scholarly journals Chemical and biochemical strategies for the randomization of protein encoding DNA sequences: library construction methods for directed evolution

2004 ◽  
Vol 32 (4) ◽  
pp. 1448-1459 ◽  
Author(s):  
C. Neylon
Keyword(s):  
Directed Evolution ◽  
Dna Sequences ◽  
Library Construction ◽  
Construction Methods ◽  
Protein Encoding

A semi-rational design strategy of directed evolution combined with chemical synthesis of DNA sequences

2007 ◽  
Vol 388 (12) ◽  
pp. 1291-1300 ◽  
Author(s):  
Ai-Sheng Xiong ◽  
Ri-He Peng ◽  
Jing Zhuang ◽  
Jin-Ge Liu ◽  
Feng Gao ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Dna Sequences ◽  
Active Sites ◽  
Rational Design ◽  
Specific Activity ◽  
Dna Shuffling ◽  
Gene Encoding ◽  
Library Construction ◽  
Key Sites

Abstract Directed evolution in vitro is a powerful molecular tool for the creation of new biological phenotypes. It is unclear whether it is more efficient to mutate an enzyme randomly or to mutate just the active sites or key sites. In this study, the strategy of a semi-rational design of directed evolution combined with whole sequence and sites was developed. The 1553 bp gene encoding the thermostable β-galactosidase of Pyrococcus woesei was chemically synthesized and optimized for G+C content and mRNA secondary structures. The synthesized gene product was used as a template or as a wild-type control. On the basis of the first round of DNA shuffling, library construction and screening, one mutant of YH6754 was isolated with higher activity. Eight potential key sites were deduced from the sequence of the shuffled gene, and 16 degenerate oligonucleotides were designed according to those eight amino acids. Two variants of YG6765 and YG8252 were screened in the second part of DNA shuffling, library construction and screening. For comparison, one mutant of YH8757 was screened through the same routine rounds of directed evolution with YH6754 as template. The purified β-galactosidase from YH8757 exhibited a lower specific activity at 25°C than those purified from mutated YG6755 and YG8252.

scholarly journals Antigen Presentation of mRNA-Based and Virus-Vectored SARS-CoV-2 Vaccines

Vaccines ◽  
2021 ◽  
Vol 9 (8) ◽  
pp. 848
Author(s):  
Ger T. Rijkers ◽  
Nynke Weterings ◽  
Andres Obregon-Henao ◽  
Michaëla Lepolder ◽  
Taru S. Dutt ◽  
...  
Keyword(s):  
Antigen Presentation ◽  
Conformational Changes ◽  
Dna Sequences ◽  
Neutralizing Antibodies ◽  
Large Scale ◽  
Viral Vector ◽  
Adenovirus Vector ◽  
Platelet Factor ◽  
Immune Mediated ◽  
Protein Encoding

Infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causes Coronavirus Disease 2019 (COVID-19), which has reached pandemic proportions. A number of effective vaccines have been produced, including mRNA vaccines and viral vector vaccines, which are now being implemented on a large scale in order to control the pandemic. The mRNA vaccines are composed of viral Spike S1 protein encoding mRNA incorporated in a lipid nanoparticle and stabilized by polyethylene glycol (PEG). The mRNA vaccines are novel in many respects, including cellular uptake and the intracellular routing, processing, and secretion of the viral protein. Viral vector vaccines have incorporated DNA sequences, encoding the SARS-CoV-2 Spike protein into (attenuated) adenoviruses. The antigen presentation routes in MHC class I and class II, in relation to the induction of virus-neutralizing antibodies and cytotoxic T-lymphocytes, will be reviewed. In rare cases, mRNA vaccines induce unwanted immune mediated side effects. The mRNA-based vaccines may lead to an anaphylactic reaction. This reaction may be triggered by PEG. The intracellular routing of PEG and potential presentation in the context of CD1 will be discussed. Adenovirus vector-based vaccines have been associated with thrombocytopenic thrombosis events. The anti-platelet factor 4 antibodies found in these patients could be generated due to conformational changes of relevant epitopes presented to the immune system.

scholarly journals Combination of computational prescreening and experimental library construction can accelerate enzyme optimization by directed evolution

2005 ◽  
Vol 18 (11) ◽  
pp. 509-514 ◽  
Author(s):  
Susanne Aileen Funke ◽  
Nikolaj Otte ◽  
Thorsten Eggert ◽  
Marco Bocola ◽  
Karl-Erich Jaeger ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Library Construction ◽  
Enzyme Optimization

scholarly journals A newly formed telomere in Ascaris suum does not exert a telomere position effect on a nearby gene.

1996 ◽  
Vol 16 (1) ◽  
pp. 130-134 ◽  
Author(s):  
Y J Huang ◽  
R Stoffel ◽  
H Tobler ◽  
F Mueller
Keyword(s):  
Dna Sequences ◽  
Developmental Stages ◽  
Ascaris Suum ◽  
Chromosomal Rearrangements ◽  
Position Effect ◽  
Start Codon ◽  
Chromosomal Breakage ◽  
Chromatin Diminution ◽  
Developmentally Regulated ◽  
Protein Encoding

During the process of chromatin diminution in Ascaris suum (formerly named Ascaris lumbricoides var. suum), developmentally regulated chromosomal fragmentation and new telomere addition occur within specific chromosomal breakage regions (CBRs). The DNA sequences flanking one of these CBRs (CBR-1) were analyzed, and two protein-encoding genes were found on either side. The noneliminated gene, agp-1, whose AUG start codon is located within approximately 2 kb of the boundary of CBR-1, encodes a putative GTP-binding protein which is structurally related to eukaryotic and prokaryotic elongation factors. Northern (RNA) blot analyses revealed that transcripts of this gene are present at all developmental stages, suggesting that the massive chromosomal rearrangements associated with the process of chromatin diminution have no influence on agp-1 expression. This demonstrates that addition of new telomeres in CBR-1 does not result in a telomeric position effect, a phenomenon previously described in Saccharomyces cerevisiae.

scholarly journals Deep Impact of Random Amplification and Library Construction Methods on Viral Metagenomics Results

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 253
Author(s):  
Béatrice Regnault ◽  
Thomas Bigot ◽  
Laurence Ma ◽  
Philippe Pérot ◽  
Sarah Temmam ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Limit Of Detection ◽  
Viral Metagenomics ◽  
High Background ◽  
Deep Impact ◽  
Library Construction ◽  
Viral Genomes ◽  
Construction Methods ◽  
Random Amplification ◽  
Reference Virus

Clinical metagenomics is a broad-range agnostic detection method of pathogens, including novel microorganisms. A major limit is the low pathogen load compared to the high background of host nucleic acids. To overcome this issue, several solutions exist, such as applying a very high depth of sequencing, or performing a relative enrichment of viral genomes associated with capsids. At the end, the quantity of total nucleic acids is often below the concentrations recommended by the manufacturers of library kits, which necessitates to random amplify nucleic acids. Using a pool of 26 viruses representative of viral diversity, we observed a deep impact of the nature of sample (total nucleic acids versus RNA only), the reverse transcription, the random amplification and library construction method on virus recovery. We further optimized the two most promising methods and assessed their performance with fully characterized reference virus stocks. Good genome coverage and limit of detection lower than 100 or 1000 genome copies per mL of plasma, depending on the genome viral type, were obtained from a three million reads dataset. Our study reveals that optimized random amplification is a technique of choice when insufficient amounts of nucleic acid are available for direct libraries constructions.

scholarly journals Comparison of double-stranded and single-stranded cfDNA library construction methods for targeted genome and methylation sequencing

2022 ◽  
Author(s):  
Jianchao Zheng ◽  
Zhilong Li ◽  
Xiuqing Zhang ◽  
Hongyun Zhang ◽  
Shida Zhu ◽  
...  
Keyword(s):  
Deep Sequencing ◽  
Mutation Detection ◽  
Therapy Response ◽  
Utilization Efficiency ◽  
Response Monitoring ◽  
Library Construction ◽  
Construction Methods ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing ◽  
Methylation Profiling

Cell-free DNA (cfDNA) profiling by deep sequencing (i.e., by next generation sequencing (NGS)) has wide applications in cancer diagnosis, prognosis, and therapy response monitoring. One key step of cfDNA deep sequencing workflow is NGS library construction, whose efficiency significantly affects the utilization efficiency of cfDNA molecules, and eventually determines effective sequencing depth and sequencing accuracy. In this study, we compared two different types of cfDNA library construction methods, namely double-stranded library (dsLib, the conventional method which captures dsDNA molecules) and single-stranded library (ssLib) preparation, which captures ssDNA molecules, for the applications of mutation detection and methylation profiling, respectively. Our results suggest that the dsLib method was suitable for mutation detection while the ssLib method proved more efficient for methylation analysis. Our findings could help researchers choose the more appropriate library construction method for corresponding downstream applications of cfDNA sequencing.

Solid-Phase Gene Synthesis for Mutant Library Construction: The Future of Directed Evolution?

ChemBioChem ◽  
2018 ◽  
Vol 19 (19) ◽  
pp. 2023-2032 ◽  
Author(s):  
Aitao Li ◽  
Zhoutong Sun ◽  
Manfred T. Reetz
Keyword(s):  
Directed Evolution ◽  
Solid Phase ◽  
Gene Synthesis ◽  
Mutant Library ◽  
Library Construction ◽  
The Future

scholarly journals Evaluation of Nanopore sequencing technology to differentiate Salmonella serotypes and serotype variants with the same or closely related antigenic formulae

2020 ◽  
Author(s):  
Feng Xu ◽  
Chongtao Ge ◽  
Shaoting Li ◽  
Silin Tang ◽  
Xingwen Wu ◽  
...  
Keyword(s):  
Sensu Stricto ◽  
Source Tracking ◽  
List Type ◽  
Snp Analysis ◽  
Nanopore Sequencing ◽  
Sequencing Data ◽  
Library Construction ◽  
Construction Methods ◽  
Snp Typing ◽  
Industry Applications

ABSTRACTOur previous study demonstrated that whole genome sequencing (WGS) data generated by Oxford Nanopore Technologies (ONT) can be used for rapid and accurate prediction of Salmnonella serotypes. However, one limitation is that established methods for WGS-based serotype prediction cannot differentiate certain serotypes and serotype variants with the same or closely related antigenic formulae. This study aimed to evaluate Nanopore sequencing and corresponding data analysis for differentiation of these serotypes and serotype variants, thus overcoming this limitation. Five workflows that combined different flow cells, library construction methods and basecaller models were evaluated and compared. The workflow that consisted of the R9 flow cell, rapid sequencing library construction kit and guppy basecaller with base modified model performed best for Single Nucleotide Polymorphism (SNP) analysis. With this workflow, as high as 99.98% matched the identity of the assembled genomes and only less than five high quality SNPs (hqSNPs) between ONT and Illumina sequencing data were achieved. SNP typing allowed differentiation of Choleraesuissensu stricto, Choleraesuis var. Kunzendorf, Choleraesuis var. Decatur, Paratyphi C, and Typhisuis that share the same antigenic formula 6,7:c:1,5. Prophage prediction further distinguished Orion var. 15+ and Orion var. 15+, 34+. Our study improves the readiness of ONT as a Salmonella subtyping and source tracking tool for food industry applications.HighlightsSalmonella serotypes or serotype variants with the same antigenic formula were differentiated by SNP typing.Nanopore sequencing followed by phage prediction identified the Salmonella serotype variants caused by phage conversion.The latest ONT technology is capable of high fidelity SNP typing of Salmonella.

Development Of Methods To Perform Next Generation Sequencing (NGS)-Based Genomics Studies On Multiple Myeloma Clinical Samples

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5347-5347
Author(s):  
Jonathan J Keats ◽  
Kevin A Kwei ◽  
Jeremiah D Degenhardt ◽  
Kristi Allen ◽  
Christopher J. Kirk ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Clinical Samples ◽  
Employment Equity ◽  
Library Construction ◽  
Rna Integrity ◽  
Construction Methods ◽  
Dna And Rna ◽  
Equity Ownership ◽  
Dna 2

Abstract NGS is a powerful tool for interrogating genomic alterations in cancer cells from the scale of single nucleotides up to whole chromosomes. While methods for performing NGS analysis on solid tumors have been widely reported in the literature, fewer methods are available for hematological malignancies, which are often collected and stored in different conditions. Here we report our efforts to overcome the unique challenges of performing a whole exome sequencing (WES) study on hundreds of multiple myeloma (MM) patient samples. As part of Onyx-sponsored clinical trials of the proteasome inhibitor, carfilzomib, bone marrow aspirates were collected from patients and subjected to bead-based CD138 positive selection to enrich tumor cells. The yield of CD138+ cells after enrichment varied greatly from 200,000 to >10,000,000, depending primarily on the disease state and the volume and quality of aspirate received. CD138+ cells were banked in a variety of formats that differed in their suitability for downstream DNA and RNA analysis, including as pellets or re-suspended in reagents such as TRIzol®, RNAlater and Buffer RLT. At the time of abstract submission we have performed extensive analyses of samples stored in TRIzol. TRIzol is renowned for its excellence in preserving RNA integrity while providing a means to simultaneously extract DNA and protein. However, the standard DNA extraction method yields material that is not of sufficient quality for DNA analyses such as WES. We attempted to remedy this problem through three approaches: (1) modifying extraction conditions to improve the quality of the DNA, (2) rescuing the extracted DNA with double-stranding steps prior to library preparation, and (3) applying newer, low input and single-strand library construction methods. Our efforts to modify the extraction conditions were unsuccessful, but by combining DNA rescue and newer library construction methods, we could ultimately generate high quality exomes (as judged by comparison to exomes generated from matched, pelleted samples) from the whole range of clinical samples in our biobank. We believe these methods create new opportunities for the community to interrogate, with the latest NGS approaches, vast numbers of clinical samples banked in the era of gene-expression profiling (GEP). Disclosures: Kwei: Onyx Pharmaceuticals: Employment, Equity Ownership. Degenhardt:Onyx Pharmaceuticals: Employment, Equity Ownership. Kirk:Onyx Pharmaceuticals: Employment, Equity Ownership. Tuch: Onyx Pharmaceuticals: Employment, Equity Ownership.

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