Download Using GenomiPhi DNA Amplification Kit for the Representative

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
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This poster was presented at the Thermophiles 2003 Exeter, UK on Sep
15-19, 2003
*To whom all correspondence should be addressed.
[email protected]