一个RNAi载体上反向重复片段的测序策略

相邻的反向重复DNA片段有形成单链内二级结构的倾向,属于一种测序困难的DNA模板。解决RNAi载体插入的反向重复片段的测序问题,为该类载体正确性的测序验证奠定基础。采用常规分子克隆方法构建表达小麦TaATG2串联反向重复片段的RNAi载体,设计2种策略对经菌落PCR初步鉴定的载体进行测序验证:一种是以完整的载体质粒为模板进行测序;另一种是先对载体进行酶切处理,切除反向重复片段中的一个后对保留另一个片段的线性载体进行测序。结果表明,第一种测序策略受到串联反向重复片段形成的单链内部二级结构的影响,测序信号在反向重复片段处出现衰减或乱峰,无法读取序列。第二种测序策略排除了2个反向重复片段之间的干扰,保留在载体上的片段测序信号清晰,序列准确。采用酶切切除一个片段后进行测序的方法,经过2次酶切和2次测序可以有效地对载体上的2个反向重复片段分别进行序列测定,进而确认构建载体的正确性。     

Rapid and reliable fluorescent cycle sequencing of double-stranded templates.

Automated DNA sequencing is an extremely valuable technique which requires very high quality DNA templates to be carried out successfully. While it has been possible to readily produce large numbers of such templates from M13 or other single-stranded vectors for several years, the sequencing of double-stranded DNA templates using the ABI 373 DNA Sequencer has had a considerably lower success rate. We describe how the combination of a new fluorescent, dideoxy sequencing method, called cycle-sequencing, coupled with modifications to template isolation procedures based on Qiagen columns, makes fluorescent sequencing of double-stranded templates a reliable procedure. From a single five milliliter culture enough DNA can be isolated (up to 20 micrograms) to do 4-8 sequencing reactions, each of which yields 400-500 bases of high quality sequence data. These procedures make the routine use of double-stranded DNA templates a viable strategy in automated DNA sequencing projects.     

A Sequence-Tagged Site Map of Human Chromosome 11

We report the construction of 370 sequence-tagged sites (STSs) that are detectable by PCR amplification under sets of standardized conditions and that have been regionally mapped to human chromosome 11. DNA sequences were determined by sequencing directly from cosmid templates using primers complementary to T3 and T7 promoters present in the cloning vector. Oligonucleotide PCR primers were predicted by computer and tested using a battery of genomic DNAs. Cosmids were regionally localized on chromosome 11 by using fluorescence in situ hybridization or by analyzing a somatic cell hybrid panel. Additional STSs corresponding to known genes and markers on chromosome 11 were also produced under the same series of standardized conditions. The resulting STSs provide uniform coverage of chromosome 11 with an average spacing of 340 kb. The DNA sequence determined for use in STS production corresponds to about 0.1% (116 kb) of chromosome 11 and has been analyzed for the presence of repetitive sequences, similarities to known genes and motifs, and possible exons. Computer analysis of this sequence has identified and therefore mapped at least eight new genes on chromosome 11.     

Direct sequencing of baculovirus genomic DNA: sequence determination of the engineered respiratory syncytial virus chimeric FG gene.

Primer-directed enzymatic sequencing has proven to be an efficient and effective method for sequencing various size double-stranded DNA templates. We previously developed a primer-directed sequencing procedure for using double-stranded cosmid (50 kb) DNAs as template. We are interested in using this method to directly sequence larger DNA templates. Towards this goal we applied this method to directly sequence an engineered gene that had been transferred and integrated into the 130-kb baculovirus genome. Both crudely prepared and CsCl gradient-banded baculovirus DNAs were tested and reasonable sequencing ladders were obtained for both types of DNA templates. As little as 3 micrograms of gradient-banded baculovirus DNA were found to be sufficient to obtain film exposure times similar to those observed for cosmid size templates, 24 to 48 h. Effectiveness of the described method was demonstrated by obtaining the complete sequence of the engineered respiratory syncytial virus chimeric FG gene (2.5 kb in length) directly from the recombinant baculovirus "Baculo-FG" genome. Thus, our results demonstrate first, that double-stranded DNA templates as large as 130 kb can be sequenced directly and second, that the nucleotide sequence of engineered genes integrated within the baculovirus genome can be determined without the use of any intermediate steps of procedures.     

Effective Isolation of Retrotransposons and Repetitive DNA Families from the Wheat Genome

New classes of repetitive DNA elements were effectively identified by isolating small fragments of the elements from the wheat genome. A wheat A genome library was constructed from Triticum monococcum by degenerate cleavage with EcoO109I, the recognition sites of which consisted of 5'-PuGGNCCPy-3'multi-sequences. Three novel repetitive sequences pTm6, pTm69 and pTm58 derived from the A genome were screened and tested for high copy number using a blotting approach. pTm6 showed identity with integrase domains of the barley Ty1-Copia-retrotransposon BARE-1 and pTm58 showed similarity to the barley Ty3-gypsy-like retrotransposon Romani. pTm69, however, constituted a tandem array with useful genomic specificities, but did not share any identity with known repetitive elements. This study also sought to isolate wheat D-genome-specific repetitive elements regardless of the level of methylation, by genomic subtraction. Total genomic DNA of Aegilops tauschii was cleaved into short fragments with a methylation-insensitive 4 bp cutter, Mbol, and then common DNA sequences between Ae. tauschii and Triticum turgidum were subtracted by annealing with excess T. turgidum genomic DNA. The D genome repetitive sequence pAt1 was isolated and used to identify an additional novel repetitive sequence family from wheat bacterial artificial chromosomes with a size range of 1 395-1 850 bp. The methods successfully led pathfinding of two unique repetitive families.     

A shotgun approach to discovering and reconstructing consensus retrotransposons ex novo from dense contigs of short sequences derived from Genbank Genome Survey Sequence database records

Retrotransposons constitute the majority of pseudogenic protein coding regions of most eukaryotic genomes. Most genomes carry tens to thousands of retrotransposon copies derived from dozens of distinct families, but most if not all of these copies are non-functional and contain disabling mutations, including large numbers of indels. Until recently, most regions rich in these elements were virtually ignored in all but the most complete genome sequencing projects, and the full extent of their impact on the structure and function of the genomes of higher eukaryotes was under-appreciated. Even when new retrotransposons are encountered and annotated by automated gene finding programs and similarity searches, coding regions are treated as exons and invariably and not surprisingly mistranslated because of numerous frameshift mutations and large indels. Very few functional retrotransposons contain introns, as in silico annotations imply. While many repetitive DNA consensus sequences have been assembled from collections of largely full-length copies using full-length templates, we have shown that repetitive DNA consensus sequence contigs representing long, moderately high copy-number elements can also be generated ex novo in the absence of templates from very short overlapping sequences. We have devised an in silico strategy to recover and reconstruct consensus sequences of elements up to 20,000 bp by building dense contigs of hundreds of overlapping 400 to 900-bp records found in the Genbank Genome Survey Sequence database. The results are hypothetical ancestral sequences that encode elements that appear to be fully functional with intact open reading frames and other conserved features.     

Sequence organisation in nuclear DNA from Physarum polycephalum. Arrangement of highly-repeated sequences

Recombinant plasmids containing highly repetitive Physarum DNA segments were identified by colony hybridisation using a radioactively-labelled total Physarum DNA probe. A large number of these clones also hybridised to a foldback DNA probe purified from Physarum nuclear DNA. The foldback DNA probe was characterised by reassociation kinetic analysis. About one-half of this component was shown to consist of highly repeated sequences with a kinetic complexity of 1100 bp and an average repetition frequency of 5200. Direct screening of 67 recombinant plasmids for foldback sequences using the electron microscope revealed that about one-half were located in segments of DNA containing highly repetitive sequences; the remainder were present in clones containing low-copy number repeated elements. Analysis of two DNA clones showed that they contained repetitive elements located in over half of all DNA segments containing highly repetitive DNA and that the foci containing these highly repetitive sequences had different sequence arrangements. The results are consistent with the hypothesis that the most highly repeated DNA sequence families in the Physarum genome are few in number and are clustered together in different arrangements in about one-sixth of the genome. Over one-half of the foldback DNA complement in the Physarum genome is derived from these segments of DNA.     

六倍体大小麦tritordeum花药组织特异性启动子的鉴定与分离

启动子是基因表达调控的重要顺式元件,也是基因工程表达载体的一个重要元件。一个无启动子的带有UidA基因的质粒pPLGUS通过基因枪转化进tritordeum材料中,对转基因材料的多种不同组织进行了X-gluc显色来检测不同组织中的GUS活性,有一个株系的花药组织特异性启动子已被证明成功捕获,并通过PCR方法将其分离。提取叶片的总DNA作模板,上游使用水稻花药启动子分离的引物P1,以UidA基因的部分序列为下游引物P2,PCR扩增UidA基因的上游旁侧序列。已经获得一条长667 bp的目的片断,含有部分UidA基因的序列和一段UidA基因的上游旁侧序列,该序列中具有植物启动子的一些必备元件,初步断定它是一段花药组织特异性启动子序列。     

Fundamentals of sequencing of difficult templates--an overview.

Despite enormous progress, the DNA sequencing of difficult regions, whether they are part of complex genomes or individual constructs, still presents a significant challenge and may require many trials, brute-force, or the intervention of a very experienced sequencer (and sometimes all of the above). Very early on, it was realized that sequencing of various types of difficult templates requires distinct treatments. To sequence through GC-rich regions, the addition of DMSO, NP-40/Tween-20 detergents, or the mix (4:1) of BD3.0:dGTP3.0 was sometimes helpful. To get through long poly-A/T tails, sometimes one would be successful using tailored poly- A/T (V/B) N primers or primers that spanned part of pre-tail and tail regions. A few years ago invitrogen introduced a set of sequencing additives that proved to be useful for many different types of difficult templates.     

Genomic Subtraction Recovers Rye-Specific DNA Elements Enriched in the Rye Genome

Repetitive DNA sequence families have been identified in methylated relic DNAs of rye. This study sought to isolate rye genome-specific repetitive elements regardless of the level of methylation, using a genomic subtraction method. The total genomic DNAs of rye-chromosome-addition-wheat lines were cleaved to short fragments with a methylation-insensitive 4-bp cutter, MboI, and then common DNA sequences between rye and wheat were subtracted by annealing with excess wheat genomic DNA. Four classes of rye-specific repetitive elements were successfully isolated from both the methylated and non-methylated regions of the genome. Annealing of the DNA mixture at a ratio of the enzyme-restricted fragments:the sonicated fragments (1:3–1:5) was key to this success. Two classes of repetitive elements identified here belong to representative repetitive families: the tandem 350-family and the dispersed R173 family. Southern blot hybridization patterns of the two repetitive elements showed distinct fragments in methylation-insensitive EcoO109I digests, but continuous smear signals in the methylation-sensitive PstI and SalI digests, indicating that both of the known families are contained in the methylated regions. The subtelomeric tandem 350-family is organized by multimers of a 380-bp-core unit defined by the restriction enzyme EcoO109I. The other two repetitive element classes had new DNA sequences (444, 89 bp) and different core-unit sizes, as defined by methylation-sensitive enzymes. The EcoO109I recognition sites consisting of PyCCNGGPu-multi sequences existed with high frequency in the four types of rye repetitive families and might be a useful tool for studying the genomic organization and differentiation of this species.     

PCR template preparation for capillary DNA sequencing

Fluorescence-based capillary DNA sequencing has facilitated the early completion of several complex sequencing projects. While capillary systems offer great benefits in terms of ease of use and automation, we find that they are sufficiently different from slab gel separation methodologies, demanding re-examination of the protocols used to generate and use DNA sequencing templates. We have recently initiated a large-scale Human Open Reading Frame EST project involving 30 laboratories feeding 11 MegaBace 1000 capillary sequencers. The group has already produced more than 300,000 valid sequences. The most successful template preparation protocol we have found is described here. We have found that a crucial step is the standardization of the quantity and quality of the templates, which have been achieved by overnight bacterial culture followed by PCR using limiting amounts of primers. Using this protocol, there is no need for post-PCR purification, and the final preparation cost is US $0.09/template. After sequencing 10,848 templates using this protocol, 78% of the reads were accepted (after discarding vectors without inserts and inserts smaller than 100 nucleotides), and 85% of the total number of bases had Phred scores of 15 or above.     

Sequence organisation in nuclear DNA from Physarum polycephalum: Arrangement of highly-repeated sequences

Recombinant plasmids containing highly repetitive Physarum DNA segments were identified by colony hybridisation using a radioactively-labelled total Physarum DNA probe. A large number of these clones also hybridised to a foldback DNA probe purified from Physarum nuclear DNA. The foldback DNA probe was characterised by reassociation kinetic analysis. About one-half of this component was shown to consist of highly repeated sequences with a kinetic complexity of 1100 bp and an average repetition frequency of 5200. Direct screening of 67 recombinant plasmids for foldback sequences using the electron microscope revealed that about one-half were located in segments of DNA containing highly repetitive sequences; the remainder were present in clones containing low-copy number repeated elements. Analysis of two DNA clones showed that they contained repetitive elements located in over half of all DNA segments containing highly repetitive DNA and that the foci containing these highly repetitive sequences had different sequence arrangements. The results are consistent with the hypothesis that the most highly repeated DNA sequence families in the Physarum genome are few in number and are clustered together in different arrangements in about one-sixth of the genome. Over one-half of the foldback DNA complement in the Physarum genome is derived from these segments of DNA.     

The ABRF Edman Sequencing Research Group 2008 Study: Investigation into Homopolymeric Amino Acid N-Terminal Sequence Tags and Their Effects on Automated Edman Degradation

The Edman Sequence Research Group (ESRG) of the Association of Biomolecular Resource designs and executes interlaboratory studies investigating the use of automated Edman degradation for protein and peptide analysis. In 2008, the ESRG enlisted the help of core sequencing facilities to investigate the effects of a repeating amino acid tag at the N-terminus of a protein. Commonly, to facilitate protein purification, an affinity tag containing a polyhistidine sequence is conjugated to the N-terminus of the protein. After expression, polyhistidine-tagged protein is readily purified via chelation with an immobilized metal affinity resin. The addition of the polyhistidine tag presents unique challenges for the determination of protein identity using Edman degradation chemistry. Participating laboratories were asked to sequence one protein engineered in three configurations: with an N-terminal polyhistidine tag; with an N-terminal polyalanine tag; or with no tag. Study participants were asked to return a data file containing the uncorrected amino acid picomole yields for the first 17 cycles. Initial and repetitive yield (R.Y.) information and the amount of lag were evaluated. Information about instrumentation and sample treatment was also collected as part of the study. For this study, the majority of participating laboratories successfully called the amino acid sequence for 17 cycles for all three test proteins. In general, laboratories found it more difficult to call the sequence containing the polyhistidine tag. Lag was observed earlier and more consistently with the polyhistidine-tagged protein than the polyalanine-tagged protein. Histidine yields were significantly less than the alanine yields in the tag portion of each analysis. The polyhistidine and polyalanine protein-R.Y. calculations were found to be equivalent. These calculations showed that the nontagged portion from each protein was equivalent. The terminal histidines from the tagged portion of the protein were demonstrated to be responsible for the high lag during N-terminal sequence analysis.     

Strategy and methods for directly sequencing cosmid clones

The primer-directed enzymatic sequencing method for sequencing double-stranded DNA templates has made possible the development of new strategies for directly sequencing large DNA molecules. Toward this goal, we have developed a strategy and the necessary techniques to obtain the complete sequence of cosmid clones (double-stranded DNA molecules in the size range of 50 kb). Our present strategy uses the chemical sequencing method to obtain sequence initiation points internal to a cosmid insert and the primer-directed enzymatic DNA sequencing method to extend these sequence contigs. As part of this development we added a nucleotide "chase" solution to the standard T7 sequencing protocol and included the use of both [alpha-32P]-dATP and -dCTP for labeling. With these modifications our double-stranded cosmid DNA sequencing reactions routinely extend well beyond 1000 bp, and film exposure times are kept to a minimum (24 to 48 h). We can routinely separate sequenced DNA fragments, using a 1-m gel system, which can be accurately read (with less than 0.5% error) to distances of 800 bp or more, from the oligomer primer. The strategy and procedures presented here allow the complete sequence of a cosmid clone to be obtained without subcloning.     

An improved protocol for sequencing of repetitive genomic regions and structural variations using mutagenesis and next generation sequencing

The rise of Next Generation Sequencing (NGS) technologies has transformed de novo genome sequencing into an accessible research tool, but obtaining high quality eukaryotic genome assemblies remains a challenge, mostly due to the abundance of repetitive elements. These also make it difficult to study nucleotide polymorphism in repetitive regions, including certain types of structural variations. One solution proposed for resolving such regions is Sequence Assembly aided by Mutagenesis (SAM), which relies on the fact that introducing enough random mutations breaks the repetitive structure, making assembly possible. Sequencing many different mutated copies permits the sequence of the repetitive region to be inferred by consensus methods. However, this approach relies on molecular cloning in order to isolate and amplify individual mutant copies, making it hard to scale-up the approach for use in conjunction with high-throughput sequencing technologies. To address this problem, we propose NG-SAM, a modified version of the SAM protocol that relies on PCR and dilution steps only, coupled to a NGS workflow. NG-SAM therefore has the potential to be scaled-up, e.g. using emerging microfluidics technologies. We built a realistic simulation pipeline to study the feasibility of NG-SAM, and our results suggest that under appropriate experimental conditions the approach might be successfully put into practice. Moreover, our simulations suggest that NG-SAM is capable of reconstructing robustly a wide range of potential target sequences of varying lengths and repetitive structures.     

Use of a chemically modified T7 DNA polymerase for manual and automated sequencing of supercoiled DNA

Procedures are presented for reliable and accurate nucleotide sequence analysis using as template supercoiled DNA prepared by a modified rapid boiling minipreparation protocol. This method yields DNA templates suitable for sequencing within 1 h of bacterial harvest. We describe optimal reaction conditions for supercoiled miniprep DNA sequencing using a modified T7 DNA polymerase (Sequenase) in dideoxynucleotide chain termination reactions. We demonstrate that under these conditions, the sequencing data obtained with miniprep DNA is indistinguishable from that obtained with CsCl purified supercoiled DNA or from that obtained using single stranded DNA templates. We further show that the supercoiled DNA sequencing reactions can be analyzed on a commercially available automated DNA sequencing system that detects 32P labeled DNA during its electrophoretic separation. Taken together, these developments represent a significant improvement in the process of nucleotide sequence analysis.     

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.