Multiple Strand Displacement Amplification Research Articles

Au-quantum dot nanocluster electrochemiluminescence coupled with cycling-amplification for sensitive microRNA detection

A novel polyamidoamine (PAMAM) dendrimer-Au nanocluster composite was synthesized, and used to fabricate a new amplified electrochemiluminescence (ECL) signal probe for sensitive detection of microRNA by multiple strand displacement amplification (SDA) strategy. The as prepared PAMAM-Au nanocluster with many amino groups could assemble a large number of quantum dots (QDs) to greatly amplify ECL of the probe. In addition, a new sliver nanocluster (NC) with excellent conductivity and many reactive carboxyl groups was prepared, and used to immobilize a large amount of capture (c1) DNA molecules on the electrode. Moreover, by using bifunctional DNA strand displacement reaction-mediated multiple cycling-amplification technique, a small number of target miRNA could induce to generate abundant DNA (t1) fragments, which was used as a linker to hybridize with c1 DNA on the electrode, and then conjugate many amplified QDs probe. Thus an amplified ECL analytical method for detecting target miRNA was designed, and highly sensitive detection of miRNA was achieved. This newly established strategy paves a new way for homogeneous microRNA detection, which hold great potential for application in early clinical diagnosis.

Inverted mirror image molecular beacon-based three concatenated logic gates to detect p53 tumor suppressor gene

Mutation of p53 tumor suppressor gene represents one of the early molecular events in tumor initiation and progression. Although molecular computing holds tremendous potential with important applications in diagnosis, prognosis and treatment of human diseases at the molecular level, designing molecular logic gates to implement cascade amplification via operating autonomously for the detection of point mutations still remains challenging. In this contribution, we developed a three concatenated logic gates (TCLG) to perform multiple strand displacement amplification (m-SDA) for screening the cancer-related point mutations only via designing an innovative molecular beacon (MB). Specifically, using p53 gene as model target, extending the two ends of a MB via adding two fragments with the same sequence achieves two unique terminal single-stranded (ss) overhangs. After self-folding of MB into hairpin structure, the two overhangs exhibit a near inverted mirror image (IM) relationship if taking the base nature and direction into account. For this, the probe is called IM-MB. Because cascade SDAs can occur on IM-MB and promote each other, the target gene can be detected down to 10 pM. Along this line, the TCLG circuit was proposed, and two primers and target gene serve as the indispensable input signals. Utilizing this logic circuit, the point mutation or absence of target gene can be sensitively screened. Moreover, its potential application in the recognition of point mutations in complex biomatrix has been demonstrated via blind test. The proof-of-concept scheme is expected to provide new insight into the development of DNA-based molecular logic gates and their applications in basic research, medical diagnosis and precise treatment and treatment of genetic diseases.

Limited reverse transcriptase activity of phi29 DNA polymerase.

Phi29 (Φ29) DNA polymerase is an enzyme commonly used in DNA amplification methods such as rolling circle amplification (RCA) and multiple strand displacement amplification (MDA), as well as in DNA sequencing methods such as single molecule real time (SMRT) sequencing. Here, we report the ability of phi29 DNA polymerase to amplify RNA-containing circular substrates during RCA. We found that circular substrates with single RNA substitutions are amplified at a similar amplification rate as non-chimeric DNA substrates, and that consecutive RNA pyrimidines were generally preferred over purines. We observed RCA suppression with higher number of ribonucleotide substitutions, which was partially restored by interspacing RNA bases with DNA. We show that supplementing manganese ions as cofactor supports replication of RNAs during RCA. Sequencing of the RCA products demonstrated accurate base incorporation at the RNA base with both Mn2+ and Mg2+ as cofactors during replication, proving reverse transcriptase activity of the phi29 DNA polymerase. In summary, the ability of phi29 DNA polymerase to accept RNA-containing substrates broadens the spectrum of applications for phi29 DNA polymerase-mediated RCA. These include amplification of chimeric circular probes, such as padlock probes and molecular inversion probes.

Structural Alterations from Multiple Displacement Amplification of a Human Genome Revealed by Mate-Pair Sequencing

Comprehensive identification of the acquired mutations that cause common cancers will require genomic analyses of large sets of tumor samples. Typically, the tissue material available from tumor specimens is limited, which creates a demand for accurate template amplification. We therefore evaluated whether phi29-mediated whole genome amplification introduces false positive structural mutations by massive mate-pair sequencing of a normal human genome before and after such amplification. Multiple displacement amplification led to a decrease in clone coverage and an increase by two orders of magnitude in the prevalence of inversions, but did not increase the prevalence of translocations. While multiple strand displacement amplification may find uses in translocation analyses, it is likely that alternative amplification strategies need to be developed to meet the demands of cancer genomics.

Use of In Vivo Biotinylation for Chromatin Immunoprecipitation

This unit describes a system for expression of biotinylated proteins in mammalian cells in vivo, and its application to chromatin immunoprecipitation (ChIP). The system is based on co-expression of the target protein fused to a short biotin acceptor domain, together with the biotinylating enzyme BirA from Escherichia coli. The superior strength of the biotin-avidin interaction in the modified ChIP protocol presented here allows one to employ more stringent washing conditions, resulting in a better signal/noise ratio. Methods for interpreting the data obtained from ChIP samples analyzed by qPCR, and methods for testing the efficiency of biotinylation using a streptavidin gel-shift are also presented. In addition, a complementary method, based on isothermal multiple strand displacement amplification (IMDA) of circular concatemers generated from the DNA fragments obtained after ChIP, is described. This method helps to decrease bias in DNA amplification and is useful for the analysis of complex mixtures of DNA fragments typically generated in miniscale ChIP experiments.

Multiple strand displacement amplification of mitochondrial DNA from clinical samples

BackgroundWhole genome amplification (WGA) methods allow diagnostic laboratories to overcome the common problem of insufficient DNA in patient specimens. Further, body fluid samples useful for cancer early detection are often difficult to amplify with traditional PCR methods. In this first application of WGA on the entire human mitochondrial genome, we compared the accuracy of mitochondrial DNA (mtDNA) sequence analysis after WGA to that performed without genome amplification. We applied the method to a small group of cancer cases and controls and demonstrated that WGA is capable of increasing the yield of starting DNA material with identical genetic sequence.MethodsDNA was isolated from clinical samples and sent to NIST. Samples were amplified by PCR and those with no visible amplification were re-amplified using the Multiple Displacement Amplificaiton technique of whole genome amplification. All samples were analyzed by mitochip for mitochondrial DNA sequence to compare sequence concordance of the WGA samples with respect to native DNA. Real-Time PCR analysis was conducted to determine the level of WGA amplification for both nuclear and mtDNA.ResultsIn total, 19 samples were compared and the concordance rate between WGA and native mtDNA sequences was 99.995%. All of the cancer associated mutations in the native mtDNA were detected in the WGA amplified material and heteroplasmies in the native mtDNA were detected with high fidelity in the WGA material. In addition to the native mtDNA sequence present in the sample, 13 new heteroplasmies were detected in the WGA material.ConclusionGenetic screening of mtDNA amplified by WGA is applicable for the detection of cancer associated mutations. Our results show the feasibility of this method for: 1) increasing the amount of DNA available for analysis, 2) recovering the identical mtDNA sequence, 3) accurately detecting mtDNA point mutations associated with cancer.

Multiple-strand displacement and identification of single nucleotide polymorphisms as markers of genotypic variation of Pasteuria penetrans biotypes infecting root-knot nematodes

Pasteuria species are endospore-forming obligate bacterial parasites of soil-inhabiting nematodes and water-inhabiting cladocerans, e.g. water fleas, and are closely related to Bacillus spp. by 16S rRNA gene sequence. As naturally occurring bacteria, biotypes of Pasteuria penetrans are attractive candidates for the biocontrol of various Meloidogyne spp. (root-knot nematodes). Failure to culture these bacteria outside their hosts has prevented isolation of genomic DNA in quantities sufficient for identification of genes associated with host recognition and virulence. We have applied multiple-strand displacement amplification (MDA) to generate DNA for comparative genomics of biotypes exhibiting different host preferences. Using the genome of Bacillus subtilis as a paradigm, MDA allowed quantitative detection and sequencing of 12 marker genes from 2000 cells. Meloidogyne spp. infected with P. penetrans P20 or B4 contained single nucleotide polymorphisms (SNPs) in the spoIIAB gene that did not change the amino acid sequence, or that substituted amino acids with similar chemical properties. Individual nematodes infected with P. penetrans P20 or B4 contained SNPs in the spoIIAB gene sequenced in MDA-generated products. Detection of SNPs in the spoIIAB gene in a nematode indicates infection by more than one genotype, supporting the need to sequence genomes of Pasteuria spp. derived from single spore isolates.

Large fragment Bst DNA polymerase for whole genome amplification of DNA from formalin-fixed paraffin-embedded tissues

BackgroundFormalin-fixed paraffin-embedded (FFPE) tissues represent the largest source of archival biological material available for genomic studies of human cancer. Therefore, it is desirable to develop methods that enable whole genome amplification (WGA) using DNA extracted from FFPE tissues. Multiple-strand Displacement Amplification (MDA) is an isothermal method for WGA that uses the large fragment of Bst DNA polymerase. To date, MDA has been feasible only for genomic DNA isolated from fresh or snap-frozen tissue, and yields a representational distortion of less than threefold.ResultsWe amplified genomic DNA of five FFPE samples of normal human lung tissue with the large fragment of Bst DNA polymerase. Using quantitative PCR, the copy number of 7 genes was evaluated in both amplified and original DNA samples. Four neuroblastoma xenograft samples derived from cell lines with known N-myc gene copy number were also evaluated, as were 7 samples of non-small cell lung cancer (NSCLC) tumors with known Skp2 gene amplification. In addition, we compared the array comparative genomic hybridization (CGH)-based genome profiles of two NSCLC samples before and after Bst MDA. A median 990-fold amplification of DNA was achieved. The DNA amplification products had a very high molecular weight (> 23 Kb). When the gene content of the amplified samples was compared to that of the original samples, the representational distortion was limited to threefold. Array CGH genome profiles of amplified and non-amplified FFPE DNA were similar.ConclusionLarge fragment Bst DNA polymerase is suitable for WGA of DNA extracted from FFPE tissues, with an expected maximal representational distortion of threefold. Amplified DNA may be used for the detection of gene copy number changes by quantitative realtime PCR and genome profiling by array CGH.

Multiple strand displacement amplification of DNA isolated from human archival plasma/serum: Identification of cytokine polymorphism by pyrosequencing analysis

BackgroundDNA isolation from formalin-fixed paraffin-embedded tissue appears to be problematic due to degradation caused by fixative. Our aim was to investigate if the isolated genomic DNA from archival plasma/serum, combined with multiple strand displacement amplification (MDA) can be used for genotyping. MethodsNine archival plasma/serum samples and freshly frozen gastric biopsies from the same nine H. pylori-infected subjects were used for DNA isolation. Subsequently, MDA-DNA derived from the plasma/serum samples and DNA isolated from the antrum biopsies were analyzed by PCR amplification and pyrosequencing for the presence of interleukin-1beta gene (IL-1B) single nucleotide polymorphism (SNP). In addition, Southern blot and pyrosequencing analysis of H. pylori-specific PCR amplicons were performed. ResultsIL-1B SNP profiles obtained from the plasma/serum MDA-DNA and antrum biopsy DNA were identical. A C/C genotype was observed in 7 of 9 samples, and 2 of 9 revealed a C/T genotype for IL-1B −511. Similarly, 7 of 9 had a T/T, and 2 of 9 had a C/T genotype for IL-1B −31; 4 of 9 had a C/C, 4 of 9 had a C/T, and 1 of 9 had a T/T genotype, respectively, for IL-1B +3954. Moreover, pyrosequencing analysis revealed the presence of H. pylori 26695 and J99-like 16S rDNA variable V3 region sequence motifs in the antrum biopsies but not in the plasma/serum samples. ConclusionsWe conclude that MDA combined with pyrosequencing enables a rapid and accurate molecular typing of cytokine single nucleotide polymorphisms from archival plasma/serum samples.

Evaluation of 3 Methods of Whole-Genome Amplification for Subsequent Metaphase Comparative Genomic Hybridization

A common aim in cancer research is to investigate mechanisms of malignant progression by genetic analysis of key stages, including pre-malignancy, microinvasion, and micrometastases. As such lesions are small and require microdissection from clinical samples, the amount of DNA that can be recovered is limited and frequently inadequate for commonly used techniques of genomic analysis, such as comparative genomic hybridization (CGH). There is a critical requirement for techniques of whole-genome amplification that minimize representation bias in the amplified sample. Several techniques have been described, although their relative suitability for CGH has not been examined adequately. Here we compare the abilities of degenerate oligonucleotide-primed PCR (DOP-PCR), multiple-strand displacement amplification (MDA), and balanced PCR accurately to amplify limited amounts of template DNA for use in CGH. Amplification by DOP-PCR and MDA, but not balanced PCR faithfully preserved the original genomic content following amplification, as evidenced by generally concordant CGH copy number karyograms. Whereas the amplification products of DOP-PCR were immediately available for labeling and hybridization, the products of MDA required a further digestion step to produce optimal-sized probes for CGH. Moreover, MDA was less reliable overall than DOP-PCR at the lowest starting amount of 10 pg of template DNA. We conclude that DOP-PCR is the method of choice for whole-genome amplification of minute quantities of DNA to enable global genomic analysis to be performed on limited clinical samples.

Single-Molecule Dilution and Multiple Displacement Amplification for Molecular Haplotyping

Separate haploid analysis is frequently required for heterozygous genotyping to resolve phase ambiguity or confirm allelic sequence. We demonstrate a technique of single-molecule dilution followed by multiple strand displacement amplification to haplotype polymorphic alleles. Dilution of DNA to haploid equivalency, or a single molecule, is a simple method for separating di-allelic DNA. Strand displacement amplification is a robust method for non-specific DNA expansion that employs random hexamers and phage polymerase Phi29 for double-stranded DNA displacement and primer extension, resulting in high processivity and exceptional product length. Single-molecule dilution was followed by strand displacement amplification to expand separated alleles to microgram quantities of DNA for more efficient haplotype analysis of heterozygous genes.

Isothermal Multiple Displacement Amplification: A Highly Reliable Approach for Generating Unlimited High Molecular Weight Genomic DNA from Clinical Specimens

Isothermal multiple strand displacement amplification (IMDA) of the whole human genome is a promising method for procuring abundant DNA from valuable and often limited clinical specimens. However, whether DNA generated by this method is of high quality and a faithful replication of the DNA in the original specimen, allowing for subsequent molecular diagnostic testing, requires verification. In this study, we evaluated the suitability of IMDA-generated DNA (IMDA-DNA) for detecting antigen receptor gene rearrangements, chromosomal translocations, and gene mutations using Southern blot analysis, polymerase chain reaction (PCR) methods, or sequencing methods in 28 lymphoma and leukemia clinical specimens. Molecular testing before and after whole genome amplification of these specimens using the IMDA technique showed concordance in 27 of 28 (96%) specimens. Analysis of IMDA-DNA by Southern blot analysis detected restriction fragments >12 kilobases long. No amplification bias was observed at all loci tested demonstrating that this method can be useful in generating large amounts of unbiased, high molecular weight DNA from limited clinical specimens.