Next-generation sequencing (NGS) is becoming increasingly popular because of its sensitivity and throughput, particularly for applications such as oncology and reproductive health. We can help you assemble your own library preparation kit for any available NGS platform, using a wide variety of methods and enzymes. Regardless of the platform you choose for the development of your test, you’ll find every consumable you need for library preparation in our portfolio of NGS-compatible reagents.
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SuperScript IV 1-step RT-PCR: A User Review
Dr. Eugene Wee, Head of Assay Development, Vela Diagnostics
From sample prep to assay reagents, Thermo Fisher enables commercialization of robust molecular assays with innovative tools and technologies. Dr. Wee discusses the results in the development of NGS kit with SuperScript IV reverse transcriptase.
Reverse transcriptases selection chart
For ultimate performance, particularly with challenging samples such as formalin-fixed paraffin-embedded (FFPE) tissue specimens or low DNA input, Invitrogen SuperScript IV Reverse Transcriptase is the ideal choice due to its enhanced sensitivity.
Thermo Scientific T4 DNA Polymerase—T4 DNA polymerase is an enzyme used to fill in 5´ overhangs to form blunt ends. It is a template-dependent DNA polymerase that catalyzes 5´–3´ synthesis from primed single-stranded DNA. The enzyme has a 3´–5´ exonuclease activity, but lacks 5´–3´ exonuclease activity. T4 DNA polymerase shows stronger 3´–5´ exonuclease activity on single-stranded rather than on double-stranded DNA and more than 200 times greater exonuclease activity than DNA polymerase I, E.coli, and Klenow fragment.
Invitrogen DNA Polymerase I, Large (Klenow) Fragment—This DNA polymerase enzyme is used for fill-in 5´ overhangs to form blunt ends, and for the removal of 3´ overhangs. It lacks the 5´–3´ exonuclease activity of intact DNA polymerase I, but does exhibit the 5´–3´ DNA polymerase and 3´–5´ exonuclease activities.
dNTPs—We offer high-quality nucleotides in a variety of formats, formulations, and volumes for convenience and flexibility. Our dNTPs are highly stable with greater than 99% purity, and the nucleotides are free of nuclease activities, human and E.coli DNA.
Thermo Scientific T4 Polynucleotide Kinase—this enzyme adds 5´-phosphates to oligonucleotides to allow subsequent ligation. T4 polynucleotide kinase (T4 PNK) catalyzes the transfer of the gamma-phosphate from ATP to the 5´-OH group of single- and double-stranded DNAs and RNAs, oligonucleotides, or nucleoside 3´-monophosphates (forward reaction). The reaction is reversible. In the presence of ADP, T4 polynucleotide kinase exhibits 5´-phosphatase activity and catalyzes the exchange of phosphate groups between 5´-P-oligo-polynucleotides and ATP (exchange reaction). The enzyme is also a 3´-phosphatase.
Thermo Scientific Klenow, exo—Klenow, exo is a large fragment of DNA polymerase I. It exhibits 5´–3´ polymerase activity, but lacks the 3´–5´ and 5´–3´ exonuclease activities of DNA polymerase I. The 3´–5´ exonuclease activity of the enzyme is eliminated by mutations in the 3´–5´-exonuclease active site.
dATP—We offer high-quality dATP in a variety of concentration, formulations, and volumes for convenience and flexibility. Our dATP is highly stable with greater than 99% purity, and free of nuclease activities, human and E.coli DNA.
Thermo Scientific T4 Ligase—this enzyme catalyzes the formation of a phosphodiester bond between juxtaposed 5'-phosphate and 3'-hydroxyl termini in duplex DNA or RNA. The enzyme repairs single-strand nicks in duplex DNA, RNA, or DNA/RNA hybrids. It also joins DNA fragments with either cohesive or blunt termini, but has no activity on single-stranded nucleic acids.
In most cases, your prepared library will be amplified prior to sequencing. Because NGS is such a sensitive technique, it is recommended that you use a high-fidelity proofreading enzyme to ensure that your template is error-free for the most accurate sequencing. Thermo Scientific and Invitrogen proofreading enzymes are designed to amplify DNA fragments with exceptional robustness and fidelity, and to generate PCR products with high accuracy and speed even from the most difficult templates.
Resistance to inhibitors. Amplification of a 2 kb human gDNA fragment using Platinum SuperFi DNA Polymerase or competitor high-fidelity DNA polymerases (A—Q5 Hot Start High-Fidelity, B—KAPA HiFi HotStart PCR Kit, C—KOD Hot Start, and D—PrimeSTAR GXL) in reaction mixtures containing 1—no inhibitor, 2—heparin (0.15 µg/µl), 3—xylan (0.5 µg/µl), or 4—humic acid (0.5 ng/µl). The molecular weight marker is ZipRuler Express DNA Ladder 2.
|Characteristics||Invitrogen Platinum SuperFi DNA Polymerase||Thermo Scientific Phusion Hot Start II HighFidelity DNA Polymerase|
|Fidelity compared to Taq polymerase||>100x||52x|
|Compatible with TaqMan probes||No||No|
For Research Use Only. Not for use in diagnostic procedures.