* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Using GenomiPhi DNA Amplification Kit for the Representative
Whole genome sequencing wikipedia , lookup
Genetic engineering wikipedia , lookup
Designer baby wikipedia , lookup
Zinc finger nuclease wikipedia , lookup
DNA barcoding wikipedia , lookup
Human Genome Project wikipedia , lookup
Restriction enzyme wikipedia , lookup
Mycoplasma laboratorium wikipedia , lookup
DNA sequencing wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Comparative genomic hybridization wikipedia , lookup
DNA vaccination wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
United Kingdom National DNA Database wikipedia , lookup
Metagenomics wikipedia , lookup
Transformation (genetics) wikipedia , lookup
Molecular cloning wikipedia , lookup
SNP genotyping wikipedia , lookup
DNA supercoil wikipedia , lookup
Genome editing wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Non-coding DNA wikipedia , lookup
Using GenomiPhi DNA Amplification Kit for the Representative Amplification of Microbial Metagenomes Collins, Daniel J.1*; Jones, Kristi E. 1 ; Reddy, Ponaka V.1; Mamone, J. Anthony1; Blamey, Jenny M. 2; Hamilton,Scott1; 1 Amersham Biosciences Corp, Piscataway, NJ 08855 USA 2 Fundacion Cientifica y Cultural Biociencia, Jose Domingo Canas 2280, Nunoa, Santiago, Chile Comparing BLAST Hit Distributions INTRODUCTION RESULTS AND DISCUSSIONS For roughly the last two decades, the polymerase chain reaction has enabled culture independent methods for characterizing microbial populations using phylogenetic trees constucted mainly via 16s rRNA sequence. Isothermal DNA amplification methods such as rolling-circle amplification hold promise for improving environmental collection and analysis of microbes by providing large quantities of starting material from small amount of input DNA. Whole genome amplification method can be applied to linear, genomic DNA and is the basis of our recently launched GenomiPhiTM product. This method employs the unique biochemical properties of Phi29 DNA polymerase, a highly processive enzyme with excellent strand displacement activity, along with random-sequence hexamer primers to amplify DNA. Most notable features include simplicity of use, high sensitivity, preservation of genetic heterogeneity, and easy compatibility of GenomiPhi products with downstream applications such as PCR. Our research has shown excellent representational amplification of genomic material even after 5 cycles of amplification. Best matches generated for each sequence (NCBI MegaBLAST) were compiled for all successful sequences. Of roughly 100 transformed colonies picked for each condition, 50-60 sequences of sufficient length and quality were generated. Genome Amplification by Phi29 DNA Polymerase Comparisons of BLAST hit and phylogenetic distributions indicate similar content of template populations. Alignment analysis of 20 sequences that showed hits to the same strain of Limnobacter showed no differences over a 400+ base common stretch for both the PCR and GenomiPhi amplified samples. Species Limnobacter Thioxidans R. sphaeroides Aminomonas aminovorous Sphingomonas sp. Ralstonia sp. Agrobacterium tumefaciens Arthrobacter oxydans Aquaspirilium autotrophicum Acidivorax avenae Burkholderia solanacearum Haplosroridium sp. Flavobacteriales bacterium L. rubrilucens Methylophilus methylotrophus Mesorhizobium sp. Proprionobacterium acnes Pseudomonas sp. Ricksettia montana Azospirillum sp. Uncultured or Un-ID’d Bacterial Clones Physical Appearance 1 um Figure 2: Electron Micrograph of GenomiPhi Products from Salmon Sperm DNA. Sample was prepared using the “aqueous basic film” technique (Kleinschmidt and Zahn, 1959). Image (5000X) was captured by JOEL JEM-1230 at an accelerating voltage of 80kV. Planctomyces G’Phi Soil 13 13 4 3 2 1 8 7 5 4 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16 17 Representative Amplification of a Thermophile Genome 12000 10000 Gap size 8000 6000 4000 Phylogenetic Distribution of Selected Sequences Parachlamydia acanthamoebae PCR Soil 2000 Green Sulfur Bacteria 0 Pelodictyon luteolum 0 200000 400000 600000 800000 1000000 1200000 1400000 1600000 Genome position Gram-positive Bacteria Streptomyces thermovulgaris Gamma Proteobacteria E. coli Figure 1: Schematic of whole genome amplification using GenomiPhi DNA amplification method. Random hexamer primers anneal to the template DNA at multiple sites. Phi29 DNA polymerase initiates simultaneous replication and strand displacement at multiple sites on the denatured linear DNA generating new single-stranded DNA. This cycle repeats itself resulting in a double-stranded DNA. Green Non-Sulfur Bacteria Thermus aquaticus Cyanobacteria Cyanothece sp. Figure 5: Shotgun cloning of an extremophile, Thermoplasma volcanium using GenomiPhi amplification. ATCC glycerol stock culture (51530) was heat lysed, amplified by GenomiPhi and then cloned in to pUC18 vector. 1305 clones were isolated and sequenced on MegaBACE 4000. Sequence alignment and assembly was done by NCBI-BLAST (RefSeq NC 002689). Thermotogales Thermatoga subterranea Aquificales Aquifex pyrophilus CONCLUSIONS = 1 clone METHOD Samples of soil measured at 95°C and pH 3.0 collected in Solfatara, Naples, Italy were subjected either to DNA extraction directly, via the SoilMaster™ DNA Extraction Kit (Epicentre), or DNA was extracted from an enrichment using the same soil as innoculant, incubated at 95°C, pH 6.5. Both samples were used to generate 16s sequences and these were analyzed using MegaBLAST (NCBI) and Phylip phylogenetic tree generator software (Ribosomal Database II, Michigan State University). Universal bacterial primers were used to directly amplify 16s fragments of ~1.2kb via Pfu-catalyzed PCR or were first amplified using GenomiPhi DNA Amplification Kit prior to PCR. PCR products were cleaned using GFX for PCR Purification Kit (Amersham Biosciences), restriction digested to generate cohesive ends and cloned into pBluescript II KS+ Phagemid (Stratagene) using New England Biolabs’ Quick Ligation Kit. Ligated DNA was then used to transform Z-Competent™ (Zymo) XL10-Gold Ultracompetent Cells (Stratagene) which were color screened and used as input for TempliPhi reactions (Amersham Biosciences). DNA products of these reactions were used as inout into cycle sequencing reactions and the resultant Sanger fragments were run on MegaBACE™ 1000 capillary sequencers (Amersham Biosciences) and analyzed using Cimarron 3.12 basecaller software. GenomiPhi DNA Amplification Kit and the use thereof for DNA synthesis is covered by US patent application number 09/920,571 and US patents 5,854,033, 5,198,543, 5,576,204 and 5,001,050 licensed exclusively to Amersham Biosciences Corp. GenomiPhi is a trademarks of Amersham Biosciences Limited. Amersham and Amersham Biosciences are trademarks of Amersham plc. Figure 3: Phylogenetic Distribution of High Consensus Sequence For Direct PCR from Soil Amplification Strategy = 5 clones = 9 clones Archea 0.1 • GenomiPhi provides a simple and highly sensitive amplification method for retaining precious sample DNA. • GenomiPhi generates primer independant, representative amplification products of microbial genomes, allowing library construction and ample archival material for future work. • Method works on fresh cells, paper immobilised DNA or extracted DNA. • Products are greater than 10 kb and are unchanged over serial rounds. • Representation of the input gDNA is maintained based on gene copy and full genome sequence coverage. • Literature describing diverse applications for GenomiPhi products such as real time PCR, STR genotyping, multiplex PCR and SNP typing is available at: www.genomiphi.com Pyrococcus furiosis Planctomyces Parachlamydia acanthamoebae Green Sulfur Bacteria Pelodictyon luteolum Gram-positive Bacteria Streptomyces thermovulgaris Gamma Proteobacteria E. coli Green Non-Sulfur Bacteria Thermus aquaticus Cyanobacteria Cyanothece sp Thermotogales Thermatoga subterranea Aquificales Aquifex pyrophilus = 1 clone Figure 4: Phylogenetic Distribution of High Consensus Sequence for GenomiPhi Amplification Strategy = 4 clones Archea 0.1 Pyrococcus furiosis All goods and services are sold subject to terms and conditions of sale of the company within the Amersham Biosciences group that supplies them. A copy of these terms and conditions are available on request. Amersham Biosciences Corp, 2002 - All rights reserved. Amersham Biosciences UK Limited Amersham Place Little Chalfont Buckinghamshire England U.K. HP7 9NA. Amersham Biosciences AB SE-751 84 Uppsala Sweden. Amersham Biosciences Corp 800 Centennial Avenue PO Box 1327 Piscataway This poster was presented at the Thermophiles 2003 Exeter, UK on Sep 15-19, 2003 *To whom all correspondence should be addressed. [email protected]