| 1. |
- Bar, Tzachi, 1970, et al.
(författare)
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Kinetics quality assessment for relative quantification by real-time PCR
- 2005
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Ingår i: BioTechniques. - 0736-6205 .- 1940-9818. ; 39:3, s. 333-
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Tidskriftsartikel (refereegranskat)abstract
- For proper relative quantification by real-time PCR, compared samples should have similar PCR efficiencies. To test this prerequisite, we developed two quality tests: (i) adjustment of a test for kinetic outlier detection (KOD) to relative quantification; and (ii) comparison of the efficiency variance of test samples with the efficiency variance of samples with highly reproducible quantification. The tests were applied on relative quantification of two genes in 30 sets of 5 replicate samples (same treatment, different animals). Ten low-quality sets and 28 outliers were identified. The low-quality sets showed higher coefficient of variation (cv)% of DNA quantities in replicate experiments than high-quality sets (63% versus 26%; P = 0.001) and contained a higher proportion of outlying quantities (35% versus 5.9%; P = 0.001) when individual samples were detected by adjusted KOD. Outlier detection with adjusted KOD reduced thefalse detection of outliers by 213 compared with the previous, nonadjusted version of KOD (20% versus 5.9%; P = 0.001). We conclude that the presented tests can be used to assign technical reasons to outlying observations.
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| 2. |
- Darmanis, Spyros, et al.
(författare)
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Self-assembly of proximity probes for flexible and modular proximity ligation assays
- 2007
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Ingår i: BioTechniques. - : Future Science Ltd. - 0736-6205 .- 1940-9818. ; 43:4, s. 443-450
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Tidskriftsartikel (refereegranskat)abstract
- Proximity ligation assay (PLA) is a recently developed strategy for protein analysis in which antibody-based detection of a target protein via a DNA ligation reaction of oligonucleotides linked to the antibodies results in the formation of an amplifiable DNA strand suitable for analysis. Here we describe a faster and more cost-effective strategy to construct the antibody-based proximity ligation probes used in PLA that is based on the noncovalent interaction of biotinylated oligonucleotides with streptavidin followed by the interaction of this complex with biotinylated antibodies.
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| 3. |
- Gidlöf, Olof, et al.
(författare)
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Complete discrimination of six individuals based on high-resolution melting of hypervariable regions I and II of the mitochondrial genome
- 2009
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Ingår i: BioTechniques. - : Future Science Ltd. - 0736-6205 .- 1940-9818. ; 47:2, s. 671-678
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Tidskriftsartikel (refereegranskat)abstract
- Analysis of mitochondrial DNA in forensic samples is routinely carried out by direct sequencing of hypervariable regions within the non-coding displacement loop. Although the accuracy and sensitivity of this method cannot be questioned, it is both time-consuming and labor intensive. Finding a way to rapidly pre-screen forensic samples-prior to sequencing, to reduce the number of samples that need to be sequenced-would greatly benefit forensic laboratories. Herein, we describe an assay for discrimination of DNA from different individuals based on high-resolution melting analysis of the two hypervariable regions HVI and HVII of the mitochondrial genome. By clearly distinguishing the DNA melting curves of six different individuals, we show that this assay has the potential to function as a rapid and inexpensive pre-screening method for forensic samples prior to DNA sequencing.
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| 4. |
- Hedman, Johannes, et al.
(författare)
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Improved forensic DNA analysis through the use of alternative DNA polymerases and statistical modeling of DNA profiles
- 2009
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Ingår i: BIOTECHNIQUES. - : Eaton Publishing. - 0736-6205 .- 1940-9818. ; 47:5, s. 951-958
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Tidskriftsartikel (refereegranskat)abstract
- DNA evidence, linking perpetrators to crime scenes, is central to many legal proceedings. However, DNA samples from crime scenes often contain PCR-inhibitory substances, which may generate blank or incomplete DNA profiles. Extensive DNA purification can be required to rid the sample of these inhibitors, although these procedures increase the risk of DNA loss. Most forensic laboratories use commercial DNA amplification kits (e.g., AmpFlSTR SGM Plus) with the DNA polymerase AmpliTaq Gold as the gold standard. Here, we show that alternative DNA polymerase-buffer systems can improve the quality of forensic DNA analysis and efficiently circumvent PCR inhibition in crime scene samples, without additional sample preparation. DNA profiles from 20 of 32 totally or partially inhibited crime scene saliva samples were significantly improved using Bio-X-Act Short, ExTaq Hot Start, or PicoMaxx High Fidelity instead of AmpliTaq Gold. A statistical model for unbiased quality control of forensic DNA profiles was developed to quantify the results. Our study demonstrates the importance of adjusting the chemistry of the PCR to enhance forensic DNA analysis and diagnostic PCR, providing an alternative to laborious sample preparation protocols.
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| 5. |
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| 6. |
- Lind, Kristina, 1977, et al.
(författare)
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Combining sequence-specific probes and DNA binding dyes in real-time PCR for specific nucleic acid quantification and melting curve analysis
- 2006
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Ingår i: BioTechniques. - : Future Science Ltd. - 0736-6205 .- 1940-9818. ; 40:3, s. 315-319
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Tidskriftsartikel (refereegranskat)abstract
- Currently, in real-time PCR, one often has to choose between using a sequence-specific probe and a nonspecific double-stranded DNA (dsDNA) binding dye for the detection of amplified DNA products. The sequence-specific probe has tire advantage that it only detects the targeted product, while the nonspecific dye has the advantage that melting curve analysis can be performed after completed amplifcation, which reveals what kind of products have been formed. Here we present a new strategy based on combining a sequence-specific probe and a nonspecific dye, BOXTO, in the swine reaction, to take the advantage of both chemistries. We show that BOXTO can be used together with both TaqMan((R)) probes and locked nucleic acid (LNA) probes without interfering with the PCR. The probe signal reflect formation of target product, while melting curve analysis of the BOXTO signal reveals primer-dinter formation and the presence of any other anomalous products.
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| 7. |
- Lindskog, Mats, et al.
(författare)
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Selection of protein epitopes for antibody production
- 2005
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Ingår i: BioTechniques. - 0736-6205 .- 1940-9818. ; 38:5, s. 723-727
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Tidskriftsartikel (refereegranskat)abstract
- Protein functional analysis in the post-genomic era is a huge task that has to be approached by different methods in parallel. The use of protein-specific antibodies in conjunction with tissue microarrays has proven to be one important technology. In this study, we present a strategy for the optimized design of protein subfragments for subsequent antibody production. The fragments are selected based on a principle of lowest sequence similarity to other human proteins, optimally to generate antibodies with high selectivity. Furthermore, the fragments should have properties optimized for efficient protein production in Escherichia coli. The strategy has been implemented in Bishop, which is a Java-based software enabling the high-throughput production of protein fragments. Bishop allows for the avoidance of certain restriction enzyme sites, transmembrane regions, and signal peptides. A Basic Local Alignment Search Tool (BLAST) scanning procedure permits the selection of fragments of a selected size with a minimal sequence similarity to other proteins. The software and the strategy were evaluated on a human test data set and verified to fulfill the requested criteria.
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| 8. |
- Uhlén, Mathias
(författare)
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Affinity as a tool in life science
- 2008
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Ingår i: BioTechniques. - : Future Science Ltd. - 0736-6205 .- 1940-9818. ; 44:5, s. 649-654
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Forskningsöversikt (refereegranskat)abstract
- The use of affinity-based tools has become invaluable as a platform for basic research and in the development of drugs and diagnostics. Applications include affinity chromatography and affinity tag fusions for efficient purification of proteins as well as methods to probe the protein network interactions on a whole-proteome level. A variety of selection systems has been described for in vitro evolution of affinity reagents using combinatorial libraries, which make it possible to create high-affinity reagents to virtually all biomolecules, as exemplified by generation of therapeutic antibodies and new protein scaffold binders. The strategies for high-throughput generation of affinity reagents have also opened up the possibility of generating specific protein probes on a whole-proteome level. Recently, such affinity proteomics have allowed the detailed analysis of human protein expression in a comprehensive manner both in normal and disease tissue using tissue microarrays and confocal microscopy.
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| 9. |
- Vennström, Lina, et al.
(författare)
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Real-time viability assay based on 51Cr retention in adherent cells
- 2008
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Ingår i: BioTechniques. - : Future Science Ltd. - 0736-6205 .- 1940-9818. ; 44:2, s. 237-240
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Tidskriftsartikel (refereegranskat)abstract
- The chromium (51Cr) release assay has been widely used for viability measurements, even though it has major disadvantages such as high manual workload and poor time resolution. By the use of LigandTracer 51Cr release viability measurements on adherent cells can be significantly simplified and improved. LigandTracer enables a time-resolved detection of 5SCr in target cells, with the result that the effect of toxic material is updated continuously throughout the experiment. Here we explain the principle behind this novel real-time viability assay and show viability curves for known toxic compounds on A431 and U343MGaCl2:6 cell lines.
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| 10. |
- Wilkening, S, et al.
(författare)
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Determination of allele frequency in pooled DNA: comparison of three PCR-based methods
- 2005
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Ingår i: BioTechniques. - : Future Science Ltd. - 0736-6205 .- 1940-9818. ; 39:6, s. 853-858
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Tidskriftsartikel (refereegranskat)abstract
- Determination of allele frequency in pooled DNA samples is a powerful and efficient tool for large-scale association studies. In this study, we tested and compared three PCR-based methods for accuracy, reproducibility, cost, and convenience. The methods compared were: (i) real-time PCR with allele-specific primers, (ii) real-time PCR with allele-specific TaqMan® probes, and (iii) quantitative sequencing. Allele frequencies of three single nucleotide polymorphisms in three different genes were estimated from pooled DNA. The pools were made of genomic DNA samples from 96 cases with basal cell carcinoma of the skin and 96 healthy controls with known genotypes. In this study, the allele frequency estimation made by real-time PCR with allele-specific primers had the smallest median deviation (MD) from the real allele frequency with 1.12% (absolute percentage points) and was also the cheapest method. However, this method required the most time for optimization and showed the highest variation between replicates (SD = 6.47%). Quantitative sequencing, the simplest method, was found to have intermediate accuracies (MD = 1.44%, SD = 4.2%). Real-time PCR with TaqMan probes, a convenient but very expensive method, had an MD of 1.47% and the lowest variation between replicates (SD = 3.18%).
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