Integrated system for high throughput capture of genetic diversity

#Methods to capture biological diversity in the form of genes encoding novel enzymes and proteins of commercial value are provided. Additionally, novel methods to rapidly sample and screen bacterial genomes for novel genes of interest are described.

#Increasingly, bacterial genes are being used in various industrial and agricultural applications such as insect resistant crops, herbicide tolerant crops, or improved industrial processes. Bacteria are capable of carrying out virtually every known biochemical process and are therefore a good source of proteins and enzymes for use in a wide variety of commercial processes. Bacterial genes of utility include those that encode proteins with insecticidal activity, those that catalyze industrial processes, proteins responsible for antibiotic resistance and virulence factors. While use of biologically derived genes and proteins is increasing, it remains a cumbersome process to discover and characterize genes encoding proteins which are viable for commercial application. Traditional approaches to identify commercially viable genes and proteins have relied on following the function of interest. Newer genomics approaches have attempted to sequences genes as quickly as possible and identify their function by homology to known genes. It remains unclear how efficient it is to sequence entire genomes of a given organism to identify new genetic activities. Efforts to characterize the genomes of organisms have been ongoing since tools of molecular biology became available for this purpose. These studies often look at the relatedness of different species or at the degree of difference between two or more organisms. There have been no systematic efforts to characterize the specific genes carried by plasmids, small discrete genetic elements of bacteria, and to use such characterization as a means to rapidly identify bacterial genes with commercial applications.

#Bacterial species often carry genetic elements called plasmids that include a variety of genes. Often these plasmid encoded genes give the strain of a given bacterium commercially important characteristics. For instance, many Bacillus thuringiensis (Bt) strains are used as microbial pesticides. The genes responsible for producing the insecticidal proteins of these strains are plasmid encoded. Bt strain HD-1 has been used for decades as a microbial spray against various lepidopteran pests. Since many genes of commercial utility reside on the plasmids, not within the chromosomal DNA, whole-genome based genomics approaches to discover new genes are inefficient because one repeatedly sequences the chromosomal DNA. A number of techniques have been developed to increase the efficiency of gene discovery.

#FIG. 1 provides a diagram of Phase I of an improved sequence capture strategy.

#FIG. 2 provides a diagram of two methods for Phase II of an improved sequence capture strategy.

#FIG. 3 provides an example of a sequence capture strategy to isolate novel clones.

#The invention describes a method to rapidly characterize the genetic diversity in microorganisms and identify genes and nucleotide sequences of commercial interest, without the need for sequencing the entire genome. This method involves a unique coupling of several techniques to create an integrated strategy; generation of libraries with inserts of specific sizes, sampling of sequences, use of algorithms to pick clones most likely to have novel sequences, followed by methods for efficient sequencing of novel clones. The method provides for the rapid sampling of genetic diversity and permits identification of genes and nucleic acid molecules that may not be identified by hybridization or other available methods. Use of the method provides for rapid discovery of new genes and proteins.

#Rapid methods for identifying novel nucleotide sequences from extrachromosomal DNA in a host organism are provided. While the methods are described generally in terms of characterizing bacterial extrachromosomal DNA, the method is applicable to any host organism as well as to direct isolation of DNA from environmental sources such as soil, water, and the like. Direct isolation removes the necessity of culturing the organism or strain prior to isolation of DNA. Host organisms from which the libraries may be prepared include prokaryotic microorganisms, such as Eubacteria and Archaebacteria, lower eukaryotic microorganisms such as fungi, some algae and protozoa, as well as mixed populations of plants, plant spores and pollen.

#The method involves an integrated strategy for isolation and identification of novel nucleotide sequences. By “novel nucleotide sequences” is intended nucleotide sequences that share less than about 30% homology, preferably less than about 60% homology, more preferably less than about 80% homology, most preferably less than about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to any sequence in the database used for comparison.