies derived from one parental species, making the recovery of hybrid history difficult or impossible. Methods such as direct sequencing and cloning are typically used to find ITS sequences contributed from parental species, but are limited in their ability to detect rare repeat types. Here we report that repeat-specific polymerase chain reaction primers can recover rare parental ITS sequences in the Glycine tomentella polyploid complex. In three allopolyploid lineages of this complex, repeat-specific primers reliably detected rare repeats that both direct sequencing and the screening of many cloned sequences failed to detect. Other strategies, such as the use of exclusion primers, may detect rare parental repeat types in hybrids when previous hypotheses regarding the second parental species are lacking.     

DNA Sequencing Directly from a Mixture Using Terminal-Base-Selective Primers

DNA cloning is often used to select and amplify one DNA speciesfrom a mixture. However, the cloning process is complex andlabor-intensive. We have developed a new two-step method forDNA sequencing directly from a mixture. The first is the introductionof a known oligonucleotide (common part) into the terminus ofunknown DNA by ligation. The second is selective DNA sequencingusing primers with two additional nucleotides at the 3' terminusin addition to the common part (terminal-base-selective primers).The primers work only for templates on which the primers perfectlyhybridized. This method was found to be effective for the HindIIIdigestion products of phage.     

Optimization of Protocol for Sequencing of Difficult Templates

In this paper, we have fine-tuned a DNA sequencing protocol suitable for a wide range of difficult templates. The primary goal was to evaluate a number of parameters—such as various dye terminator mixes in the presence or absence of additives, the amount of DNA or primer, and cycling protocols—about the effectiveness of reading through complex regions. We showed that the modification of a published protocol leads to significant (75%) cost reduction without forfeiting quality of the data. In the recommended protocol, we used betaine as a standard additive, but better results can be obtained when betaine and Reagent A are mixed in an equivalent ratio.     

A site-directed mutagenesis method particularly useful for creating otherwise difficult-to-make mutants and alanine scanning

Site-directed mutagenesis has become routine in molecular biology. However, many mutants can still be very difficult to create. Complicated chimerical mutations, tandem repeats, inverted sequences, GC-rich regions, and/or heavy secondary structures can cause inefficient or incorrect binding of the mutagenic primer to the target sequence and affect the subsequent amplification. In theory, these problems can be avoided by introducing the mutations into the target sequence using mutagenic fragments and so removing the need for primer-template annealing. The cassette mutagenesis uses the mutagenic fragment in its protocol; however, in most cases it needs to perform two rounds of mutagenic primer-based mutagenesis to introduce suitable restriction enzyme sites into templates and is not suitable for routine mutagenesis. Here we describe a highly efficient method in which the template except the region to be mutated is amplified by polymerase chain reaction (PCR) and the type IIs restriction enzyme-digested PCR product is directly ligated with the mutagenic fragment. Our method requires no assistance of mutagenic primers. We have used this method to create various types of difficult-to-make mutants with mutagenic frequencies of nearly 100%. Our protocol has many advantages over the prevalent QuikChange method and is a valuable tool for studies on gene structure and function.     

SNPsFinder--a web-based application for genome-wide discovery of single nucleotide polymorphisms in microbial genomes

SUMMARY: Single nucleotide polymorphisms (SNPs) are the most abundant form of genetic variations in closely related microbial species, strains or isolates. Some SNPs confer selective advantages for microbial pathogens during infection and many others are powerful genetic markers for distinguishing closely related strains or isolates that could not be distinguished otherwise. To facilitate SNP discovery in microbial genomes, we have developed a web-based application, SNPsFinder, for genome-wide identification of SNPs. SNPsFinder takes multiple genome sequences as input to identify SNPs within homologous regions. It can also take contig sequences and sequence quality scores from ongoing sequencing projects for SNP prediction. SNPsFinder will use genome sequence annotation if available and map the predicted SNP regions to known genes or regions to assist further evaluation of the predicted SNPs for their functional significance. SNPsFinder can generate PCR primers for all predicted SNP regions according to user's input parameters to facilitate experimental validation. The results from SNPsFinder analysis are accessible through the World Wide Web. AVAILABILITY: The SNPsFinder program is available at http://snpsfinder.lanl.gov/. SUPPLEMENTARY INFORMATION: The user's manual is available at http://snpsfinder.lanl.gov/UsersManual/     

Evaluation of methods for sequence analysis of highly repetitive DNA templates.

The DNA Sequencing Research Group (DSRG) of the ABRF conducted a study to assess the ability of DNA sequencing core facilities to successfully sequence a set of well-defined templates containing difficult repeats. The aim of this study was to determine whether repetitive templates could be sequenced accurately by using equipment and chemistries currently utilized in participating sequencing laboratories. The effects of primer and template concentrations, sequencing chemistries, additives, and instrument formats on the ability to successfully sequence repeat elements were examined. The first part of this study was an analysis of the results of 361 chromatograms from participants representing 40 different laboratories who attempted to sequence a panel of difficult-to-sequence templates using their best in-house protocols. The second part of this study was a smaller multi-laboratory evaluation of a single robust protocol with the same panel of templates. This study provides a measure of the potential success of different approaches to sequencing across homopolymer tracts and repetitive elements.     

Automated system for purification of dye-terminator sequencing products eliminates up-stream purification of templates.

The quality of sequencing results is to a large extent determined by the purity of the template and the purification of the sequencing products. Fragments that can act as unspecific primers and templates are removed before gel analysis, and the background of unspecific signals is highly reduced. Purification of the sequencing products is needed to remove salts, nucleotides, proteins and template DNA that can interfere with the gel separation. We have developed a product, DYNAPURE Dye Terminator Removal, that specifically isolates and purifies the sequencing products in 10 min. The method is based on biotinylated sequencing primers and super-paramagnetic streptavidin beads. A PCR product is sequenced using a biotinylated sequencing primer, and the sequencing products are then bound to streptavidin beads in a 5-min reaction. The bead-DNA complexes are magnetically separated from the rest of the solution, and the remaining buffer constituents are washed away with TE buffer or with 70% ethanol. The whole procedure can be automated on liquid-handling robots fitted with a magnet station. The method eliminates purification of templates before cycle sequencing.     

Efficient mutagenesis method for producing the templates of single nucleotide polymorphisms

DNA templates harboring specific single nucleotide polymorphism (SNP) sites are largely needed as positive controls in practical SNP analysis and in determination of the reliability of newly developed methods in high-throughput screening assays. Here we report a one-step method to produce SNP templates by amplifying a wild-type sequence with primers having single nucleotide mismatches at or near their 3′ ends. A short amplicon harboring an EcoRI site was used to evaluate the feasibility of our strategy. Perfectly matched primers and primers with a single base mismatch occurring from the first base to the sixth base of the EcoRI site were used for primer extension. By using polymerase without a proofreading function, we kept mismatched nucleotides from occurring in extended primer products, as confirmed by EcoRI digestion and sequencing analysis. The strategy of using primers with a single mismatched base and exo- polymerase was shown to be an efficient one-step method for preparing SNP templates, either for application in the development of SNP screening assays or as positive controls in practical SNP assays.     

RAPD analysis of isolated mitochondrial DNA reveals heterogeneity in elite wild abortive (WA) cytoplasm in rice

 RAPD profiles were generated using mitochondrial DNA (mtDNA) isolated from two cytoplasmic male-sterile lines, two restorer lines and four maintainer lines of rice. Of the 40 primers tested, 25 generated consistent and easily scoreable patterns that were used for the computation of pairwise similarities as well as UPGMA analyses. The different lines of rice, including lines IR58025A and IR62829A that contained the same wild abortive (WA) cytoplasm, were distinguishable on the basis of RAPD profiles. These latter two lines were not distinguishable from each other by mtDNA RFLP analyses with as many as 16 mtDNA probes. The data illustrate the utility of the RAPD technique as a powerful tool for distinguishing different cytoplasms that by other techniques appear to be similar. To our knowledge, this is the first report wherein RAPD profiles obtained with isolated mtDNA templates enable the distinction between two or more types of cytoplasms in rice. Received: 1 April 1997 / Accepted: 2 June 1997     

NGS-PrimerPlex: High-throughput primer design for multiplex polymerase chain reactions

Multiplex polymerase chain reaction (PCR) has multiple applications in molecular biology, including developing new targeted next-generation sequencing (NGS) panels. We present NGS-PrimerPlex, an efficient and versatile command-line application that designs primers for different refined types of amplicon-based genome target enrichment. It supports nested and anchored multiplex PCR, redistribution among multiplex reactions of primers constructed earlier, and extension of existing NGS-panels. The primer design process takes into consideration the formation of secondary structures, non-target amplicons between all primers of a pool, primers and high-frequent genome single-nucleotide polymorphisms (SNPs) overlapping. Moreover, users of NGS-PrimerPlex are free from manually defining input genome regions, because it can be done automatically from a list of genes or their parts like exon or codon numbers. Using the program, the NGS-panel for sequencing the LRRK2 gene coding regions was created, and 354 DNA samples were studied successfully with a median coverage of 97.4% of target regions by at least 30 reads. To show that NGS-PrimerPlex can also be applied for bacterial genomes, we designed primers to detect foodborne pathogens Salmonella enterica, Escherichia coli O157:H7, Listeria monocytogenes, and Staphylococcus aureus considering variable positions of the genomes.     

PCR and DNA sequencing

Specific DNA segments defined by the sequence of two oligonucleotides can be enzymatically amplified up to a millionfold using the polymerase chain reaction (PCR). One of the most significant uses of this technique is for generation of sequencing templates, either from cloned inserts or directly from genomic DNA. To avoid the problem of reassociation of the linear DNA strands in the sequencing reaction, ssDNA templates can be produced directly in the PCR or generated directly from dsDNA by enzymatic treatment, electrophoretic separation or affinity purification. By combining PCR with direct sequencing, both the amplification and the sequencing reaction can be performed in the same vial. Finally, use of fluorescently labeled terminators or sequencing primers will allow the whole procedure to be amenable to complete automation.