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| [[BISC 219/F10: Gene Mapping Info | Gene Mapping Info]]<br> | | [[BISC 219/F10: Gene Mapping Info | Background: Classical Forward Genetics and Gene Mapping]]<br> |
| [[BISC 219/F10: Lab 2 | Lab 2: Mutant Hunt]]<br> | | [[BISC 219/F10: Lab 2 Mutant Hunt | Lab 2: Mutant Hunt]]<br> |
| [[BISC 219/F10: Lab 3 | Lab 3: Linkage Test Part 1]]<br> | | [[BISC 219/F10: Lab 3 | Lab 3: Linkage Test Part 1]]<br> |
| [[BISC 219/F10: Lab 4 | Lab 4: Linkage Test Part 2, Mapping and Complementation]]<br> | | [[BISC 219/F10: Lab 4 | Lab 4: Linkage Test Part 2, Mapping and Complementation]]<br> |
| [[BISC 219/F10: Lab 5 | Lab 5: Mapping Part 2]]<br> | | [[BISC 219/F10: Lab 5 | Lab 5: Mapping con't; complete complementation]]<br> |
| [[BISC 219/F10: Lab 6 | Lab 6: Score]]<br> | | [[BISC 219/F10: Lab 6 | Lab 6: DNA sequencing analysis; Mapping con't ]]<br> |
| | [[BISC 219/F10: Lab 7 | Lab 7: Complete Mapping: Score!]] |
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| | =='''Schedule of Experiments for Series 2'''== |
| | {| border="1" |
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| | ! Lab # !! Dates !! Activity !! Outside Activity |
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| | ! 2 |
| | | 9/14 - 9/20 |
| | | Mutant Hunt |
| | | Confirm mutant phenotype and set up first cross |
| | |- |
| | ! 3 |
| | |9/21 - 9/27 |
| | | '''Linkage''' crosses - heterozygous males with Unc hermaphrodites |
| | | Pick hermaphrodites for selfing |
| | |- |
| | ! 4 |
| | | 9/28 - 10/4 |
| | | Examine phenotypes to determine linkage<br> |
| | '''Mapping:''' Pick Unc non-Dpy to separate plates;<BR>'''Complementation:''' cross unknown Dpy worms to wild type males |
| | | '''Mapping:''' Pick putative double mutants<br>'''Complementation:''' cross heterozygous males with known Dpy mutants |
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| | |- |
| | ! 5 |
| | | 10/5 - 10/12 |
| | | '''Mapping:''' Maintain double mutants (d u/d u): pick a few to individual plates<br>'''Complementation:''' examine crosses for Dpy '''MALES''' |
| | | Nothing! |
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| == Gene Mapping: Mutant Hunt ==
| | |- |
| | | ! 6 |
| In Series 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. Then we find out through linkage analysis whether or not our mutated gene is sex-linked or autosomal and, if autosomal, on which autosome the defect is found. Our eventual goal is to locate this gene mutation (to ''map'' it on a particular chromosome and gene). To conclude our forward genetics study, we hope to find the exact change in the gene sequence that causes the phenotypic abnormality through DNA sequencing of the mutated gene and comparing it to the wild type sequence.
| | | 10/13 - 10/19 |
| | | | ''' DNA Sequencing Analysis''':Examine sequencing data to determine the lesion in your gene of interest & significance in gene product;<br>'''Mapping''':Cross N2 males(++/++) with double mutant hermaphrodites (d u/ du) |
| To begin, you will first perform a '''mutant hunt''': scanning a plate for rare mutants that occur among the background of wild-type animals. You will then pick your mutants to a separate plate, confirm their mutant phenotype, and begin genetic analyses that culminate with mapping the mutation to a particular gene and sequencing that gene. The first step, the mutant hunt, is usually a long, tedious process that requires applying a mutagen (UV or some mutagenic chemical) 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 secreted on the plate by your devious instructor. Thus, while rare, they will be more frequently encountered than had you sifted through the second-generation progeny from mutagenized worms (as is the case with a real mutant hunt).<br>
| | | '''Bioinformatics''':Use Wormbase and BLAST to learn more about the gene in other species;<br>'''Mapping:'''Cross herterozygous males (++/ d u) with L4 double mutant hermaphrodites (d u/ d u) |
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| | |- |
| Once you have recovered your mutant and confirmed its phenotype (by examining its progeny) you will next perform '''linkage testing''': determining on which of the five autosomes (linkage groups) your mutation is located. This task is a prerequisite to mapping: determining the exact location of the mutation on the chromosome/gene. Linkage Analysis is accomplished by determining the segregation behavior of your unmapped mutation relative to standard reference markers (e.g., mutations whose location is already known). Recall that unlinked mutations will segregate independently (your basic dihybrid inheritance as first observed by Gregor Mendel) whereas linked mutations will not.<br>
| | ! 7 |
| <br>
| | | 10/20 - 10/26 |
| In practice, linkage tests are performed using the following steps (where "d" (''dpy'') represents your recessive mutant tested with reference marker "u" (''unc'')). The markers d and u must be visibly distinguishable. Since homozygous mutant males usually will not mate, the desired double heterozygote is constructed by mating males heterozygous for your ''dpy'' mutation but wild type for all other genes including the reference mutation (d/+;+/+) with hermaphrodites homozygous for the reference mutation ''unc'' (+/+; u/u). The genotypes of the F1 hybrids will be (+/d;u/+) and (+/+;u/+). We are only interested in the double heterozygote (+/d;u/+). The F1 hybrids containing only u are not useful. To select the (+/d;u/+) heterozygotes, we let 4 to 5 individual F1's self fertilize on their own individual plates (one on each plate). We score the progeny of the F1 individuals (the F2) for linkage. Only F1 worms which produce d/d homozygotes are scored, since those are the (+/d;u/+) parents. The d/d homozygotes should be found on 50% of the plates.<br>
| | | ''' Complete Mapping'''<br> |
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| | SCORE! Find recombinant frequency |
| F2 progeny of each class are counted in the (+/d;u/+) plates: wild-type (+/+;+/+); d (d/d;+/+); u (+/+;u/u) and du double (d/d;u/u). If assortment is independent, progeny will be:<br>
| | | Determine map distance between your Dpy and Unc marker<br> |
| <center>[[Image:Independent assortment.tif]]</center>
| | |- |
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| Or 9/16 wild; 3/16 d, 3/16 u; 1/16 du (that is our good old friend the 9:3:3:1 ratio)<br>
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| On the other hand, if the markers are closely linked, double homozygotes (d u/d u) would occur only through a two recombination events. Such an event might occur in either the sperm or the occyte. If the probability of a recombination event is p, and if the event produces wild-type and double mutant recombinant chromosomes, then the probability of getting the double mutant chromosome in an individual gamete is p/2. The chance of an individual gamete which is a (d u) recombinant combining with another (d u) recombinant is (p/2) x (p/2). If the map distance between 2 mutations is 10%, then the probability (P) of recombination occurring is p = 0.1. p/2 is 0.05 and (p/2) x (p/2) is 0.0025. Consequently only about 2 - 3 worms in a thousand will be double mutants if the genes were 10MU apart. That is significantly lower than the 63/1000 one would expect if the genes were not linked (1/16 of the progeny). Therefore, the test for linkage is usually the virtual if not complete absence of the double mutant class (d u/d u). You will use this first assesment to determine which chromosome dpy mutation is on (the chromosome location of each of the unc mutations is known). <br>
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| Once you determined in which chromosome your dpy and unc are (for successful mapping they must be on the same chromosome), we will perform a two factor cross, which in ''C. elegans'' requires that we first construct a double mutant (du/du). As discussed above, the appearance of a double mutant in the F2 requires the union of 2 recombinant gametes -- a very rare event. It is unlikely therefore that we will find a double mutant among the F2 progeny if the mutants are linked. The probability of a recombination event having occurred on one of the two homologues is much better; that is, there will be many more progeny that are genotypically du/d+ than du/du. It would exhibit the dpy mutation's phenotype and would segregate double mutants (du/du) as one quarter of its progeny. To find a double mutant for mapping, we choose 5 individuals of the dpy phenotype from the F2 linkage testing plate to self fertilize and look for the segregation of the du/du double mutant among their progeny. <br>
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| To map, one crosses a homozygous double mutant hermaphrodite (du/du) with wild type males. The heterozygous F1 hermaphrodites self fertilize and the number of F2 individuals of each different phenotype are then counted. As discussed above, four phenotypes will be observed: wild type, dpy, unc, and dpy unc. The map distance can be calculated any number of ways. You will determine map distances using the formula: RF (recombinant frequency) = the number of single mutants (dpy and unc single mutants totals) divided by the total number of worms counted * 100 (to obtain it in % recombinants and thus in map units).<br>
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| '''To Do Today (per group)'''<br>
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| #Choose one of the six plates at the front of the room <br>
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| #Scan your “mutagenized” plate for visible dumpy mutants
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| #Transfer 1 putative mutant hermaphrodite to each of three new plates. Take care not to transfer any other animals except your putative mutant.<br>
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| #Label these identical plates with the hermaphrodite mutant's strain information and your group's information. Use your <font color= blue><b>BLUE </b></font color> Sharpie for labeling mapping experiment plates.
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| #Put an elastic around the three mapping plates. Place them in your group's plastic box (make sure your box is labeled with your names and lab section).
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| #Place your box on the shelf in the incubator for your lab section.<br>
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| #Incubate your worms at 23°C for 3 days.<br>
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| '''3 Days After Lab'''<br>
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| #Examine your three plates containing your dpy worms from the mutant hunt. Are all the worms on the plates the dpy phenotype? If '''YES''', then start a cross between 4 dpy L4 hermaphrodites (still have the clearing about 1/2 way down the body of the worm) and 3-4 wild type male worms. If '''NO''' contact your instructor ASAP!<br>
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| #Set up two identical mating plates - 3-4 L4 hermaphrodites and 3-4 wild type males.<br>
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| #Label these identical plates with the hermaphrodite mutant's strain information and your group's information. Use your <font color= blue><b>BLUE </b></font color> Sharpie for labeling mapping experiment plates. <br>
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| #Incubate your worms at 23°C until next lab period.
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