Advanced Techniques in Molecular Biology

Fall 2012

Objective: To learn how to use PCR to genotype a plant at an arbitrary genetic locus using bioinformatics, oligonucleotide primer design software, qualitative PCR and agarose gel electrophoresis.

Background: Our lab has performed a series of cross-pollination experiments with various mutants in the gravity response pathway. Some mutants show an obvious phenotype that is readily visible through observation, but others have much more subtle phenotypes that don’t lend themselves easily to visual screening. For these mutants it is necessary to rely on genotyping to obtain unambiguous screening results. You will be designing and performing a PCR screen for pin3–2 and pin3–3 alleles that have been crossed into a pgm–1 (starchless) mutant background.

Weeks 1 & 2: Both mutant alleles in PIN3 are the result of the insertion of a transposon, or T-DNA element, into the coding sequence. The precise point of insertion is described in Friml et al. (2002) Nature 415: 806–809. The T-DNA insertion will allow us to detect the presence of the mutant alleles using PCR with carefully-designed primers. Your first goal is to design primers in a way that allows us to differentiate between insertion mutants and the wild-type genotype at this locus. Using these primers, your second goal is to design a PCR experiment (including positive and negative controls) that allows us to screen for individuals that are homozygous for either the pin3–2 or pin3–3 allele.