Biomedical subjects

Cynthia Pfannkoch

Publications and source records attributed to Cynthia Pfannkoch.

4 recordsLinked to original sources

PubMed2005

Cell-free cloning using phi29 DNA polymerase.

We describe conditions for rolling-circle amplification (RCA) of individual DNA molecules 5-7 kb in size by >10(9)-fold, using phi29 DNA polymerase. The principal difficulty with amplification of small amounts of template by RCA using phi29 DNA polymerase is "background" DNA synthesis that usually occurs when template is omitted, or at low template concentrations. Reducing the reaction volume while keeping the amount of template fixed increases the template concentration, resulting in a suppression of background synthesis. Cell-free cloning of single circular molecules by using phi29 DNA polymerase was achieved by carrying out the amplification reactions in very small volumes, typically 600 nl. This procedure allows cell-free cloning of individual synthetic DNA molecules that cannot be cloned in Escherichia coli, for example synthetic phage genomes carrying lethal mutations. It also allows cell-free cloning of genomic DNA isolated from bacteria. This DNA can be sequenced directly from the phi29 DNA polymerase reaction without further amplification. In contrast to PCR amplification, RCA using phi29 DNA polymerase does not produce mutant jackpots, and the high processivity of the enzyme eliminates stuttering at homopolymer tracts. Cell-free cloning has many potential applications to both natural and synthetic DNA. These include environmental DNA samples that have proven difficult to clone and synthetic genes encoding toxic products. The method may also speed genome sequencing by eliminating the need for biological cloning.

Bacillus Phages↗

PubMed2004

Environmental genome shotgun sequencing of the Sargasso Sea.

We have applied "whole-genome shotgun sequencing" to microbial populations collected en masse on tangential flow and impact filters from seawater samples collected from the Sargasso Sea near Bermuda. A total of 1.045 billion base pairs of nonredundant sequence was generated, annotated, and analyzed to elucidate the gene content, diversity, and relative abundance of the organisms within these environmental samples. These data are estimated to derive from at least 1800 genomic species based on sequence relatedness, including 148 previously unknown bacterial phylotypes. We have identified over 1.2 million previously unknown genes represented in these samples, including more than 782 new rhodopsin-like photoreceptors. Variation in species present and stoichiometry suggests substantial oceanic microbial diversity.

Archaea↗

PubMed2003

Generating a synthetic genome by whole genome assembly: phiX174 bacteriophage from synthetic oligonucleotides.

We have improved upon the methodology and dramatically shortened the time required for accurate assembly of 5- to 6-kb segments of DNA from synthetic oligonucleotides. As a test of this methodology, we have established conditions for the rapid (14-day) assembly of the complete infectious genome of bacteriophage X174 (5386 bp) from a single pool of chemically synthesized oligonucleotides. The procedure involves three key steps: (i). gel purification of pooled oligonucleotides to reduce contamination with molecules of incorrect chain length, (ii). ligation of the oligonucleotides under stringent annealing conditions (55 degrees C) to select against annealing of molecules with incorrect sequences, and (iii). assembly of ligation products into full-length genomes by polymerase cycling assembly, a nonexponential reaction in which each terminal oligonucleotide can be extended only once to produce a full-length molecule. We observed a discrete band of full-length assemblies upon gel analysis of the polymerase cycling assembly product, without any PCR amplification. PCR amplification was then used to obtain larger amounts of pure full-length genomes for circularization and infectivity measurements. The synthetic DNA had a lower infectivity than natural DNA, indicating approximately one lethal error per 500 bp. However, fully infectious X174 virions were recovered after electroporation into Escherichia coli. Sequence analysis of several infectious isolates verified the accuracy of these synthetic genomes. One such isolate had exactly the intended sequence. We propose to assemble larger genomes by joining separately assembled 5- to 6-kb segments; approximately 60 such segments would be required for a minimal cellular genome.

Bacteriophage phi X 174↗

PubMed2002

The genome sequence of the malaria mosquito Anopheles gambiae.

Anopheles gambiae is the principal vector of malaria, a disease that afflicts more than 500 million people and causes more than 1 million deaths each year. Tenfold shotgun sequence coverage was obtained from the PEST strain of A. gambiae and assembled into scaffolds that span 278 million base pairs. A total of 91% of the genome was organized in 303 scaffolds; the largest scaffold was 23.1 million base pairs. There was substantial genetic variation within this strain, and the apparent existence of two haplotypes of approximately equal frequency ("dual haplotypes") in a substantial fraction of the genome likely reflects the outbred nature of the PEST strain. The sequence produced a conservative inference of more than 400,000 single-nucleotide polymorphisms that showed a markedly bimodal density distribution. Analysis of the genome sequence revealed strong evidence for about 14,000 protein-encoding transcripts. Prominent expansions in specific families of proteins likely involved in cell adhesion and immunity were noted. An expressed sequence tag analysis of genes regulated by blood feeding provided insights into the physiological adaptations of a hematophagous insect.

Animals↗