PCR-based IVT Template

Gene to Template starting from 8BDs^FAST

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Overview

GenScript's advanced PCR-based IVT template is tailor-made for researchers in the early stages of mRNA therapeutics development seeking a rapid and small-scale mRNA IVT solution. Committed to refining molecular processes, we've innovated beyond the traditional plasmid preparation and linearization steps, greatly reducing the processing time. Now from Gene Synthesis to IVT ready template in as little as 8 business days.

This innovation involves a single PCR step with a long primer featuring a polyA tail. Our revamped method not only elevates the purity and uniformity of the poly(A) tail but also ensures that the mRNA product's purity remains at par with the traditional linearized IVT template.

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Molecular Biology

Specification

Gene Length	≤ 8kb
Turnaround Time	Starting from 12 BD
Quantity	10 - 50 µg
Delivery Format	Lyophilized or Liquid
Poly(A) Tract	100(A)
Endotoxin Level	< 100 EU/mg
QC Test	Sanger Sequencing Restriction Enzyme Digestion

Case Study

PCR-based Template vs Traditional Template

GenScript's PCR-based IVT Template provides enhanced DNA template and poly(A) tail purity over traditional linearized plasmids, guaranteeing a successful IVT reaction for top-tier mRNA quality.

Agarose Gel Electrophoresis

Figure 1. Agarose Gel Electrophoresis was conducted to identify the PCR template. Linearized plasmids were utilized for comparison.

DNA Template Purity - HPLC

Figure 2. PCR-based IVT template and the Traditional template undergo high-performance liquid chromatography (HPLC) elution to evaluate their purity. The Purity levels are deduced from the ratio of the primary peak area to any impurities. Results suggest that the mRNA product from both the PCR-based and Traditional templates showcase similar eGFP expression.

Poly(A) Tail Purity – Bioanalyzer 5200

Figure 3. Poly(A) tail purity of PCR-based IVT template and the Traditional template are analyzed on a Bioanalyzer 5200. Results reveal that the poly(A) tail of PCR-based template achieves a purity of 95.6%, surpassing the traditional template’s 89.3%.
Pure and high quality mRNA product

GenScript's PCR-based IVT Templates have been demonstrated to produce mRNA products of high purity and quality when compare to traditional methods. Purity and quality of eGFP encoding IVT mRNA were examined.

IVT mRNA yield & purity– Bioanalyzer 5200

Figure 1. IVT mRNA samples originating from both PCR-based and Traditional templates were evaluated with a Bioanalyzer 5200. For a 20µL IVT reaction, 1 µg of ready-to-use template from both sources was employed. The resulting mRNA was then checked for its purity and quantity using the Bioanalyzer 5200. Results suggest that mRNAs from both the PCR-based and Traditional templates showcase comparable purity and yield levels.

eGFP expression

Figure 2. 293T cells were transfected with 0.1 µg of the mRNA using the Lipofectamine 2000 reagent (Invitrogen). GFP expression was observed 48 hours after transfection. The data indicates that the mRNA derived from the BspQI-linearized template exhibited roughly 1.9 times higher GFP expression compared to the XbaI version.