Cross priming amplification: mechanism and optimization for isothermal DNA amplification.
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ABSTRACT:
SUBMITTER: Xu G
PROVIDER: S-EPMC3271364 | biostudies-other | 2012
REPOSITORIES: biostudies-other
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Project description:The application of isothermal amplification technologies is rapidly expanding and currently covers different areas such as infectious disease, genetic disorder and drug dosage adjustment. Meanwhile, many of such technologies have complex reaction processes and often require a fine-tuned primer set where existing primer design tools are not sufficient. We have developed a primer selection system for one important primer, the turn-back primer (TP), which is commonly used in loop-mediated amplification (LAMP) and smart amplification process (SmartAmp). We chose 78 parameters related to the primer and target sequence, and explored their relationship to amplification speed using experimental data for 1344 primer combinations. We employed the least absolute shrinkage and selection operator (LASSO) method for parameter selection and estimation of their numerical coefficients. We subsequently evaluated our prediction model using additional independent experiments and compared to the LAMP primer design tool, Primer Explorer version4 (PE4). The evaluation showed that our approach yields a superior primer design in isothermal amplification and is robust against variations in the experimental setup. Our LASSO regression analysis revealed that availability of the 3'- and 5'-end of the primer are particularly important factors for efficient isothermal amplification. Our computer script is freely available at: http://gerg.gsc.riken.jp/TP_optimization/.
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Project description:Polymerase chain reaction is the most widely used method for in vitro DNA amplification. However, it requires thermocycling to separate two DNA strands. In vivo, DNA is replicated by DNA polymerases with various accessory proteins, including a DNA helicase that acts to separate duplex DNA. We have devised a new in vitro isothermal DNA amplification method by mimicking this in vivo mechanism. Helicase-dependent amplification (HDA) utilizes a DNA helicase to generate single-stranded templates for primer hybridization and subsequent primer extension by a DNA polymerase. HDA does not require thermocycling. In addition, it offers several advantages over other isothermal DNA amplification methods by having a simple reaction scheme and being a true isothermal reaction that can be performed at one temperature for the entire process. These properties offer a great potential for the development of simple portable DNA diagnostic devices to be used in the field and at the point-of-care.
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Project description: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 antisense 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.
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Project description:Visceral leishmaniasis (VL), one of the most important neglected tropical diseases, is caused by Leishmania donovani eukaryotic protozoan parasite of the genus Leishmania, the disease is prevalent mainly in the Indian sub-continent, East Africa and Brazil. VL can be diagnosed by PCR amplifying ITS1 and/or kDNA genes. The current study involved the optimization of Loop-mediated isothermal amplification (LAMP) for the detection of Leishmania DNA in human blood or tissue samples. Three LAMP systems were developed; in two of those the primers were designed based on shared regions of the ITS1 gene among different Leishmania species, while the primers for the third LAMP system were derived from a newly identified repeated region in the Leishmania genome. The LAMP tests were shown to be sufficiently sensitive to detect 0.1pg of DNA from most Leishmania species. The green nucleic acid stain SYTO16, was used here for the first time to allow real-time monitoring of LAMP amplification. The advantage of real time-LAMP using SYTO 16 over end-point LAMP product detection is discussed. The efficacy of the real time-LAMP tests for detecting Leishmania DNA in dried blood samples from volunteers living in endemic areas, was compared with that of qRT-kDNA PCR.
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Project description:Isothermal nucleic acid amplification is becoming increasingly important for molecular diagnostics. Therefore, new computational tools are needed to facilitate assay design. In the isothermal EXPonential Amplification Reaction (EXPAR), template sequences with similar thermodynamic characteristics perform very differently. To understand what causes this variability, we characterized the performance of 384 template sequences, and used this data to develop two computational methods to predict EXPAR template performance based on sequence: a position weight matrix approach with support vector machine classifier, and RELIEF attribute evaluation with Naïve Bayes classification. The methods identified well and poorly performing EXPAR templates with 67-70% sensitivity and 77-80% specificity. We combined these methods into a computational tool that can accelerate new assay design by ruling out likely poor performers. Furthermore, our data suggest that variability in template performance is linked to specific sequence motifs. Cytidine, a pyrimidine base, is over-represented in certain positions of well-performing templates. Guanosine and adenosine, both purine bases, are over-represented in similar regions of poorly performing templates, frequently as GA or AG dimers. Since polymerases have a higher affinity for purine oligonucleotides, polymerase binding to GA-rich regions of a single-stranded DNA template may promote non-specific amplification in EXPAR and other nucleic acid amplification reactions.
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Project description:Experiment 1: U133A arrays (2) hybridized to duplicate sscDNA samples prepared from 20 ng Clontech UHR RNA Experiment 2: Mu6500A arrays (6) hybridized to triplicate sscDNA samples prepared from 3 x 5 ng mouse liver RNA and 3 x 100 ng mouse liver RNA Experiment 3: U95Av2 arrays (6) hybridized to triplicate sscDNA samples prepared from 3 x 10 ng K562 RNA and 3 x 10 ng Stratagene UHR RNA Experiment 4: U95Av2 array (1) hybridized to sscDNA sample prepared from template minus reaction (negative control) Keywords: parallel sample
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Project description:Background/aimTuberculosis is still one of the most contagious diseases around the world. Key factors of tuberculosis control are rapid diagnostic, efficient treatment, and prevention of contamination by surveillance and monitoring. However, culture is the gold standard method for laboratory diagnosis of tuberculosis; the results are several weeks to obtain. In order to prevent contamination of tuberculosis, diagnosis must be made in short time and treatment should be started as soon as possible. The aim of this study is to optimize the loop-mediated isothermal amplification (LAMP) method, which provides a much faster and more sensitive result than the polymerase chain reaction (PCR) method and allows the replication of target nucleic acid sequences under isothermal conditions without the need for laboratory infrastructure.Materials and methodsSputum samples were homogenized with 5% trypsin solution in CaCl2 to obtain DNA.DNA was purified using QIAGEN QIAamp DNA mini kit. LAMP primers were design using Primer explorer V5 program according to IS6110 gene of Mycobacterium tuberculosis. NEB Bst 3.0 DNA polymerase kit was used for LAMP reactions. Besides, LAMP reactions were compared with TaqMan based RT-PCR method using NEB’s Taq polymerase kit. Finally, for visualization of LAMP products, lateral flow dipsticks that produced by Milenia Biotec, colorimetric 2X LAMP master mix that produced by NEB and 2% w/v agarose gel electrophoresis methods were used.ResultsOptimum amplification temperature for LAMP was found to be 71.4 °C. The detection limit of the method was 102 CFU/mL and sensitivity was determined 100% compared to five different Mycobacterium species.ConclusionThe current study indicated that the LAMP-LFD and colorimetric LAMP protocol optimized with sputum samples can be reliable used as a rapid, sensitive and specific assay in the diagnosis of tuberculosis in the field.
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