An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol

Background: Mutagenesis plays an essential role in molecular biology and biochemistry. It has also been used in enzymology and protein science to generate proteins which are more tractable for biophysical techniques. The ability to quickly and specifically mutate a residue(s) in protein is important for mechanistic and functional studies. Although many site-directed mutagenesis methods have been developed, a simple, quick and multi-applicable method is still desirable. Results: We have developed a site-directed plasmid mutagenesis protocol that preserved the simple one step procedure of the QuikChange (TM) site-directed mutagenesis but enhanced its efficiency and extended its capability for multi-site mutagenesis. This modified protocol used a new primer design that promoted primer-template annealing by eliminating primer dimerization and also permitted the newly synthesized DNA to be used as the template in subsequent amplification cycles. These two factors we believe are the main reasons for the enhanced amplification efficiency and for its applications in multi-site mutagenesis. Conclusion: Our modified protocol significantly increased the efficiency of single mutation and also allowed facile large single insertions, deletions/truncations and multiple mutations in a single experiment, an option incompatible with the standard QuikChange (TM). Furthermore the new protocol required significantly less parental DNA which facilitated the DpnI digestion after the PCR amplification and enhanced the overall efficiency and reliability. Using our protocol, we generated single site, multiple single-site mutations and a combined insertion/deletion mutations. The results demonstrated that this new protocol imposed no additional reagent costs (beyond basic QuikChange T) but increased the overall success rates.Publisher PDFPeer reviewe

Optimizing a PCR protocol for cpn60-based microbiome profiling of samples variously contaminated with host genomic DNA.

The current recommended protocol for chaperonin-60 (cpn60) universal target based microbiome profiling includes universal PCR of microbiome samples across an annealing temperature gradient to maximize the diversity of sequences amplified. However, the value of including this gradient approach has not been formally evaluated since the optimization of a modified universal PCR primer cocktail for cpn60 PCR. PCR conditions that maximize representation of the microbiome while minimizing PCR-associated distortion of the community structure, especially in samples containing large amounts of host genomic DNA are critical. The goal of this study was to measure the effects of PCR annealing temperature and the ratio of host to bacterial DNA on the outcome of microbiota analysis, using pig microbiota as a model environment.Six samples were chosen with an anticipated range of ratios of pig to bacterial genomic DNA, and universal cpn60 PCR amplification with an annealing temperature gradient was used to create libraries for pyrosequencing, resulting in 426,477 sequences from the six samples. The sequences obtained were classified as target (cpn60) or non-target based on the percent identity of their closest match to the cpnDB reference database, and target sequences were further processed to create microbiome profiles for each sample at each annealing temperature. Annealing temperature affected the amount of PCR product generated, with more product generated at higher temperatures. Samples containing proportionally more host genomic DNA yielded more non-target reads, especially at lower annealing temperatures. However, microbiome composition for each sample across the annealing temperature gradient remained consistent at both the phylum and operational taxonomic unit levels. Although some microbial sequences were detected at only one annealing temperature, these sequences accounted for a minority of the total microbiome.These results indicate that PCR annealing temperature does have an affect on cpn60 based microbiome profiles, but that most of the differences are due to differences in detection of low abundance sequences. Higher annealing temperatures resulted in larger amounts of PCR product and lower amounts of non-target sequence amplification, especially in samples containing proportionally large amounts of host DNA. Taken together these results provide important information to guide decisions about experimental design for cpn60 based microbiome studies

Rapid, solid-phase based automated analysis of chromatin structure and transcription factor occupancy in living eukaryotic cells

Transcription factors, chromatin components and chromatin modification activities are involved in many diseases including cancer. However, the means by which alterations in these factors influence the epigenotype of specific cell types is poorly understood. One problem that limits progress is that regulatory regions of eukaryotic genes sometimes extend over large regions of DNA. To improve chromatin structure–function analysis over such large regions, we have developed an automated, relatively simple procedure that uses magnetic beads and a capillary sequencer for ligation-mediated-PCR (LM-PCR). We show that the procedure can be used for the rapid examination of chromatin fine-structure, nucleosome positioning as well as changes in transcription factor binding-site occupancy during cellular differentiation

A genotyping protocol for multiple tissue types from the polyploid tree species Sequoia sempervirens (Cupressaceae).

Premise of the studyIdentifying clonal lineages in asexually reproducing plants using microsatellite markers is complicated by the possibility of nonidentical genotypes from the same clonal lineage due to somatic mutations, null alleles, and scoring errors. We developed and tested a clonal identification protocol that is robust to these issues for the asexually reproducing hexaploid tree species coast redwood (Sequoia sempervirens).MethodsMicrosatellite data from four previously published and two newly developed primers were scored using a modified protocol, and clones were identified using Bruvo genetic distances. The effectiveness of this clonal identification protocol was assessed using simulations and by genotyping a test set of paired samples of different tissue types from the same trees.ResultsData from simulations showed that our protocol allowed us to accurately identify clonal lineages. Multiple test samples from the same trees were identified correctly, although certain tissue type pairs had larger genetic distances on average.DiscussionThe methods described in this paper will allow for the accurate identification of coast redwood clones, facilitating future studies of the reproductive ecology of this species. The techniques used in this paper can be applied to studies of other clonal organisms as well

Single cell transcriptome analysis using next generation sequencing.

The heterogeneity of tissues, especially in cancer research, is a central issue in transcriptome analysis. In recent years, research has primarily focused on the development of methods for single cell analysis. Single cell analysis aims at gaining (novel) insights into biological processes of healthy and diseased cells. Some of the challenges in transcriptome analysis concern low abundance of sample starting material, necessary sample amplification steps and subsequent analysis. In this study, two fundamentally different approaches to amplification were compared using next-generation sequencing analysis: I. exponential amplification using polymerase-chain-reaction (PCR) and II. linear amplification. For both approaches, protocols for single cell extraction, cell lysis, cDNA synthesis, cDNA amplification and preparation of next-generation sequencing libraries were developed. We could successfully show that transcriptome analysis of low numbers of cells is feasible with both exponential and linear amplification. Using exponential amplification, the highest amplification rates up to 106 were possible. The reproducibility of results is a strength of the linear amplification method. The analysis of next generation sequencing data in single cell samples showed detectable expression in at least 16.000 genes. The variance between samples results in a need to work with a greater amount of biological replicates. In summary it can be said that single cell transcriptome analysis with next generation sequencing is possible but improvements leading to a higher yield of transcriptome reads is required. In the near future by comparing single cancer cells with healthy ones for example, a basis for improved prognosis and diagnosis can be realised

Optimizing Genetic Manipulation of Methanogens through Faster Cloning Techniques

Methanogenesis is the biological production of methane. Only anaerobic archaea known as methanogens are capable of such a metabolic feat. They have strict living conditions and substrate sources which determine their rate of metabolism. This is of particular importance from a greenhouse gas reduction perspective or biogas capturing perspective. One of the best ways to optimize methanogen methane production is via genetic manipulation. The current procedures are timely though, therefore a faster cloning processes should be developed. The objective of this study was to optimize a premade genetic transformation kit known as the Gibson Kit. The Gibson Kit was supposed to ease the work of genetic manipulation by combining several linear chunks of DNA together at once via the use of sequence overhangs. The resulting plasmid from the Gibson could then be transformed into E. coli where E. coli is supposed to replicate the plasmids extremely quickly. Afterwards, that plasmid could then transform methanogens. Several baseline PCR (polymerase chain reaction) transformations were performed to linearize and amplify the desired plasmid of pNB72.3. PCR amplifications of the desired gene segments which would be added to the plasmid were also performed. The desired gene segments being assembled were supposed to take out the production of Cytochrome C within the methanogen electron transport system by deleting the ccmf gene. Several PCR experiments were carried through without success. The cause of the failures included the primers “hair pinning” at the recommended annealing temperatures, primers being too nonspecific, and primers unable to perform efficiently at the recommend annealing temperatures. After several tries of no success, the Gibson Kit was tested without PCR linearization of the circular plasmid. Instead, standard digestion was relied on. With standard electro competent cell transformation and antibiotic screening, the Gibson product was then tested for successful transmission of the plasmid to the E. coli. Results were negative; therefore, the optimization of faster cloning techniques was unsuccessful, but it will help guide future efforts

The Human Pregnancy-Specific Glycoprotein Genes are Tightly Linked on the Long Arm of Chromosome 19 and are Coordinately Expressed

The pregnancy-specific glycoprotein (PSG) genes encode a group of proteins which are found in large amounts in placenta and maternal serum. In situ hybridization analyses of metaphase chromosomes reveal that all the human pregnancy-specific glycoprotein (PSG) genes are located on the long arm of chromosome 19 (19q13.2–13.3), overlapping the region containing the closely-related carcinoembryonic antigen (CEA) gene subgroup. Higher resolution analyses indicate that the PSG genes are closely linked within an 800kb SacII restriction endonuclease fragment. This has been confirmed through restriction endonuclease mapping and DNA sequence analyses of isolated genomic clones, which show that at least some of these genes are located in very close proximity. Further, these studies have helped to identify a new member of the PSG gene sub-family (PSG7). DNA/RNA hybridization analyses, using gene-specific oligonucleotide probes based on published sequences, showed that five from six PSG genes tested are coordinately transcribed in the placenta. Due to the close proximity of these genes and their coordinated expression pattern, common transcriptional regulatory elements may exist

Magic-State Functional Units: Mapping and Scheduling Multi-Level Distillation Circuits for Fault-Tolerant Quantum Architectures

Quantum computers have recently made great strides and are on a long-term path towards useful fault-tolerant computation. A dominant overhead in fault-tolerant quantum computation is the production of high-fidelity encoded qubits, called magic states, which enable reliable error-corrected computation. We present the first detailed designs of hardware functional units that implement space-time optimized magic-state factories for surface code error-corrected machines. Interactions among distant qubits require surface code braids (physical pathways on chip) which must be routed. Magic-state factories are circuits comprised of a complex set of braids that is more difficult to route than quantum circuits considered in previous work [1]. This paper explores the impact of scheduling techniques, such as gate reordering and qubit renaming, and we propose two novel mapping techniques: braid repulsion and dipole moment braid rotation. We combine these techniques with graph partitioning and community detection algorithms, and further introduce a stitching algorithm for mapping subgraphs onto a physical machine. Our results show a factor of 5.64 reduction in space-time volume compared to the best-known previous designs for magic-state factories.Comment: 13 pages, 10 figure