Gene Analysis Introduction

When a geneticist is given an unknown DNA sequence and is tasked with finding out its function, there are two approaches.  Ab initio, or “from the beginning” involves using programs that analyze the sequence for known trends in gene expression.  These trends include translation initiation occurring at ATG, and intron boundaries being defined by GT at the beginning and AG at the end.  Stop codons include TAG, TAA, and TGA.  Using these in combination with more complex trends of gene expression, ab initio programs can make a prediction about the coding sequence and protein sequence of a gene.  The other method is homology-based searches.  These include comparing a query sequence to sequences of nucleic acids of a known origin.  One database includes expressed sequence tags (ESTs), which are sequences derived from cDNA clones.  A set of ESTs can be joined together to form a consensus “contig” sequence, which can then be used to find an mRNA for the gene.  In this lab, we begin by building two predictions of the protein our gene encodes: one using ab initio methods and another using homology-based methods.  For the ab initio method, we use the program FGENESH.  For the homology-based searches, we use Phytozome and NCBI BLAST.  Both programs output predicted intron-exon boundaries as well as a predicted protein sequence.  We compare the two predictions and finalize our working map with intron-exon boundaries and a predicted protein sequence, keeping in mind the differences between ab initio and homology-based searched.  We then proceed to research our gene of interest and provide an assessment of function of our gene.