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Genetic analysis of the embryo
Published in David K. Gardner, Ariel Weissman, Colin M. Howles, Zeev Shoham, Textbook of Assisted Reproductive Techniques, 2017
Yuval Yaron, Liran Hiersch, Veronica Gold, Sagit Peleg-Schalka, Mira Malcov
Multiplex PCR refers to the simultaneous amplification of more than one fragment in the same PCR reaction using more than one pair of unrelated primers (12,15,16,22). One or more primer pairs amplify the DNA fragment con- taining the locus to be tested, while the other(s) serve as a positive control within the same reaction. Amplification of multiple loci within the same multiplex PCR reaction is possible in single blastomeres. This requires careful primer design and reaction optimization to ensure that all primer sets amplify efficiently under the same conditions including annealing temperatures and concentrations of the different reagents in the PCR buffer, such as MgCl2. Careful design of primers is mandated in order to avoid primer dimer formation, interaction between different PCR products, and interaction of primers with products. The primers should be designed such that the product of each PCR primer pair is of a different size so that it may be distinguished by gel electrophoresis. Alternatively, differ- ent fluorescent tags can be used for each primer pair.
Experimental Protocols for Generation and Evaluation of Articular Cartilage
Published in Kyriacos A. Athanasiou, Eric M. Darling, Grayson D. DuRaine, Jerry C. Hu, A. Hari Reddi, Articular Cartilage, 2017
Kyriacos A. Athanasiou, Eric M. Darling, Grayson D. DuRaine, Jerry C. Hu, A. Hari Reddi
While multiple chemistries are available for quantitative real-time PCR, SYBR Green and probe-based primer chemistries are the most common. SYBR Green binds to double-stranded DNA and has the advantage of requiring only forward and reverse primers, without needing a probe (reduced cost). However, significant primer optimization is required to prevent primer dimers and other false signals. Given the commercial availability of many primer-probe combinations, these are often the preferred choice.
Target Amplification-Based Techniques
Published in Attila Lorincz, Nucleic Acid Testing for Human Disease, 2016
Antoinette A.T.P. Brink, Peter J.F. Snijders, Chris J.L.M. Meijer
Amplification primer design can be performed with commercially available software (e.g., Oligo, [Molecular Biology Insights, Cascade, Colorado, USA, http://www.oligo.net], Vector NTI [Invitrogen, http://www.invitrogen.com], Primer Express [Applied Biosystems, Foster City, California, USA, http://www.appliedbiosystems.com]) or shareware on the internet (e.g., Primer3, http://frodo.wi.mit.edu/primer3/primer3_code.html). These programs calculate the optimal primer pair for the sequence to be amplified, taking into account sequence-specific parameters such as GC and repeat contents and user-defined parameters such as desired amplicon lengths. For most amplification methods, primers are approximately 20 bases in length and have GC contents approaching 50%. Furthermore, they should not form internal secondary structures such as hairpin loops and the 3′ ends of the two primers should not be complementary because this would lead to the generation of primer dimers.
Ocimum sanctum, Zingiber officinale, and Piper nigrum extracts and their effects on gut microbiota modulations (prebiotic potential), basal inflammatory markers and lipid levels: oral supplementation study in healthy rats
Published in Pharmaceutical Biology, 2022
Narendra Babu Kondapalli, Rajkumar Hemalatha, Satyanarayana Uppala, Srinivas Reddy Yathapu, Shujauddin Mohammed, Mullapudi Venkata Surekha, Ananthan Rajendran, Dinesh Kumar Bharadwaj
The primers used in the investigation are listed in Table 1. RT-PCR reactions were carried out on MicroAmp Fast Optical 96-Well Reaction Plates using a StepOnePlus Real-Time PCR System (Applied Biosystems, CA, and U. S. A). Each reaction contained 2 µL of diluted DNA, 0.5 µL of 10 µM solution of each primer, and 5 µL of Power SYBR Green PCR Master Mix (Applied Biosystems) in a final volume of 10 µL. The PCR amplification reactions were carried out as follows: 6 min at 50 °C, 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 30 s at 55 °C for Lactobacillus and 1 min at 60 °C for Bifidobacterium. The PCR amplification reactions were carried out as follows: 6 min at 50 °C, 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C for Bacteroide and Firmicutes. Detection was carried out on a StepOnePlus Real-Time PCR System (Applied Biosystems, CA, USA). To rule out the possibility of primer dimer formation, the amplified product obtained with each primer pair was checked by agarose gel electrophoresis (2% agarose) followed by melting curve analysis of the qRT-PCR amplified products. Each assay was performed in triplicate in the same run. To determine the sensitivity and specificity of the assays, the PCR assays were confirmed using controls. Bacterial estimation was done by qRT-PCR and their relative expression was determined using the ΔΔCt method with all bacterial as an endogenous control gene. The data were presented as the fold change in gene expression normalized to the endogenous reference gene (all bacterial primer) relative to the baseline time-point.
Design, optimization, and application of multiplex rRT-PCR in the detection of respiratory viruses
Published in Critical Reviews in Clinical Laboratory Sciences, 2022
Jing Yang, Dandan Li, Jie Wang, Rui Zhang, Jinming Li
For multiplex rRT-PCR, it is necessary to reduce the secondary structure and interaction among primers, probes and the template. The secondary structure of the target region will markedly damage its affinity in hybridization, thus damaging the detection performance (Figure 4(B-a,b)) [37,38]. Fan et al. designed primers covering different positions for the hairpin structure template containing a 9 bp stem and 15 nt loop, and specifically described the effect of the secondary structure of the template on amplification [39]. The results showed that Ct values from big to small were primer 3 > primer 4 > primer 5 > primer 2 > primer 6 > primer 1 > primer F (Figure 4(B-c)). Compared with primer F, primer 6 was only 5 nt complementary to the stem, but the Ct value increased significantly. Primers 3 and 4 covered a large area of the hairpin structure, and thus the amplification was inhibited. The coverage areas of primers 1 and 2 were close to the 5′ end of the hairpin structure and had little effect on amplification. Additionally, if two primers are complementary at the 3′ end, DNA polymerase can bind to them and extend the primers in both directions to generate primer dimers. However, if two primers are bound at the 5′ end or in the middle, they will not be bound by DNA polymerase and extended [40]. The 3′ end complementarity of primers is an important parameter for evaluating the formation of primer dimers; it is critical to analyze the position and secondary structure of amplification before design (Figure 4(B)) [41].
Smartphone technology facilitates point-of-care nucleic acid diagnosis: a beginner’s guide
Published in Critical Reviews in Clinical Laboratory Sciences, 2021
Vinoth Kumar Rajendran, Padmavathy Bakthavathsalam, Peter L. Bergquist, Anwar Sunna
A label-free DNA detection method that used white light illumination, was based on the electrochemical dissolution activity of a chromium microelectrode and was monitored by a smartphone was described by Huang and Ugaz [122]. The unique advantage of the procedure was that no labeling probes were employed for detection and the system produced a sensitive, label-free, visible signature representative of the presence of the analyte. This approach was analogous to SYBR Green-based PCR detection that may not provide discrimination between the specific target and any primer-dimers that may be present in the reaction, leading to limitations in specificity. Therefore, this method needs further improvement of its specificity of detection without increases in cost and complexity of the device rather than simple improvement in the visible signature representative of the analyte.