BISC 219/F10: Lab 3

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(Lab 3: Gene Mapping: Linkage Analysis)
Current revision (20:52, 30 September 2010) (view source)
(Links to Labs& Project Info)
 
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[[BISC 219/F10:Gene Mapping  | Lab 2: Gene Mapping]]<br>
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== Lab 3: Forward Genetics Project: Linkage Analysis ==
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[[BISC 219/F10: Lab 4  | Lab 4: Linkage Test Part 2 and Mapping]]<br>
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[[BISC 219/F10: Lab 5  | Lab 5: Mapping Part 2]]<br>
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[[BISC 219/F10: Lab 6  | Lab 6: Score]]<br>
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== Lab 3: Gene Mapping: Linkage Analysis ==
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Now that you have found from your mutant hunt a visible marker of a gene defect (phenotype alteration from wild type), you will determine on which of the five autosomes (linkage groups) the gene mutation responsible for your aberrant phenotype is located. This task is a prerequisite to mapping the mutation (locating where on a particular chromosome the mutation is likely to be found). Linkage testing is accomplished by determining the segregation behavior of your unmapped mutation relative to standard reference markers (e.g., mutations whose locations are already known). Recall that unlinked mutations will segregate independently (your basic dihybrid inheritance as first observed by Gregor Mendel) whereas linked mutations will not.  
Now that you have found from your mutant hunt a visible marker of a gene defect (phenotype alteration from wild type), you will determine on which of the five autosomes (linkage groups) the gene mutation responsible for your aberrant phenotype is located. This task is a prerequisite to mapping the mutation (locating where on a particular chromosome the mutation is likely to be found). Linkage testing is accomplished by determining the segregation behavior of your unmapped mutation relative to standard reference markers (e.g., mutations whose locations are already known). Recall that unlinked mutations will segregate independently (your basic dihybrid inheritance as first observed by Gregor Mendel) whereas linked mutations will not.  
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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 visually distinguishable. Since homozygous mutant males usually will not mate, the desired double heterozygote is constructed by mating males that are heterozygous for your dpy mutation [wild type for all other genes including the reference mutation (d/+;+/+)] with hermaphrodites that are homozygous for the reference mutation unc(+/+; u/u) and have no dpy mutation. 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 separate plates (one animal 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/+). You should find d/d homozygotes on 50% of the plates. Why?  
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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 visually distinguishable. Since homozygous mutant males usually will not mate, the desired double heterozygote is constructed by mating males that are heterozygous for your ''dpy'' mutation [wild type for all other genes including the reference mutation (d/+;+/+)] with hermaphrodites that are homozygous for the reference mutation ''unc'' (+/+; u/u) and have no ''dpy'' mutation. 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 separate plates (one animal 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/+). You should find d/d homozygotes on 50% of the plates. Why?  
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: 9/16 wild type; 3/16 d, 3/16 u; 1/16 du (that is the 9:3:3:1 ratio)!
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: 9/16 wild type; 3/16 d, 3/16 u; 1/16 du (that is the 9:3:3:1 ratio)!
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'''To Do Today:'''
'''To Do Today:'''
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1. For linkage testing set up four different crosses. Each cross will contain 3 heterozygous males (d/+; +/+) from the cross you initiated using your mutant Dpy worms. Make sure that these are the only animals that you transfer from that plate by transferring the males to a transfer plate and letting them crawl around for a minute - away from any contaminating worms - then pick a second time to the mating plate. <bR>
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1. For linkage testing set up five different crosses. Each cross will contain 3 heterozygous males (d/+; +/+) from the cross you initiated using your mutant Dpy worms. Make sure that these are the only animals that you transfer from that plate by transferring the males to a transfer plate and letting them crawl around for a minute - away from any contaminating worms - then pick a second time to the mating plate. <bR>
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2. Each heterozygous (d/+; +/+) male will be mated to three L4 hermaphrodites that are homozygous for one of 5 known Unc (+/+; u/u) mutations on a mating plate. The strains and their reference mutations are: <br>
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2. Each heterozygous (d/+; +/+) male will be mated to three L4 hermaphrodites that are homozygous for one of 5 known ''unc''(+/+; u/u) mutations on a mating plate. The strains and their reference mutations are: <br>
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3. Label your five plates with your PURPLE Sharpie. With the genotype of the strain - for example:  +/+; unc-13/unc-13 (H) X d/+; +/+ (M) with your initials and date. <br>
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3. Label your five plates with your <font color=purple>PURPLE </font color=purple> Sharpie. With the genotype of the strain - for example:  +/+; ''unc-13/unc-13'' (H) X d/+; +/+ (M) with your initials and date. <br>
4. Incubate all of the worms at 23°C for 3 days in your team's worm box.<br>
4. Incubate all of the worms at 23°C for 3 days in your team's worm box.<br>
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#For linkage testing, transfer 2 wild type cross progeny (heterozygous for both traits) that are L4 stage hermaphrodites from each of your 5 crosses to each of 2 new plates per cross for a total of 10 plates.  
#For linkage testing, transfer 2 wild type cross progeny (heterozygous for both traits) that are L4 stage hermaphrodites from each of your 5 crosses to each of 2 new plates per cross for a total of 10 plates.  
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#Label your 10 (5 sets of duplicates) plates with your PURPLE Sharpie. Label each plate with your initials, the genotype of your worms and the date. In each case, why is it important that you transfer L4’s and not adults? What is the genotype and phenotype of your expected F2 progeny?
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#Label your 10 (5 sets of duplicates) plates with your <font color=purple>PURPLE </font color=purple> Sharpie. Label each plate with your initials, the genotype of your worms and the date. In each case, why is it important that you transfer L4’s and not adults? What is the genotype and phenotype of your expected F2 progeny?
#Incubate all worms at 23°C until the next lab period.
#Incubate all worms at 23°C until the next lab period.
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== Assignment ==
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Remember to check the Assignment section of the wiki for instructions about the graded assignment due in the next lab and check the Weekly Calendar for other work to accomplish before the next lab.
 +
==Links to Labs& Project Info==
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Series1:<BR>
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[[BISC 219/F10: Worm Info| Worm Info]] <br>
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[[BISC 219/F10: Gene Linkage| Lab 1: Worm Boot Camp & Sex-Linked or Autosomal Start]]<BR>
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[[BISC 219/F10: Lab 2  | Lab 2: Sex-Linked or Autosomal Finale]]<br>
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Series2:<BR>
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[[BISC 219/F10: Gene Mapping Info | Background: Classical Forward Genetics and Gene Mapping]]<br>
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[[BISC 219/F10: Lab 2 Mutant Hunt | Lab 2: Mutant Hunt]]<br>
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[[BISC 219/F10: Lab 3  | Lab 3: Linkage Test Part 1]]<br>
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[[BISC 219/F10: Lab 4  | Lab 4: Linkage Test Part 2, Mapping and Complementation]]<br>
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[[BISC 219/F10: Lab 5  | Lab 5: Finish Complementation; Mapping Con't]]<br>
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[[BISC 219/F10: Lab 6 | Lab 6: DNA sequence analysis; Mapping Con't]]<BR>
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[[BISC 219/F10: Lab 7  | Lab 7: Complete Mapping: Score]]<br>
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Series3:<BR>
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[[BISC 219/F10:RNA interference | Schedule of Reverse Genetics Project]]<BR>
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[[BISC 219/F10:RNAi General Information| RNAi General Information]] <br>
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[[BISC 219/F10:Media Recipes | Media Recipes]]<br>
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[[BISC 219/F10: RNAi Lab 5  | Lab 5: Picking your gene to RNAi]]<br>
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[[BISC 219/F10: RNAi Lab 6  | Lab 6: Cloning your gene of interest]]<br>
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[[BISC 219/F10: RNAi Lab 7  | Lab 7: Picking your transformant]]<br>
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[[BISC 219/F10: RNAi Lab 8  | Lab 8: Plasmid purification and transformation]]<br>
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[[BISC 219/F10: RNAi Lab 9  | Lab 9: Induction of bacteria for RNAi]]<br>
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[[BISC 219/F10: RNAi Lab 10 | Lab 10: Scoring your worms and RNA purification]]<br>
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[[BISC 219/F10: RNAi Lab 11 | Lab 11: RT PCR reactions]]<br><br>

Current revision

Lab 3: Forward Genetics Project: Linkage Analysis

Now that you have found from your mutant hunt a visible marker of a gene defect (phenotype alteration from wild type), you will determine on which of the five autosomes (linkage groups) the gene mutation responsible for your aberrant phenotype is located. This task is a prerequisite to mapping the mutation (locating where on a particular chromosome the mutation is likely to be found). Linkage testing is accomplished by determining the segregation behavior of your unmapped mutation relative to standard reference markers (e.g., mutations whose locations are already known). Recall that unlinked mutations will segregate independently (your basic dihybrid inheritance as first observed by Gregor Mendel) whereas linked mutations will not.

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 visually distinguishable. Since homozygous mutant males usually will not mate, the desired double heterozygote is constructed by mating males that are heterozygous for your dpy mutation [wild type for all other genes including the reference mutation (d/+;+/+)] with hermaphrodites that are homozygous for the reference mutation unc (+/+; u/u) and have no dpy mutation. 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 separate plates (one animal 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/+). You should find d/d homozygotes on 50% of the plates. Why?

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: 9/16 wild type; 3/16 d, 3/16 u; 1/16 du (that is the 9:3:3:1 ratio)!

On the other hand, if the markers are closely linked double homozygotes (d u/d u) will occur only through a very rare recombination event; therefore, you are not likely to observe the double mutant class.

To Do Today:

1. For linkage testing set up five different crosses. Each cross will contain 3 heterozygous males (d/+; +/+) from the cross you initiated using your mutant Dpy worms. Make sure that these are the only animals that you transfer from that plate by transferring the males to a transfer plate and letting them crawl around for a minute - away from any contaminating worms - then pick a second time to the mating plate.
2. Each heterozygous (d/+; +/+) male will be mated to three L4 hermaphrodites that are homozygous for one of 5 known unc(+/+; u/u) mutations on a mating plate. The strains and their reference mutations are:


Chromosome Strain Phenotype
Chromosome 1 unc-13 coiler
Chromosome 2 unc-52 immobile
Chromosome 3 unc-32 coiler
Chromosome 4 unc-17 coiler
Chromosome 5 unc-60 immobile

3. Label your five plates with your PURPLE Sharpie. With the genotype of the strain - for example: +/+; unc-13/unc-13 (H) X d/+; +/+ (M) with your initials and date.
4. Incubate all of the worms at 23°C for 3 days in your team's worm box.

3-4 days after lab:

  1. For linkage testing, transfer 2 wild type cross progeny (heterozygous for both traits) that are L4 stage hermaphrodites from each of your 5 crosses to each of 2 new plates per cross for a total of 10 plates.
  2. Label your 10 (5 sets of duplicates) plates with your PURPLE Sharpie. Label each plate with your initials, the genotype of your worms and the date. In each case, why is it important that you transfer L4’s and not adults? What is the genotype and phenotype of your expected F2 progeny?
  3. Incubate all worms at 23°C until the next lab period.

Assignment

Remember to check the Assignment section of the wiki for instructions about the graded assignment due in the next lab and check the Weekly Calendar for other work to accomplish before the next lab.

Links to Labs& Project Info

Series1:
Worm Info
Lab 1: Worm Boot Camp & Sex-Linked or Autosomal Start
Lab 2: Sex-Linked or Autosomal Finale
Series2:
Background: Classical Forward Genetics and Gene Mapping
Lab 2: Mutant Hunt
Lab 3: Linkage Test Part 1
Lab 4: Linkage Test Part 2, Mapping and Complementation
Lab 5: Finish Complementation; Mapping Con't
Lab 6: DNA sequence analysis; Mapping Con't
Lab 7: Complete Mapping: Score
Series3:
Schedule of Reverse Genetics Project
RNAi General Information
Media Recipes
Lab 5: Picking your gene to RNAi
Lab 6: Cloning your gene of interest
Lab 7: Picking your transformant
Lab 8: Plasmid purification and transformation
Lab 9: Induction of bacteria for RNAi
Lab 10: Scoring your worms and RNA purification

Lab 11: RT PCR reactions

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