Strand displacement amplification--an isothermal, in vitro DNA amplification technique (original) (raw)
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Novel Strategies to Optimize the Amplification of Single-Stranded DNA
Frontiers in Bioengineering and Biotechnology, 2020
The generation of single stranded DNA plays a key role in in vitro selection of DNA aptamers and in other molecular techniques such as DNA sequencing and microarrays. Here we describe three novel methodologies for ssDNA production and amplification. Furthermore, we describe some previously unnoticed aspects of random DNA amplification. Our results showed that in asymmetric PCR the addition of a high melting temperature reverse primer blocked at its 3 end by a dideoxy nucleotide drives the reaction further toward ssDNA production. We demonstrated also that incorporation of internally inverted nucleotide/(s) in one primer can be used as a new method of polymerization termination. Using such modified primer, the PCR product includes two complementary DNA strands having different lengths and separable from one another by denaturing gel electrophoresis. In addition, we showed that nicking enzymes can be used to cleave the undesirable strand allowing the isolation of the target ssDNA strand.
Analytical Biochemistry, 2005
Whole genome ampliWcation, WGA 1 , is widely used in forensic analyses but also extends its application to pathology, molecular diagnostics, and environmental microbiology. Recently, two new methods for WGA have been introduced. The Wrst is based on strand displacement synthesis [3] and termed multipledisplacement ampliWcation. It uses the highly processive phi 29 DNA polymerase [4] and random primers in an isothermal ampliWcation reaction . The second, OmniPlex method, converts randomly fragmented genomic DNA into a library of ampliWable DNA fragments using ligation of linkers, followed by ampli-Wcation with linker-speciWc oligonucleotides . The phi 29-based WGA procedure is technically more convenient.
2000
Whole genome ampliWcation, WGA 1 , is widely used in forensic analyses but also extends its application to pathology, molecular diagnostics, and environmental microbiology. Recently, two new methods for WGA have been introduced. The Wrst is based on strand displacement synthesis [3] and termed multipledisplacement ampliWcation. It uses the highly processive phi 29 DNA polymerase [4] and random primers in an isothermal ampliWcation reaction . The second, OmniPlex method, converts randomly fragmented genomic DNA into a library of ampliWable DNA fragments using ligation of linkers, followed by ampli-Wcation with linker-speciWc oligonucleotides . The phi 29-based WGA procedure is technically more convenient.
2000
Whole genome ampliWcation, WGA 1 , is widely used in forensic analyses but also extends its application to pathology, molecular diagnostics, and environmental microbiology. Recently, two new methods for WGA have been introduced. The Wrst is based on strand displacement synthesis [3] and termed multipledisplacement ampliWcation. It uses the highly processive phi 29 DNA polymerase [4] and random primers in an isothermal ampliWcation reaction . The second, OmniPlex method, converts randomly fragmented genomic DNA into a library of ampliWable DNA fragments using ligation of linkers, followed by ampli-Wcation with linker-speciWc oligonucleotides . The phi 29-based WGA procedure is technically more convenient.
Terminal Protein-Primed DNA Amplification
Proceedings of The National Academy of Sciences, 1994
By using appropriate amounts of four bacteriophage 429 DNA replication proteins-terminal protein, DNA polymerase, protein p6 (double-stranded DNA-binding protein), and protein p5 (single-stranded DNA-binding protein)-it has been possible to amplify limited amounts of the 19,285-bp-long 429 DNA molecule by three orders of magnitude after 1 hr of incubation at 300C. Moreover, the quality of the amplified material was demonstrated by transfection experiments, in which infectivity of the synthetic (amplified) 429 DNA, measured as the ability to produce phage particles, was identical to that of the natural 429 DNA obtained from virions. The results presented in this paper establish some of the requisites for the development ofisothermal DNA amplification strategies based on the bacteriophage 429 DNA replication machinery that are suitable for the amplification of very large (>70 kb) segments of DNA.
Selective DNA amplification from complex genomes using universal double-sided adapters
Nucleic Acids Research, 2004
There is a rapidly developing need for new technologies to amplify millions of different targets from genomic DNA for high throughput genotyping and population gene-sequencing from diverse species. Here we describe a novel approach for the speci®c selection and ampli®cation of genomic DNA fragments of interest that eliminates the need for costly and time consuming synthesis and testing of potentially millions of amplicon-speci®c primers. This technique relies upon Type IIs restriction enzyme digestion of genomic DNA and ligation of the fragments to double-sided adapters to form closed-circular DNA molecules. The novel use of double-sided adapters, assembled through the combinatorial use of two small universal sets of oligonucleotide building blocks, provides greater selection capacity by utilizing both sides of the adapter in a sequence-speci®c ligation event. As demonstrated, formation of circular structures results in protection of the desired molecules from nuclease treatment and enables a level of selectivity high enough to isolate single, or multiple, pre-de®ned fragments from the human genome when digested at over ®ve million sites. Priming sites incorporated into the adapter allows the utilization of a common pair of primers for the ampli®cation of any adapter-captured DNA fragment of interest.
Sequence-independent, single-primer amplification (SISPA) of complex DNA populations
Molecular and Cellular Probes, 1991
Sequence-Independent, Single-Primer Amplification (SISPA) is a primer initiated technique that requires target sequence modification to achieve the non-selective logarithmic amplification of heterogeneous DNA populations. The method contrasts with the polymerase chain reaction (PCR), and its modified approaches, that have as their objective the amplification of unique or homologous sequences. SISPA requires the directional ligation of an asymmetric linkeffprimer oligonucleotide onto the target population of blunt ended DNA molecules. The common end sequence allows one strand of the double-stranded linker/primer to be used in repeated rounds of annealing, extension and denaturation in the presence of thermostable Taq DNA polymerase. The linker/primers contain restriction endonuclease sites to facilitate the molecular cloning of as little as I pg of starting material after amplification. SISPA is especially useful when the nucleotide sequence of the desired molecule is both unknown and present in limited amounts making its recovery by standard cloning procedures technically difficult. These conditions are present in the initial isolation and cloning of previously uncharacterized viral genomes. The application and quantitation of SISPA is described, together with its utility in the cloning and recovery of low abundance genetic sequences, as illustrated here with the hepatitis C virus.
PLoS ONE, 2014
Isothermal nucleic acid amplification technologies offer significant advantages over polymerase chain reaction (PCR) in that they do not require thermal cycling or sophisticated laboratory equipment. However, non-target-dependent amplification has limited the sensitivity of isothermal technologies and complex probes are usually required to distinguish between non-specific and target-dependent amplification. Here, we report a novel isothermal nucleic acid amplification technology, Strand Invasion Based Amplification (SIBA). SIBA technology is resistant to non-specific amplification, is able to detect a single molecule of target analyte, and does not require target-specific probes. The technology relies on the recombinase-dependent insertion of an invasion oligonucleotide (IO) into the double-stranded target nucleic acid. The duplex regions peripheral to the IO insertion site dissociate, thereby enabling target-specific primers to bind. A polymerase then extends the primers onto the target nucleic acid leading to exponential amplification of the target. The primers are not substrates for the recombinase and are, therefore unable to extend the target template in the absence of the IO. The inclusion of 29-O-methyl RNA to the IO ensures that it is not extendible and that it does not take part in the extension of the target template. These characteristics ensure that the technology is resistant to non-specific amplification since primer dimers or mis-priming are unable to exponentially amplify. Consequently, SIBA is highly specific and able to distinguish closelyrelated species with single molecule sensitivity in the absence of complex probes or
Loop-mediated isothermal amplification of DNA
We have developed a novel method, termed loop-mediated isothermal amplification (LAMP), that amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions. This method employs a DNA polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA. An inner primer containing sequences of the sense and anti-sense strands of the target DNA initiates LAMP. The following strand displacement DNA synthesis primed by an outer primer releases a single-stranded DNA. This serves as template for DNA synthesis primed by the second inner and outer primers that hybridize to the other end of the target, which produces a stem–loop DNA structure. In subsequent LAMP cycling one inner primer hybridizes to the loop on the product and initiates displacement DNA synthesis, yielding the original stem–loop DNA and a new stem–loop DNA with a stem twice as long. The cycling reaction continues with accumulation of 10 9 copies of target in less than an hour. The final products are stem–loop DNAs with several inverted repeats of the target and cauliflower-like structures with multiple loops formed by annealing between alternately inverted repeats of the target in the same strand. Because LAMP recognizes the target by six distinct sequences initially and by four distinct sequences afterwards, it is expected to amplify the target sequence with high selectivity.