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Search results for: MyTaq HS DNA Polymerase

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#32974545   2019/08/28 To Up

Assessing the comparability of different DNA extraction and amplification methods in gut microbial community profiling.

Automated, high-throughput technologies are becoming increasingly common in microbiome studies to decrease costs and increase efficiency. However, in microbiome studies, small differences in methodology - including storage conditions, wet lab methods, sequencing platforms and data analysis - can influence the reproducibility and comparability of data across studies. There has been limited testing of the effects of high-throughput methods, including microfluidic PCR technologies. In this paper, we compare two extraction methods (the QIAamp DNA Stool Mini Kit and the MoBio PowerSoil DNA Isolation kit), two taq polymerase enzymes (MyTaq HS Red Mix and Accustart II PCR ToughMix), two primer sets (V3-V4 and V4-V5) and two amplification methods (a common two-step PCR protocol and amplicon library preparation on the Fluidigm Access Array system that allows automated multiplexing of primers). Gut microbial community profiles were significantly affected by all variables. While there were no significant differences in alpha diversity measured between the two extraction methods, there was an effect of extraction method on community composition measured by unweighted UniFrac distances. Both amplification method and primers had a significant effect on both alpha diversity and community composition. The relative abundance of was significantly lower when using the MoBio kit or Fluidigm amplification method, and the relative abundance of was lower when using the Qiagen kit. Microbial community profiles based on Fluidigm-generated amplicon libraries were not comparable to those generated with more commonly used methods. Researchers should carefully consider the limitations and biases that different extraction and amplification methods can introduce into their results. Additionally, more thorough benchmarking of automated and multiplexing methods is necessary to determine the magnitude of the potential trade-off between the quality and the quantity of data.
Elizabeth K Mallott, Ripan S Malhi, Katherine R Amato

1274 related Products with: Assessing the comparability of different DNA extraction and amplification methods in gut microbial community profiling.

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#30403593   2018/10/30 To Up

Optimization of DNA isolation method from Formalin-Fixed-Paraffin-Embedded tissues (FFPE) and comparative performance of four different Polymerase Chain Reaction (PCR) kits.

The tissue sample may have important genetic information in diagnostic, prognostic and counselling issues. Formalin-Fixed-Paraffin-Embedded (FFPE) is a routine method for preserving tissues. However, DNA isolated from FFPE tissue is often difficult to be amplified in PCR due to fragmentation and DNA-protein crosslinks. This study aimed to optimize the DNA isolation method from FFPE tissue and compare the performance of four different PCR ready-to-use kits. Genomic DNA was isolated from FFPE tissue colon of Short-segment Hirschsprung (S-HSCR) patients and prostate cancer tissue using Quick-DNA™ FFPE Kit (Zymo Research) with and without pre-heating treatment in KOH/NOH solution. Primers for Androgen Receptor (AR) gene and four different PCR kits: MyTaq HS Red Mix 2X (BioLine), FastStart Taq DNA Polymerase (Roche), KAPA2G fast PCR Kit 2X (KAPA Biosystem) and KOD FX Neo (Toyobo) were used for amplification. DNA electrophoresis was performed to compare the PCR results. BioLine and Toyobo kits gave better PCR results than those of Roche and KAPA Biosystem. Increasing amount of Taq polymerase and dNTPs of Roche kit by two-fold could increase the quality of PCR results. Toyobo could amplify DNA up to 417 bp, however, none of these PCR kits could amplify DNA above 450 bp. Pre-heated treatment of FFPE tissue in NaOH/KOH did not improve the DNA quality and PCR results. Toyobo PCR ready-to-use kit gave the best result among the other three PCR kits used in this study in amplifying DNA isolated from FFPE tissue. Designing the primers producing amplicon not more than 450 bp is suggested.
Almira Zada, Puspasari S Susanto, Raden L Putri, Edhyana K Sahiratmadja, Bethy S Hernowo, Andri Rezano, Yunia Sribudiani

2508 related Products with: Optimization of DNA isolation method from Formalin-Fixed-Paraffin-Embedded tissues (FFPE) and comparative performance of four different Polymerase Chain Reaction (PCR) kits.

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