We present a method of rapid isothermal amplification of DNA without initial heat denaturation of the template, and methods and probes for (a) real-time fluorescence detection and (b) lateral flow detection of amplicons. Isothermal strand displacement amplification (iSDA) can achieve >10(9)-fold amplification of the target sequence in <20 minutes at 49 °C, which makes it one of the fastest exis...

Aptamers, or single stranded oligonucleotides, are produced by systematic evolution of ligands by exponential enrichment, abbreviated as SELEX. In the amplification and regeneration step of SELEX technique, dsDNA is conversed to ssDNA. Asymmetric PCR is one of the methods used for the generation of ssDNA. The purpose of this study was to design a random DNA library for selection of aptamers wit...

AIM To generate complete DNA profiles from blood and saliva samples deposited on FTA® and non-FTA® paper substrates following a direct amplification protocol. METHODS Saliva samples from living donors and blood samples from deceased individuals were deposited on ten different FTA® and non-FTA® substrates. These ten paper substrates containing body fluids were kept at room temperature for vary...

Molecular beacons represent a new family of fluorescent probes for nucleic acids, and have found broad applications in recent years due to their unique advantages over traditional probes. Detection of nucleic acids using molecular beacons has been based on hybridization between target molecules and molecular beacons in a 1:1 stoichiometric ratio. The stoichiometric hybridization, however, puts ...

Aptamers are DNA oligonucleotides capable of binding different classes of targets with high affinity and selectivity. They are particularly attractive as affinity probes in multiplexed quantitative analysis of proteins. Aptamers are typically selected from large libraries of random DNA sequences in a general approach termed systematic evolution of ligands by exponential enrichment (SELEX). SELE...

Rolling circle amplification (RCA) of plasmid or genomic DNA using random hexamers and bacteriophage phi29 DNA polymerase has become increasingly popular in the amplification of template DNA in DNA sequencing. We have found that the mutant protein of single-stranded DNA binding protein (SSB) from Thermus thermophilus (Tth) HB8 enhances the efficiency of amplification of DNA templates. In additi...

In this paper, we developed a metal micro-fluidic chip with advanced surface processing for ultra-small volume molecular isothermal amplification. This method takes advantages of the nucleic acid amplification with good stability and consistency, high sensitivity about 31 genomic DNA copies and bacteria specific gene identification. Based on the advanced surface processing, the bioreaction assa...

We previously reported that multiply-primed rolling circle amplification (MRPCA) using modified random RNA primers can amplify tiny amounts of circular DNA without producing any byproducts. However, contaminating DNA in recombinant Phi29 DNA polymerase adversely affects the outcome of MPRCA, especially for negative controls such as non-template controls. The amplified DNA in negative control ca...

In vitro DNA amplification methods, such as polymerase chain reaction (PCR), rely on synthetic oligonucleotide primers for initiation of the reaction. In vivo, primers are synthesized on-template by DNA primase. The bacteriophage T7 gene 4 protein (gp4) has both primase and helicase activities. In this study, we report the development of a primase-based Whole Genome Amplification (pWGA) method,...

Gene amplification is one of the most frequent genome anomalies observed in tumor cells, whereas it has never been detected in cells of normal origin. A large body of evidence indicates that DNA double-strand breaks (DSBs) play a key role in initiating gene amplification. In mammals, DSBs are mainly repaired through the nonhomologous end-joining pathway (NHEJ) that requires a functional DNA-dep...