Optimisation and alternatives to the DNA amplification system TruePrime®

• Autores: Ana Martínez Carrón
• Directores de la Tesis: Luis Blanco Dávila (dir. tes.), Ángel J. Picher Serantes (dir. tes.)
• Lectura: En la Universidad Autónoma de Madrid ( España ) en 2023
• Idioma: inglés
• Número de páginas: 192
• Títulos paralelos:
  • Optimización y alternativas al sistema de amplificación de DNA TruePrime®
• Enlaces
  • Tesis en acceso abierto en: Biblos-e Archivo
• Resumen

  • The development of several isothermal DNA amplification methods has brought improvements in terms of simplicity, sensitivity, or yields. Many of these have become fundamental tools in genomic research, clinical diagnostics, and forensic studies. Moreover, with the recent approval of gene therapy products and nucleic acid vaccines, one of the main challenges being addressed is the increased demand for large-scale production and manufacturing of highly specific genetic material. This leaves room for enzymatically produced clinical grade synthetic DNA. TruePrimeÒ is an isothermal DNA amplification method which does not require external primers (random or specific) as it combines two DNA-synthesizing enzymes (Picher et al. 2016). A DNA primase enzyme from Thermus thermophilus named PrimPol (TthPrimPol), uses dNTPs as substrates to produce DNA primers at different initiation sites of the given template. These DNA primers are then efficiently elongated by the high-fidelity and highly-processive DNA polymerase of bacteriophage Phi29 (Phi29 DNApol). The outstanding stranddisplacement capacity of Phi29 DNApol allows the continuous exposure of single-stranded DNA (ssDNA) for new priming events, thus resulting on an exponential cascade of multiple displacement amplification (MDA). We have identified and characterised a new PrimPol from T. thermophilus SG0.5JP17- 16 strain, TthPrimPol2, which is endowed with DNA primase and DNA polymerase activities, triggered by both magnesium or manganese ions. Its enzymatic activities were superior to those of TthPrimPol and although it has a similar preference for template initiation sites, it processively synthesizes longer DNA primers, endures higher temperatures, and most importantly, has a stronger ssDNA binding affinity. Its combination with Phi29 DNApol enabled the detection and amplification of lower input DNA than TruePrimeÒ, valuable for its application when DNA is found in limiting amounts, such as in liquid biopsies. Furthermore, we have assessed the ability of a new B-family DNA polymerase endowed with de novo DNA synthesis capacity, piPolB, to perform DNA amplification as a single enzyme. No DNA amplification was detectable with piPolB alone, but its combination with Phi29 DNApol did yield competitive amounts of DNA, suggesting piPolB can initiate DNA primers synthesis but requires Phi29 DNApol for proficient elongation. Detailed fidelity analysis of the amplified material showed low error rates although piPolB-primed reactions contained a higher number of indels than TruePrimeÒ, especially at homopolymeric repeats. Better genomic coverage values were obtained for lower piPolB concentrations, suggesting high amounts of piPolB could interfere with the reaction. Conversely, the presence of piPolB in the reaction, a TLS polymerase enzyme, could benefit the amplification of challenging or damaged templates, where extreme accuracy of the resulting material is not the priority