PCR Reaction Optimization

Purpose: To optimize the PCR reaction.

Advantages: Qsep100™ shows the high sensitivity and resolution to easily identify tiny differences such as residual primers and non-specific PCR products.

Qsep100™

PCR_Opt2

 


Next Generation Sequencing

Purpose: To determine the suitability of Qsep100™ to perform the QC analysis of the DNA sample to be used for next generation sequencing (NGS).

Introduction: Next generation sequencing (NGS) is a versatile technology widely used in medical diagnosis, preventative medical research, cancer biology, and many fields of scientific research to investigate disease in genetic aspects. NGS is also commonly applied to microarray technology for larger lab/clinical–scale studies. Therefore, the QC workflow is critical to ensure the best and most desirable NGS analysis result. Its data analysis commonly applied for variant detection, DNA/RNA-seq, and ChIP-seq.

Result: Qsep100™ is able to provide the precise length of a DNA sample as a QC step for the NGS workflow as shown below.

Figure 1: Electropheogram of NGS quality control checking point analysis.

Figure 1: Electropherogram of NGS quality.

 


 RFLP Detection

Purpose: To determine the utility of Qsep100™ to detect RFLPs.

Introduction: Restriction Fragment Length polymorphism (RFLP) refers to the difference in homologous DNA sequences detected from the fragments that has been digested into different lengths by the restriction enzyme. It is a common and inexpensive technique to process genetic analysis by using the restriction enzyme to digest the DNA sequence into smaller pieces in order to view the digested fragments (probes) through electrophoresis separation based on the sizes of the fragments. Sometimes, the process requires PCR to amplify the small fragment to be visible on the fragments analysis after the separation step. The RFLP analysis is frequently used in genome mapping and in variation analysis such as genotyping, forensics, localization of genes for genetic disorders, paternity tests, SNPs detecting, determination of risk for disease, and hereditary disease diagnostics, etc.

Result: Analysis of DNA digestion by a restriction enzyme into different fragment lengths, using Qsep100™, as shown below.

 Figure 1: This electropheogram shows the result of DNA being digested into different fragments by a restriction enzyme.

Figure 1: This electropheogram shows the result of DNA being digested into different fragments by a restriction enzyme.

 


Plasmid Digestion

Purpose: To investigate the utility of Qsep100™ in monitoring the digestion of pBR322 plasmid by Msp1 restriction enzyme using a custom-designed program.

Introduction: Plasmids are commonly found as small, circular, independent, self-replicated double stranded DNA separated from chromosome DNA in bacteria. They range in size from 1- 100Kbp and carry genes that confer antibiotic resistance. Plasmids can be rapidly exchanged between bacterium through pilus and insert the gene into new hosts during conjugation. They can be transmitted from one bacterium to another as well as to different species, such as archaea and eukaryotic organisms. Plasmids are widely used in fields of genetic research like gene therapy and recombinant DNA technologies.

Result: The Qsep100™ custom program can be designed for a time-course experiment to monitor plasmid digestion, to determine how the digestion enzymes affect plasmids at different times as seen below.

 

Figure1: The pBR322 plasmid DNA was digested by Msp1 enzyme in room temperature to show the mechanism of the digestion process. The results demonstrate that the plasmid was initially cleaved into a linear form of a certain length (about ≤ 4000bp) and subsequently digested further into smaller DNA fragments.

Figure1: The pBR322 plasmid DNA was digested by Msp1 enzyme in room temperature to show the mechanism of the digestion process. The results demonstrate that the plasmid was initially cleaved into a linear form of a certain length (about ≤ 4000bp) and subsequently digested further into smaller DNA fragments.


 

RNA Analysis

Purpose: To determine the utility of Qsep100 in detecting RNA.

Results: Qsep100 can aid in detecting both double-stranded (dsRNA) and single –stranded (ssRNA) fragments for various research purposes. As indicated in Figures 1 and 2, Qsep100 enables high resolution of both dsRNA and ssRNA fragments.

Figure 1: The ssRNA fragment analysis base pair numbers on these peaks have matched the sample of NEB N0364S ssRNA ladder. This has proven that Qsep100 has the analysis ability to precisely detect ssRNA sample.

Figure 1: The ssRNA fragment analysis base pair numbers on these peaks have matched the sample of NEB N0364S ssRNA ladder. This has proven that Qsep100 has the analysis ability to precisely detect ssRNA sample.

Figure 2: The electropheogram demonstrates that Qsep100 has the ability to detect dsRNA by matching the NEB N0363S dsRNA ladder with the traditional gel run analysis.

Figure 2: The electropheogram demonstrates that Qsep100 has the ability to detect dsRNA by matching the NEB N0363S dsRNA ladder with the traditional gel run analysis.