Fidelity of phi 29 DNA polymerase. Comparison between protein-primed initiation and DNA polymerization.
AuthorsEsteban JA, Salas M, Blanco L
JournalJ Biol Chem
PubMed ID8428945
'Phi 29 DNA polymerase is able to catalyze two different synthetic reactions: protein-primed initiation and DNA polymerization. We have studied the fidelity of phi 29 DNA polymerase when carrying out these two reactions. Global fidelity was dissected into three steps: insertion discrimination, mismatch elongation, and proofreading. The insertion discrimination of ... More
Whole-metagenome amplification of a microbial community associated with scleractinian coral by multiple displacement amplification using phi29 polymerase.
AuthorsYokouchi H, Fukuoka Y, Mukoyama D, Calugay R, Takeyama H, Matsunaga T
JournalEnviron Microbiol
PubMed ID16817924
'Limitations in obtaining sufficient specimens and difficulties in extracting high quality DNA from environmental samples have impeded understanding of the structure of microbial communities. In this study, multiple displacement amplification (MDA) using phi29 polymerase was applied to overcome these hindrances. Optimization of the reaction conditions for amplification of the bacterial ... More
Suitability of genomic DNA synthesized by strand displacement amplification (SDA) for AFLP analysis: genotyping single spores of arbuscular mycorrhizal (AM) fungi.
AuthorsGadkar V, Rillig MC
JournalJ Microbiol Methods
PubMed ID15936100
'Limited biological samples of microbial origin often yield insufficient amounts of genomic DNA, making application of standard techniques of genetic analysis, like amplified fragment length polymorphism (AFLP), virtually impossible. The Phi29 DNA polymerase based whole genome amplification (WGA) method has the potential to alleviate this technical bottleneck. In the present ... More
Novel application of Phi29 DNA polymerase: RNA detection and analysis in vitro and in situ by target RNA-primed RCA.
AuthorsLagunavicius A, Merkiene E, Kiveryte Z, Savaneviciute A, Zimbaite-Ruskuliene V, Radzvilavicius T, Janulaitis A
JournalRNA
PubMed ID19244362
We present a novel Phi29 DNA polymerase application in RCA-based target RNA detection and analysis. The 3'-->5' RNase activity of Phi29 DNA polymerase converts target RNA into a primer and the polymerase uses this newly generated primer for RCA initiation. Therefore, using target RNA-primed RCA, padlock probes may be targeted ... More
Towards the analysis of the genomes of single cells: further characterisation of the multiple displacement amplification.
AuthorsPanelli S, Damiani G, Espen L, Micheli G, Sgaramella V
JournalGene
PubMed ID16564650
The development of methods for the analysis and comparison of the nucleic acids contained in single cells is an ambitious and challenging goal that may provide useful insights in many physiopathological processes. We review here some of the published protocols for the amplification of whole genomes (WGA). We focus on ... More
In situ genotyping individual DNA molecules by target-primed rolling-circle amplification of padlock probes.
AuthorsLarsson C, Koch J, Nygren A, Janssen G, Raap AK, Landegren U, Nilsson M
JournalNat Methods
PubMed ID15782198
Methods are needed to study single molecules to reveal variability, interactions and mechanisms that may go undetected at the level of populations of molecules. We describe here an integrated series of reaction steps that allow individual nucleic acid molecules to be detected with excellent specificity. Oligonucleotide probes are circularized after ... More
Effect of aphidicolin and nucleotide analogs on the phage phi 29 DNA polymerase.
AuthorsBlanco L, Salas M
JournalVirology
PubMed ID3090778
The drugs aphidicolin and the nucleotide analogs butylanilino dATP, butylphenyl dGTP, and butylphenyl rGTP inhibited the protein-primed replication of phi 29 DNA-protein p3 in the presence of purified terminal protein p3 and phi 29 DNA polymerase p2. The effect of aphidicolin was mainly on the polymerization reaction by decreasing the ... More
Phosphorothioate primers improve the amplification of DNA sequences by DNA polymerases with proofreading activity.
AuthorsSkerra A
JournalNucleic Acids Res
PubMed ID1641322
Two thermostable DNA polymerases with proofreading activity--Vent DNA polymerase and Pfu DNA polymerase--have attracted recent attention, mainly because of their enhanced fidelities during amplification of DNA sequences by the polymerase chain reaction. A severe disadvantage for their practical application, however, results from the observation that due to their 3' to ... More
Terminal protein-primed DNA amplification.
AuthorsBlanco L, Lázaro JM, de Vega M, Bonnin A, Salas M
JournalProc Natl Acad Sci U S A
PubMed ID7991606
By using appropriate amounts of four bacteriophage phi 29 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 phi 29 DNA molecule by three orders of magnitude after 1 hr of incubation ... More
The bacteriophage phi 29 DNA polymerase, a proofreading enzyme.
AuthorsGarmendia C, Bernad A, Esteban JA, Blanco L, Salas M
JournalJ Biol Chem
PubMed ID1733957
The bacteriophage phi 29 DNA polymerase, involved both in the protein-primed initiation and elongation steps of the viral DNA replication, displays a very processive 3',5'-exonuclease activity acting preferentially on single-stranded DNA. This exonucleolytic activity showed a marked preference for excision of a mismatched versus a correctly paired 3' terminus. These ... More
Duality of polynucleotide substrates for Phi29 DNA polymerase: 3'-->5' RNase activity of the enzyme.
AuthorsLagunavicius A, Kiveryte Z, Zimbaite-Ruskuliene V, Radzvilavicius T, Janulaitis A
JournalRNA
PubMed ID18230765
Phi29 DNA polymerase is a small DNA-dependent DNA polymerase that belongs to eukaryotic B-type DNA polymerases. Despite the small size, the polymerase is a multifunctional proofreading-proficient enzyme. It catalyzes two synthetic reactions (polymerization and deoxynucleotidylation of Phi29 terminal protein) and possesses two degradative activities (pyrophosphorolytic and 3'-->5' DNA exonucleolytic activities). ... More
Highly efficient DNA synthesis by the phage phi 29 DNA polymerase. Symmetrical mode of DNA replication.
AuthorsBlanco L, Bernad A, Lázaro JM, Martín G, Garmendia C, Salas M
JournalJ Biol Chem
PubMed ID2498321
The results presented in this paper indicate that the phi 29 DNA polymerase is the only enzyme required for efficient synthesis of full length phi 29 DNA with the phi 29 terminal protein, the initiation primer, as the only additional protein requirement. Analysis of phi 29 DNA polymerase activity in ... More
Mutation detection and single-molecule counting using isothermal rolling-circle amplification.
AuthorsLizardi PM, Huang X, Zhu Z, Bray-Ward P, Thomas DC, Ward DC
JournalNat Genet
PubMed ID9662393
Rolling-circle amplification (RCA) driven by DNA polymerase can replicate circularized oligonucleotide probes with either linear or geometric kinetics under isothermal conditions. In the presence of two primers, one hybridizing to the + strand, and the other, to the - strand of DNA, a complex pattern of DNA strand displacement ensues ... More
Evaluation of multiple displacement amplification in a 5 cM STR genome-wide scan.
AuthorsDickson PA, Montgomery GW, Henders A, Campbell MJ, Martin NG, James MR
JournalNucleic Acids Res
PubMed ID16055919
Multiple displacement amplification (MDA) has emerged as a promising new method of whole genome amplification (WGA) with the potential to generate virtually unlimited genome-equivalent DNA from only a small amount of seed DNA. To date, genome-wide high marker density assessments of MDA-DNA have focussed mainly upon suitability for single nucleotide ... More
Genome-wide single-nucleotide polymorphism arrays demonstrate high fidelity of multiple displacement-based whole-genome amplification.
Whole-genome DNA amplification by multiple displacement (MD-WGA) is a promising tool to obtain sufficient DNA amounts from samples of limited quantity. Using Affymetrix' GeneChip Human Mapping 10K Arrays, we investigated the accuracy and allele amplification bias in DNA samples subjected to MD-WGA. We observed an excellent concordance (99.95%) between single-nucleotide ... More
Comprehensive human genome amplification using multiple displacement amplification.
AuthorsDean FB, Hosono S, Fang L, Wu X, Faruqi AF, Bray-Ward P, Sun Z, Zong Q, Du Y, Du J, Driscoll M, Song W, Kingsmore SF, Egholm M, Lasken RS
JournalProc Natl Acad Sci U S A
PubMed ID11959976
Fundamental to most genetic analysis is availability of genomic DNA of adequate quality and quantity. Because DNA yield from human samples is frequently limiting, much effort has been invested in developing methods for whole genome amplification (WGA) by random or degenerate oligonucleotide-primed PCR. However, existing WGA methods like degenerate oligonucleotide-primed ... More
Improved genome recovery and integrated cell-size analyses of individual uncultured microbial cells and viral particles.
AuthorsStepanauskas R, Fergusson EA, Brown J, Poulton NJ, Tupper B, Labonté JM, Becraft ED, Brown JM, Pachiadaki MG, Povilaitis T, Thompson BP, Mascena CJ, Bellows WK, Lubys A
JournalNat Commun
PubMed ID28729688
'Microbial single-cell genomics can be used to provide insights into the metabolic potential, interactions, and evolution of uncultured microorganisms. Here we present WGA-X, a method based on multiple displacement amplification of DNA that utilizes a thermostable mutant of the phi29 polymerase. WGA-X enhances genome recovery from individual microbial cells and ... More
In vitro evolution of phi29 DNA polymerase using isothermal compartmentalized self replication technique.
AuthorsPovilaitis T, Alzbutas G, Sukackaite R, Siurkus J, Skirgaila R
JournalProtein Eng Des Sel
PubMed ID27672049
Compartmentalized self replication (CSR) is widely used for in vitro evolution of thermostable DNA polymerases able to perform PCR in emulsion. We have modified and adapted CSR technique for isothermal DNA amplification using mezophilic phi29 DNA polymerase and whole genome amplification (WGA) reaction. In standard CSR emulsified bacterial cells are ... More