Contributing to the accuracy and efficiency of the sequencing process
Next-generation gene sequencing, also known as high-throughput sequencing or next-generation sequencing (NGS), refers to a set of modern DNA sequencing technologies that have revolutionized the field of genomics. NGS methods allow scientists to rapidly and cost-effectively determine the sequence of DNA or RNA molecules, having a profound impact on genetics, biology, and medicine, leading to numerous breakthroughs in our understanding of the genetic basis of diseases and the development of personalized medical treatments.
Sequencing by synthesis
Sequencing by synthesis is a fundamental method used in NGS technologies to determine the sequence of nucleotides (A, T, C, and G) in a DNA or RNA molecule. This approach is employed by several widely used NGS platforms.
Disulfide bond reduction
Disulfide bond reducing agents play a crucial role in gene sequencing, particularly in DNA and RNA sample preparation and library construction. These agents are used to break disulfide bonds between cysteine residues in proteins and to reduce the formation of secondary structures, helping to ensure that the genetic material is in a suitable form for sequencing. Here's how they are used and why they are important:
- Preventing Secondary Structures: DNA and RNA molecules can form secondary structures, such as hairpin loops or stem-loop structures, due to base-pairing interactions. These structures can interfere with the efficiency of downstream sequencing steps. Disulfide bond reducing agents help disrupt these secondary structures, ensuring that the nucleic acids are in a linear and accessible form for sequencing.
- Sample Denaturation: In DNA and RNA sample preparation, it is often necessary to denature the double-stranded DNA or RNA to obtain single-stranded templates for sequencing. Disulfide bond reducing agents can help in this process. By reducing the disulfide bonds in any proteins present in the sample, they prevent protein-mediated interference with the denaturation process, allowing for more effective strand separation.
- Preventing Protein Contamination: During the isolation of nucleic acids, proteins can be co-purified with the genetic material. These proteins can interfere with downstream processes, such as PCR or sequencing reactions. Disulfide bond reducing agents can be used to disrupt protein structure and help in the removal of proteins from the nucleic acid sample.
- Enhancing Efficiency: Disulfide bond reducing agents help ensure that the DNA or RNA is in a state where it can efficiently interact with enzymes and primers used in library construction and sequencing reactions. This leads to more accurate and reliable sequencing results.
CYTOP® 208 (Strem#: 97-1002) is one of the preferred reducing agents for NGS. The reagent shows exceptional stability at biological pH allowing for the rapid reduction of a wide range of differentially functionalized small-molecule disulfides with improved performance in comparison with the alternative reagents TCEP and DTT.
CYTOP® 208 (Strem#: 97-1002) is a commercial scale, relatively air-stable, water soluble tertiary phosphine that is stable under both acidic and basic conditions.
In summary, disulfide bond reducing agents are used in gene sequencing to ensure that DNA and RNA samples are in a suitable form for various molecular biology techniques. They help prevent the formation of secondary structures, promote denaturation, and disrupt proteins that may interfere with sequencing reactions, ultimately contributing to the accuracy and efficiency of the sequencing process.
To learn more about CYTOP® 208 and other specialty phosphorus products, please refer to the Solvay offering at Strem or the Solvay website.
References:
- Bioorganic Medicinal Chemistry Letters, 2015, 25, 4114-4119.
- ChemBioChem, 2020, 21, 1–10.
- ChemBioChem, 2020, 21, 1–10.
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97-1002 Tris(3-hydroxypropyl)phosphine, min. 84% [CYTOP® 208 ORGANOPHOSPHINE] (4706-17-6)