Precision will be reduced, but yield will be increased

Optimisation of a PCR reaction is often a compromise between the competing demands for precision, efficiency and yield. Although the specific effects may vary, generally, increasing the annealing temperature will increase non-specific primer binding and reduce precision. Increasing the length of the elongation phase will reduce the proportion of incomplete newly-synthesised strands and therefore increase yield. In this case, the potential effect on efficiency is unclear. Increasing the elongation phase would increase the reaction time, but the time taken to ramp down to a lower annealing temperature would be reduced.

 Which of the following is true for traditional and real time PCR?

TLS Online TPP Program

#Question id: 16134

#Unit 13. Methods in Biology

You obtain 6 BACs (of known order, as shown below) and 7 STSs (of unknown order) that derive from a region of mouse chromosome 16 whose genomic sequence has not yet been finished.   

 

By PCR (using 20-bp primers at either end of each STS), you test each of the 6 BACs for the presence (+) or absence (-) of each of the 7 STSs. You obtain the following results:

 

You determine the DNA sequences of PCR products obtained at STS5 using BACs B, C, D, E, and F as PCR templates. The DNA sequences of PCR products obtained using BACs B and C are identical, but they differ by several nucleotide substitutions from the DNA sequences of PCR products obtained using BACs E and F. Oddly, PCR product obtained using BAC D appears to be a mixture of two sequences – one corresponding to the BAC B/ BAC C sequence and the other corresponding to the BAC E/ BAC F sequence. Briefly account for these findings, and explain how a single PCR assay could amplify two different sequences.

TLS Online TPP Program

#Question id: 16135

#Unit 13. Methods in Biology

You obtain 6 BACs (of known order, as shown below) and 7 STSs (of unknown order) that derive from a region of mouse chromosome 16 whose genomic sequence has not yet been finished.   

 

By PCR (using 20-bp primers at either end of each STS), you test each of the 6 BACs for the presence (+) or absence (-) of each of the 7 STSs. You obtain the following results:

 

Would you expect the DNA sequence of PCR product obtained at STS5 using mouse genomic DNA as template to more closely resemble that obtained using BAC B, BAC D, or BAC F? Briefly explain your answer.

TLS Online TPP Program

#Question id: 16136

#Unit 13. Methods in Biology

You obtain 6 BACs (of known order, as shown below) and 7 STSs (of unknown order) that derive from a region of mouse chromosome 16 whose genomic sequence has not yet been finished.   

 

By PCR (using 20-bp primers at either end of each STS), you test each of the 6 BACs for the presence (+) or absence (-) of each of the 7 STSs. You obtain the following results:

 

Is there a second STS at which you would like to sequence PCR products obtained using BACs as templates? If so, which BACs would you test in this way, and what sequencing results might you predict for each of the BACs tested?

TLS Online TPP Program

#Question id: 16137

#Unit 13. Methods in Biology

You obtain 6 BACs (of known order, as shown below) and 7 STSs (of unknown order) that derive from a region of mouse chromosome 16 whose genomic sequence has not yet been finished.   

 

By PCR (using 20-bp primers at either end of each STS), you test each of the 6 BACs for the presence (+) or absence (-) of each of the 7 STSs. You obtain the following results:

 

How would you use the sequence information presented in to design two new STSs (with new PCR primer pairs) to replace STS5? (Call the new ones STS51 and STS52. STS51 should be present (+) in BAC B, and STS52 should be present (+) in BAC F.)

TLS Online TPP Program

#Question id: 16138

#Unit 13. Methods in Biology

You obtain 6 BACs (of known order, as shown below) and 7 STSs (of unknown order) that derive from a region of mouse chromosome 16 whose genomic sequence has not yet been finished.   

 

By PCR (using 20-bp primers at either end of each STS), you test each of the 6 BACs for the presence (+) or absence (-) of each of the 7 STSs. You obtain the following results:

 

Would you expect STS51 and STS52 to be present in BAC D? 

TLS Online TPP Program

#Question id: 16139

#Unit 13. Methods in Biology

 Many mouse genes are “tissue-specific,” that is, they are present throughout the body but are expressed in only one of the animal’s many tissue types. (Other mouse genes are expressed throughout the body, or in multiple tissues.) Geneticists can study the regulation of a mouse gene by fusing the gene’s promoter region to the LacZ coding sequence and injecting the construct to create a transgenic mouse. Fusion of the mouse amylase promoter to LacZ yielded a Pamylase-LacZ construct.              

Mice heterozygous for the resulting Pamylase-LacZ  transgene displayed the LacZ expression exclusively in the pancreas. Would you expect homozygotes for the transgene to also display LacZ expression in the pancreas?.