Effect of sequencer water on results

- Jul 09, 2020-

Gene sequencing technology is a gene detection technology, also known as DNA sequencing technology, which is the basis for molecular biology research and genetic modification, and is also one of the important ways to unlock the mysteries of human life. Gene sequencing technology has been developed for more than 40 years and has been widely used in many fields, including the genome mapping of organisms, the evolutionary succession process of species, epigenetics, the identification and diagnosis of disease-related genes, etc. In recent years, gene sequencing-related products and technologies have evolved from laboratory research to clinical use, which provides us with key information for understanding human biological characteristics and diseases and evaluating genetic diseases. Therefore, gene sequencing technology is the next technology that changes the world. Not an exaggeration.

During the new crown epidemic, this technology received more attention. At the beginning of the outbreak, the genome sequence of the new coronavirus (COVID-19) was released through gene sequencing technology in less than a month, providing first-hand information for analyzing the evolutionary source and pathogenic mechanism of the new coronavirus in Wuhan . Many countries have also initiated the COVID-19 patient gene sequencing program to understand the impact of individual genes on the viral infection response. The data of this study will provide information for patient care and drug prescriptions, and can also help vaccine design in the future, thereby saving more Many lives. In the entire sequencing link, from sample collection to on-machine sequencing, water runs through every link including routine cleaning and maintenance of the sequencer and reagent preparation. As an essential basic reagent, the quality of water will have a significant impact on the final sequencing results. Impurities in the water, such as inorganic salts, organic matter, nucleases, particles, etc., will not only contaminate the equipment, shorten the life of the sequencer capillary, but also reduce the activity of DNA polymerase, which ultimately interferes with the accuracy of the sequencing results.

The following problems often encountered in gene sequencing are likely to be problems with the water used

Q1: Which solvent is better for DNA sequencing samples?

It is best to dissolve DNA sequencing samples using ultrapure water.

Reason: The principle of DNA sequencing reaction is actually the polymerization reaction of Taq enzyme, which requires an optimal enzyme reaction condition. If the solution that dissolves the DNA sample contains too much salt, it will affect the sequencing reaction system, causing the polymerization performance of the Taq enzyme to decrease, interfering with the sequencing reaction and even failing to generate a signal.

Pure water application Tips: The ultra-pure water machine uses an ultra-purification column for gene sequencing, and a low-magnesium purification column with RephiLe's original formula packing, which can specifically remove magnesium ions in the water and reduce the impact of the blank background on the sequencing reaction system.

Q2: Why is there no peak image in the sequencing results?

Reason: The template is degraded. The culprit may be the nuclease in the water used for sequencing.

Tips for pure water application: The ultra-pure water machine for preparing ultra-pure water requires a terminal filter, such as Lephi Bio's RephiBio terminal filter, which can remove DNase and RNase.

Q3: Why is there a "waterfall effect" in the sequencing peak map? The "waterfall effect" is a phenomenon in which the baseline suddenly drops from a height.

Cause: The sample is contaminated with organic matter, which will be excited to produce fluorescence and raise the baseline.

Pure water application Tips: Use ultra-pure water machine with TOC detection system, such as Le Feng Genie series intelligent ultra-pure water system, TOC online monitoring system designed by full oxidation method, online monitoring of TOC content, organic content is less than 5 ppb , In line with ISPE Pharmaceutical Engineering Guide / USP USP and other standards for TOC testing requirements.

Q4: How to avoid "nail peak"?

The "nail peak" is the sharp peak shape formed by the four peaks of A, T, C and G being suddenly pulled up.

Reason: There may be bubbles or particles in the solution. The bubbles and particles totally reflect the laser, forming a "nail peak".

Tips for pure water application: The water inlet of the pure water machine is equipped with a 0.22μm terminal filter. Ultrasonic degassing of ultrapure water before sequencing can effectively prevent "nail peaks".