Question

You have access to a polymerase that has a maximum extension of 3kb. Below is a (not to scale) schematic from the paper depicting the location of the transposon within the cct8 gene. For our purposes, the 5' end of Primer 1 is at bp 170 of the WT allele, the 5' end of Primer 2 is at bp 2030 of the WT allele (not including the transposon). The GBT-P9 transposon is 6000 bp long, inserted at bp 300 of the WT allele with the 5' end of primer 3 at bp 5700 of the 6000 bp transposon. Given the schematic and primers below: Primer 3 GBT-P9 cct8 El E2 Primer 2 Primer 1 a. Indicate which primer pair you would use to identify the WT allele. b. Indicate which primer pair you would use to identify the mutant allele c. Draw a picture of a gel with the expected results if you were to use the primer pairs in a and b to genotype: a homozygous WT sample, a heterozygous sample, and homozygous mutant (with two copies of the transposon breaking gene). Be sure to label each lane with the sample and primer pair. Be sure to label the expected size of each band. Be sure to label the positive (anode) and negative (cathode) ends of the gel d. What controls would you want to include in your PCR reaction and why? (these do not need to be included in the picture in c)

Answer 1

You have access to a polymerase that has a maximum extension of 3kb. Below is a (not to scale) Schema g' epcting the location of the transposon wi cDeimer 2 is at bp or our 5700 bp (of transposon) 2030 of the WT allele (not including the transposon). The GBT-P9 transposon is 6000 bp long inserted at bp 300 of the WT allele with the 5' end of primer 3 at bp 5700 of the 6000 bp transposon. Given the schematic and primers below: Primer 3 Primer 3 E GBT-PS GBT-P9 cctB EI El FZ Primer 1 Primer 2 Primer 2 Primer 1 170 bp 300 bp 2030 bp a. Indicate which primer pair you would use to identify the WT allelerimer 1 and primer 2 b. Indicate which primer pair you would use to identify the mutant allele. Primer 3 and primer 2 Region amplified by primer 1 and 2 in WT allele 2030-170 1860 bp Region amplified by primer 1 and 2 in mutant allele = region amplified by them in WT allele + 6000bp transposon 7860 bp (which cannot be amplified by the polymerase whose max extension is 3000bp c. Draw a picture of a gel with the expected results if you were to use the primer pairs in a and b genotype: a homozygous WT sample, a heterozygous sample, and homozygous mutant (with two copies of the transposon breaking gene). Be sure to label each lane with the sample and primer pair. Be sure to label the expected size of cach band. Be sure to label the positive (anode) and negative (cathode) ends of the gel. Region amplified by primer 1 and primer 3 in mutant allle 300- 170+5700 bp 130+5700 5830 bp Not possible by the polymerase Region amplified by primer 3 and 3 in mutant allele (6000- 5700)+(2030-300) 2030 bp c) Homozygous Heterozygous Homozygous Mutant cathode WT Primer pair in b 2030 bp rols would you want to include in your PCR reaction and why? (these do not need to ed in the picture in c) 1860 bp d. What co Primer pair in a be inclu Anode