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The methods find use in identifying variants, particularly homologs, in complex mixtures. Compositions comprise hybridization baits that hybridize to gene families of interest, particularly agricultural interest, in order to selectively enrich the polynucleotides of interest from complex mixtures.
Bait sequences may be specific for a number of genes from distinct Sa ibl tb8e ch18 families of interest and may be designed to cover each gene of interest by at least 2-fold. Thus methods disclosed herein are drawn to an oligonucleotide hybridization gene capture approach for identification of new genes of interest from environmental samples.
This approach bypasses the need for labor-intensive microbial strain isolation, permits simultaneous discovery of genes from multiple gene families of interest, and increases the potential to discover genes from low-abundance and unculturable organisms present in complex mixtures of environmental microbes.
Description The invention is drawn to high throughput methods of gene discovery.
Despite a relative lack of exploration, several gene families of agricultural and biomedical interest have been discovered in microbes and include genes that confer resistance to herbicides and pests in plants, as well as genes for antibiotic biosynthesis and antibiotic resistance.
Current methods for new gene discovery from microbial genomes rely on screening isolated strains for activity in a bioassay and characterization of genes of interest by sequencing. However, complex samples containing mixed cultures of organisms often contain species that cannot be cultured or present other obstacles to performing traditional methods of gene discovery.
Thus, a high throughput method of new gene identification where up to millions of culturable and non-culturable microbes can be queried simultaneously would be advantageous for identifying new genes or improved variants of known genes. The methods find use in identifying variants, particularly homologs in complex mixtures.
The methods use labeled hybridization baits or bait sequences that correspond to a portion of known gene sequences to capture similar sequences from complex environmental samples. Once the DNA sequence is captured, subsequent sequencing and analysis can identify variants of the known gene sequences in a high throughput manner.
The methods of the invention are capable of identifying and isolating gene sequences, and variants thereof, from a complex sample.
In specific embodiments, the complex sample is an environmental sample, a biological sample, or a metagenomic sample. Methods in Enzymology vol. Environmental samples can be from soil, rivers, ponds, lakes, industrial wastewater, seawater, forests, agricultural lands on which crops are growing or have grown, samples of plants or animals or other organisms associated with microorganisms that may be present within or without the tissues of the plant or animal or other organism, or any other source having biodiversity.
Complex samples also include colonies or cultures of microorganisms that are grown, collected in bulk, and pooled for storage and DNA preparation. For example, colonies can be grown on plates, in bottles, in other bulk containers and collected. In certain embodiments, complex samples are selected based on expected biodiversity that will allow for identification of gene sequences, and variants thereof.
The method disclosed herein does not require purified samples of single organisms but rather is able to identify homologous sequences directly from uncharacterized mixes of populations of prokaryotic or eukaryotic organisms: In this manner, the methods described herein can identify gene sequences, and variants thereof, from unculturable organisms, or those organisms that are difficult to culture.
Genes of Interest New gene sequences of interest, variants thereof, and variants of known gene sequences can be identified using the methods disclosed herein. Known genes of interest include cry genes Hofte and Whiteley Microbiol.
Genes of interest can also be of biological, industrial, or medical interest such as genes as for antibiotic biosynthesis and antibiotic resistance, or biosynthesis of enzymes or other factors involved in bioremediation, bioconversion, industrial processes, detoxification, biofuel production, or compounds having cytotoxic, immune system priming or other therapeutic activity.
Table 1 provides examples of gene sequences that can be used in the methods and compositions disclosed herein.
The sequences and references provided herein are incorporated by reference. It is important to note that these sequences are provided merely as examples; any sequences can be used in the practice of the methods and compositions disclosed herein.
The methods disclosed herein can identify variants of known sequences from multiple gene families of interest. As used herein, the term variants can refer to homologs, orthologs, and paralogs. While the activity of a variant may be altered compared to the gene of interest, the variant should retain the functionality of the gene of interest.
For example, a variant may have increased activity, decreased activity, different spectrum of activity e.
For polynucleotides, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the native amino acid sequence of the gene of interest.
Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction PCR and hybridization techniques as outlined below.
Variant polynucleotides also include synthetically derived polynucleotides, such as those generated, for example, by using site-directed mutagenesis but which still encode the polypeptide of the gene of interest. Variants of a particular polynucleotide disclosed herein i.
Percent sequence identity between any two polypeptides can be calculated using sequence alignment programs and parameters described elsewhere herein. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties e.
When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution.
Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1.Ch18 Ketones and Aldehydes (landscape).doc Page 2 The oxygen is also sp2 hybridized, with the 2 lone pairs occupying sp2 orbitals.
This leaves one electron in a .
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