Lab 2: Examining mutant worms for Project 2
Lab 3: Linkage Test Part 1
Lab 4: Linkage Test Part 2, Mapping and Complementation
Lab 5: Mapping con't; complete complementation
Lab 6: DNA sequencing analysis; Mapping con't
Lab 7: Complete Mapping: Score!
An Investigation in Classical (Forward) Genetics: Linkage Analysis, Mapping the Mutation, Complementation Testing, DNA Sequencing Analysis, Bio-Informatics
In Project 2, you will progress through the normal sequence of events in forward (classical) genetics. Forward genetics starts with finding a worm with an aberrant phenotype that is likely to be caused by a defect in a protein encoded by a mutated gene. Your overall goal is to locate and characterize the mutated gene that causes the aberrant phenotype to facilitate understanding of the importance of this gene, gene product or functionally significant gene region in C. elegans and in other species. When we are able to make these structure/function connections by studying mutant worms and identifying the genes responsible for the defects, the main goal is not so much to understand defective gene function in worms, but rather, to be able to extrapolate the function of normal genes by seeing what their gene products are unable to do when altered. We are interested in worm genes because the genome of most eukaryotes astonishing similar. Many worm genes have homologs in other eukaryotic species, including Homo sapiens.
To do this involved forward genetics study we start with an interesting phenotypically aberrant worm. One of our main goals is to pinpoint the location of the mutation on a specific gene. In order to do this we first must find out through linkage analysis the relative location of the gene associated with the mutation: which chromosome is it on? We will be able to find out if the aberrant phenotype is caused by a mutated sex-linked or autosomal gene and, if autosomal, on which autosome the defective gene is located. Our eventual goal is to pinpoint the exact location of this mutation in a gene on a chromosome (to map the mutation). We will use complementation analysis to find out if the mutation we are studying is, possibly, a newly discovered one or a previously characterized defect. To conclude our function/structure analysis, we hope to identify the exact change in the bases of the gene that causes its product to function abnormally. We will find the exact mutation through DNA sequencing of the mutated gene and by comparing it to the wild type sequence. From this sequencing and bioinformatics work, we may be able to extract new information about functionally significant regions of the gene and its product. This structure/function relationship may be significant in a broader context than in just our nematode species. We will look for homologs/orthologs in other species.
To begin, you will first examine a plate of mutant worms and determine how they are different from wild type. You will then pick your mutants to a separate plate, confirm that they are true breeding (meaning that the worm has two copies of the defective gene), and begin our forward genetic analysis. The first step in identifying a new gene associated with an interesting mutant phenotype, is usually a long, tedious process that requires applying a mutagen (UV or some mutagenic chemical like EMS) to wild type worms and then looking through thousands of normal worms to find a good candidate mutant. To make this study easier for you, mutants have been found and isolated for you and you just have to move some to a new plate to ensure they are true breeding.
Schedule of Experiments for Series 2
| Lab # || Dates || Activity || Outside Activity
|| 9/11 - 9/17
|| Picking mutant worms for genetic mapping
|| Confirm mutant phenotype and set up first cross
|| 9/18 - 9/24
|| Linkage crosses - heterozygous males with Unc hermaphrodites
|| Pick hermaphrodites for selfing
|| 9/25 - 10/1
|| Examine phenotypes to determine linkage|
Mapping: Pick Unc non-Dpy to separate plates;
Complementation: cross unknown Dpy worms to wild type males
| Mapping: Pick putative double mutants|
Complementation: cross heterozygous males with known Dpy mutants
|| 10/2 - 10/5 (10/15 Mon. Lab)
|| Mapping: Maintain double mutants (d u/d u): pick a few to individual plates|
Complementation: examine crosses for Dpy MALES
| Nothing! Enjoy Fall Break!
|| 10/16 - 10/22
|| DNA Sequencing Analysis:Examine sequencing data to determine the lesion in your gene of interest & significance in gene product;|
Mapping:Cross N2 males(++/++) with double mutant hermaphrodites (d u/ du)
| Bioinformatics: Use Wormbase and BLAST to learn more about the gene in other species;|
Mapping:Cross herterozygous males (++/ d u) with L4 double mutant hermaphrodites (d u/ d u)
|| 10/23 - 10/29
|| Complete Mapping|
SCORE! Find recombinantion frequency
| Determine map distance between your Dpy and Unc marker|