Manual A Laboratory Guide to Genomic Sequencing: The Direct Sequencing of Native Uncloned DNA

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This modification eliminated the need to plate each LR-transfected reaction and reduced handling errors and the time-consuming screening step. The VR plasmid chosen as an expression vector for the development of DNA vaccines, expresses genes as fusion proteins with the tPA signal peptide at the N terminus. The immunogenicity of these DNA vaccines was compared by analyzing the antibody response in mice as measured by the capacity of antisera to recognize whole sporozoites parasites in an immunofluorescence antibody test IFAT.

Based on these in vitro and in vivo results, we concluded that expression of these two proteins by the VRDV destination vector was comparable to expression by the VR plasmid.

Immunogenicity of P. We set out to generate antibody reagents to individual antigens and determine their expression in the malaria parasite using a high-throughput approach. Because of the large number of sera to be analyzed, we performed the initial screen on group-pooled sera collected 2 wk after the second and third immunizations. The antibody screening was performed by the IFAT on three parasite stages, sporozoite and a mix of mature asexual erythrocytic and sexual erythrocytic stages.

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A list of the positive clones is provided as Supplemental Table 1. These results were consistent with the stage expression profiles determined by analysis of the P. All the positive genes for the gametocyte three and sporozoite three stages had high levels of RNA detected in these respective stages in the study of the transcriptome Le Roch et al. The remaining 74 DNA constructs that failed to induce antibodies to parasites generally were not identified in the proteome analysis in the parasite stages evaluated; only 24 of these genes had peptides detected from any parasite stage examined.

These results may reflect the fact that genes from Panel A were selected prior to publication of large-scale gene expression profiles. This correlation may indicate that the use of expression data as determined by proteome and transcriptome studies will be of significant value for predicting genes that will most likely induce an immune response and be useful in a reverse vaccinology approach.

Figure 4 shows examples of the stage specificity and the cellular localization of novel proteins observed by IFAT. It is possible that some of these genes showing no expression in the sporozoite and erythrocytic stages could be inducing antibodies to other stages of the parasite life cycle not tested here, including those inside of the mosquito oocysts and ookinetes and in the vertebrate host hepatocyte. Immunogenicity, stage expression, and localization of P. The murine sera were pooled and screened by IFAT against the sporozoite, asexual erythrocytic trophozoites and schizonts , and sexual erythrocytic gametocyte stages of P.

The asexual erythrocytic stage was a mixture of early and late trophozoites and schizonts. Sporozoites were isolated from mosquitos' salivary glands and gametocytes were percoll-purified from in vitro cultures. Of the 95 constructs screened, the number of positive genes were shown.

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The titer range is the lowest and the highest end-point IFA dilution scored for any positive group. Numbers on the left indicate the accession number for ORFs. Preliminary attempts to express genes from Panel A as recombinant proteins in GST- and MBP-fusion expression constructs had limited success; only seven out of 95 fusion proteins were expressed, and five of these were determined soluble. These finding are in contrast with protein expression efficiency shown for Gateway constructs encoding human ORFs using the same expression vectors Braun et al.

Subsequent to this effort, we have attempted to express recombinant proteins from genes from Panel B, cloned into a GST-fusion expression plasmid. Aguiar, K. Simmon, J. Ho, K.

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Widjaja, M. Von Rechenberg, T. Zarembinski, R. Hughes, and J. Peltier, unpubl.

The major feature noted between the expressing and nonexpressing sets of clones is the size of the genes averaging bp vs. Many malaria researchers attribute the low rate of recombinant protein expression to its toxic effect on E. We set out to bypass the E. Although this represents a slight improvement over our E. We are currently characterizing the proteins generated in both E. In an attempt to express small to mid size bp to 1.

Sharma, J.

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Bray, J. Lew, M. Vedadi, A. Edwards, and C. Arrowsmith, unpubl. Although other alternative expression systems have been shown to improve the expression of malaria proteins such as baculovirus Liang et al. Codon optimization has routinely improved protein expression, although the cost of synthesizing large numbers of codon-optimized genes limits their utility in high-throughput cloning efforts.

Improved methods of expressing P. In conclusion, we demonstrated that the Gateway cloning system provides a highly efficient method to produce recombinant plasmids containing genes from P. Noteworthy is the fact that most of the P. Using a limited number of ORFs and the DNA vaccine strategy, we have been able to further explore the functional profile and identify the stage-specific expression of novel antigens with antisera produced in mice.

These data establish the Gateway system as a platform technology, which provides for the rapid and flexible design and construction of recombinant plasmids needed for functional genomics studies and for vaccine and drug development efforts. Using this system, one will be able to perform high-throughput functional assays using a variety of expression vectors, that is, protein expression vectors to generate reagents for protein microarray experiments and animal immunization, conduct large-scale transfection experiments to assay protein localization, determine immunogenicity of candidate antigens, develop expression vectors for Y2H systems to screen for host receptors and parasite protein interactions, and develop large numbers of recombinant virus constructs that can be used in immune assays for screening protective antigens Doolan et al.

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Different ORF selection criteria were applied to evaluate the high-throughput feasibility of the Gateway technology across multiple P. The first panel of genes Panel A was comprised of single-exon ORFs from early Chromosomes 2 and 3 annotations and ranged in size from 0. The genes in Panel B were selected based on a set of credentialed data for their potential as vaccine targets Supplemental Table 2. A separate functional genomics approach led to the creation of each of the six categories.

Sacci, J. Ribeiro, F. Huang, U. Alam, J. Russell, P. Blair, A. Witney, D. Carucci, A. Azad, J. Aguiar, et al. Panel B was comprised of either single-exon genes or the major exon from genes with predicted multiexon gene structures.

An entire list of cloned genes is provided in Supplemental Table 2. The nested PCR strategy briefly described below was used to reduce the overall length of hundred of primers, thereby significantly reducing the cost incurred with primer synthesis.

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Full-length genes were amplified from P. Before proceeding with the BP reactions, PCR products were run on an agarose gel to confirm the size of the amplicons and then purified using one of two protocols. This adaptation increased the experimental throughput, limited sample handling, and reduced time without affecting DNA yield or quality. The cloning to obtain master clones was performed using the BP reaction kit Invitrogen following the protocol recommended by the manufacturer.

The latter encodes the zeocin-resistance gene, which allows the subsequent use of destination plasmids with a variety of antibiotic resistance markers and the use of M13 universal primers. For the purpose of assessing the cloning efficiency, four colonies per capture reaction BP reaction were selected for screening by PCR and by DNA sequencing using plasmid-specific primers.

A Laboratory Guide to Genomic Sequencing: The Direct Sequencing of Native Uncloned DNA (BIOMETHODS)

To sequence across the hairpin recombination segments of the clones, an optimized sequence cycling protocol was developed. Duplicate glycerol stocks were then generated from each positive Master clone. Apart from the genes selected, we also included two well-characterized malaria genes: the P. Two protocols were used; one recommended by the manufacturer that screens single-colony for recombinants is described here; the other method described later was adapted to truly high throughput by screening clones in bulk.

The screening was performed with one colony per LR reaction. In parallel to the single-colony approach described above, we also tested an alternative screening for these LR reactions. All steps for this procedure were performed in a well platform. PCR amplicons were analyzed in an agarose gel running in parallel with PCRs from single colony screening. Sign up to take part. A Nature Research Journal. Although polymerase chain reaction PCR is the most widely used method for DNA amplification, the requirement of thermocycling limits its non-laboratory applications. Isothermal DNA amplification techniques are hence valuable for on-site diagnostic applications in place of traditional PCR.

In combination with a peptide nucleic acid PNA invasion-mediated endpoint measurement, the method exhibits attomolar sensitivity and single-nucleotide specificity in detection of various DNA targets under a complex sample background. In summary, CRISDA is a powerful isothermal tool for ultrasensitive and specific detection of nucleic acids in point-of-care diagnostics and field analyses.

Moreover, some of these techniques still require an initial heat-denaturation step to unwind double-stranded DNA dsDNA targets thereby further limiting their applications. Moreover, previous investigation has revealed that the non-target strand of DNA targets in CRISPR ribonucleoprotein complexes are fully unwound and exposed to the environment formation of R-loop , which are susceptible to hydrolysis 25 , This unique conformational rearrangement may provide an ideal targeting site for various isothermal amplification techniques with enhanced robustness, specificity, and sensitivity due to the intrinsic properties of CRISPR effectors.

In combination with a robust peptide nucleic acid PNA invasion-mediated endpoint measurement 29 , 30 , the method achieves attomolar sensitivity and single-base specificity in the presence of a complex sample background. After conducting a series of proof-of-concept investigations, the attomolar sensitivity of CRISDA is examined by specific amplification and detection towards target DNA fragments as well as target regions in the genomes of human and genetically modified soybean MON The single-nucleotide specificity of CRISDA is evaluated by breast cancer-associated SNPs genotyping among various cell lines and finally, the versatility of CRISDA is demonstrated by integrating the technique with a Cas9-mediated target enrichment approach exhibiting sub-attomolar sensitivity.

CRISDA is demonstrated to be a powerful tool in ultrasensitive detection and diagnosis of nucleic acids.