Oregon State University

Molecular Breeding

Gene isolation, DNA sequencing, and molecular markers

I. Steps in genetic analysis

  1. Knowing how many genes determine a phenotype, and where the genes are located, is a first step in understanding the genetic basis of a phenotype.
  2. A second step is determining the sequence of the gene, or genes, determining the phenotype and understanding how the expression of the genes is regulated at the transcriptional level. 
  3. Subsequent steps involve analysis of post-transcriptional events, understanding how the genes fit into metabolic pathways and how these pathways interact with the environment.

II. In terms of genetic analysis, the challenge is what do with all the DNA in a higher plant.

  1. Complete genome sequences are coming, but aren't yet available for many plants.  For example, there is the $1,000 genome sequence challenge and the the Thousand Genomes Project.
  2. The trend is sequencing with multiple applications - e.g. whole genomes, specific targets within genomes, or genotyping by sequencing (GBS)
  3. Even when complete genome sequence information is available, there will always be reason to study allelic diversity and interactions at specific loci and to compare genome sequences of multiple individuals
  4. One approach to dealing with the complexity and size of eukaryotic genomes is to use recombinant DNA technology 
  • $1,000 genome sequence challenge and the the Thousand Genomes Project, L-3. Genome sequencing is no longer cost-prohibitive. The challenge is how can you use this information to answer fundamental and/or applied questions. 

III. The tools of recombinant DNA technology

  • The first step is to obtain DNA
  • The second step is to cut the DNA into smaller pieces with restriction enzymes.
  • The pieces are inserted and propagated in vectors; and collections of pieces are maintained in libraries.
  • DNA targets can be selected via hybridization and/or by amplification
  • The nucleotide sequence of the targeted DNA can then be determined. 

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