Isothermal methods, such as helicase-dependent amplification （HDA）, have an advantage over polymerase chain reaction for DNA amplification owing to their ease of operation. Here, we developed a new HDA method that i...Isothermal methods, such as helicase-dependent amplification （HDA）, have an advantage over polymerase chain reaction for DNA amplification owing to their ease of operation. Here, we developed a new HDA method that is nanoparticle-assisted, termed nanoHDA. This method uses gold nanoparticles （AuNPs） to improve the sensitivity and specificity of the isothermal method. In HDA, the denaturation of DNA templates is mediated by helicases, but this method is limited by the low denaturation efficiency of helicases. In this report, AuNPs with preferential affinity for single-stranded DNA （ssDNA） were utilized to improve the denaturation efficiency of helicases. The same affinity property of nanoparticles can also enhance specificity by suppressing primer-dimer formation. This nanoHDA method was employed to genotype the KRAS gene in genomic DNA samples from colorectal cancer patients, as achieved by the hybridization of nanoHDA amplicons using the NanoBioArray chip.展开更多
The polymerase chain reaction （PCR） has been a reliable and fruitful method for many applications in ecology. Nevertheless, unavoidable technical and instrumental require- ments of PCR have limited its widespread ap...The polymerase chain reaction （PCR） has been a reliable and fruitful method for many applications in ecology. Nevertheless, unavoidable technical and instrumental require- ments of PCR have limited its widespread application in field situations. The recent development of isothermal DNA amplifica- tion methods provides an alternative to PCR, which circumvents key limitations of PCR for direct amplification in the field. Being able to analyze DNA in the pollen cloud of an ecosystem would provide very useful ecological information, yet would require a field-enabled, high-throughput method for this potential to be realized. Here, we demonstrate the applicability of the loop- mediated DNA amplification method （LAMP）, an isothermal DNA amplification technique, to be used in pollen analysis. Wedemonstrate that LAMP can provide a reliable method to identify species from the pollen cloud, and that it can amplify successfully with sensitivity down to single pollen grains, thus opening the possibility of field-based, high-throughput analysis.展开更多
文摘Isothermal methods, such as helicase-dependent amplification （HDA）, have an advantage over polymerase chain reaction for DNA amplification owing to their ease of operation. Here, we developed a new HDA method that is nanoparticle-assisted, termed nanoHDA. This method uses gold nanoparticles （AuNPs） to improve the sensitivity and specificity of the isothermal method. In HDA, the denaturation of DNA templates is mediated by helicases, but this method is limited by the low denaturation efficiency of helicases. In this report, AuNPs with preferential affinity for single-stranded DNA （ssDNA） were utilized to improve the denaturation efficiency of helicases. The same affinity property of nanoparticles can also enhance specificity by suppressing primer-dimer formation. This nanoHDA method was employed to genotype the KRAS gene in genomic DNA samples from colorectal cancer patients, as achieved by the hybridization of nanoHDA amplicons using the NanoBioArray chip.
基金Financial support was provided by Mexus, a collaborative program between the University of California and the Mexican National Council for Science and Technology, CONACyTFurther support provided by USDA- ARS funding
文摘The polymerase chain reaction （PCR） has been a reliable and fruitful method for many applications in ecology. Nevertheless, unavoidable technical and instrumental require- ments of PCR have limited its widespread application in field situations. The recent development of isothermal DNA amplifica- tion methods provides an alternative to PCR, which circumvents key limitations of PCR for direct amplification in the field. Being able to analyze DNA in the pollen cloud of an ecosystem would provide very useful ecological information, yet would require a field-enabled, high-throughput method for this potential to be realized. Here, we demonstrate the applicability of the loop- mediated DNA amplification method （LAMP）, an isothermal DNA amplification technique, to be used in pollen analysis. Wedemonstrate that LAMP can provide a reliable method to identify species from the pollen cloud, and that it can amplify successfully with sensitivity down to single pollen grains, thus opening the possibility of field-based, high-throughput analysis.
