Image created by Dr. Michael J. Miller
A team of researchers from the Case Western Reserve University School of Medicine has developed a new method for target DNA sequence amplification, testing and analysis.
This new technique, or reaction, known as AMPLON (Amplifying DNA with Multiarm Priming and Looping Optimization of Nucleic Acid), offers an alternative to the previously accepted "gold-standard" Polymerase Chain Reaction (PCR) method, opening the opportunity for more applications in medical diagnosis.
The team's findings were recently published in the journal Advanced Materials.
"AMPLON has the potential to positively change the way molecular analysis and clinical diagnostics are performed," said Mohamed S. Draz, an assistant professor at the School of Medicine and the study's principal investigator, "from infectious-disease diagnostics to personalized medicine and environmental monitoring."
How it works
Researchers use such technology to compare the DNA of sick cells to that of healthy cells, allowing them to better understand the changes that occur as a disease progresses and how to treat it.
AMPLON provides several extensions along the DNA strand to simultaneously increase the speed and accuracy of DNA synthesis under constant temperature conditions.
Using this new simplified process eliminates the need to operate between high and low temperature extremes that can cause stress on materials. It also makes the amplification process more structured and accessible, especially in settings where precise temperature control is challenging.
Using the traditional PCR method, the DNA sample is heated so it can separate into two pieces of single-stranded DNA. Next, an enzyme builds two new strands of DNA, using the original strands as templates. The process is tedious, time consuming and expensive.
"We've developed a new method of DNA amplification that does not require bulky lab-bound equipment but can be conducted in one step and in diverse settings," Draz said. "More significantly, our approach does not weaken enzymes like the PCR method."
AMPLON's multiarmed DNA primer design can turn the shortcomings of enzymes into strengths to improve amplification efficiency and produce consistent results.
"We've been able to enhance amplification and reduce amplification time by 50%," Draz said. "Our approach has the potential to dramatically change the way nucleic acid amplification is performed, providing instead a portable, reliable and cost-effective solution for applications, ranging from point-of-care diagnostics to field-based research."
More information: Mert Tunca Doganay et al, AMPLON: Amplifying DNA with Multiarm Priming and Looping Optimization of Nucleic Acid, Advanced Materials (2024). DOI: 10.1002/adma.202311634
Abstract
Nucleic acid amplification, the bedrock of biotechnology and molecular diagnostics, surges in applications—especially isothermal approaches—heightening the demand for advanced and precisely engineered methods. Here, a novel approach for amplifying DNA with multiarm priming and looping optimization of nucleic acid (AMPLON) is presented. AMPLON relies on a novel polymeric material with unique set of multiarm polyethylene glycol–DNA primers for efficient DNA amplification under isothermal conditions. Each arm carries single-stranded DNA complementing the sense or antisense sequence of the target DNA. The amplification reaction begins with antisense arms binding to the target DNA, forming a template for sense-carrying arms to direct multiarm large DNA amplicon synthesis through successive DNA looping and unlooping steps. Using human immunodeficiency virus type 1 (HIV-1) as a model clinical target, AMPLON exhibits high sensitivity, detecting target concentrations as low as 100 copies mL^−1. Compared to a quantitative real-time polymerase chain reaction assay using sensitive primers, AMPLON reliably identifies HIV-1 RNA in plasma samples (n = 20) with a significant agreement rate of 95%. With its ability to achieve highly specific and sensitive target amplification within 30 min, AMPLON holds immense potential to transform the field of nucleic acid research and unleashing new possibilities in medicine and biotechnology.