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Lecture 10: Reading Frame Home Exercise

Problem 1: Some mutations can lead to the mislocalization or reduced expression of the CFTR protein.
You discover a candidate CF mutation directly upstream of the CFTR gene and you want to amplify the
region to insert it into a plasmid.

Design primers to amplify the entire region upstream of the CFTR gene and place it upstream of the
GFP plasmid. Bolded nucleotides are of interest and should be included in the amplified region. The
MCS of the EGFP-N1 plasmid is shown below; design two sets of primers, each of which uses different
restriction enzymes to insert the fragment.

Hint: The portion of the primers that anneal to the gene should be 19 nt long and these sequences
should be shared between the two different primer sets

CGC GGC CCA CCG TCA GGG TGG AAC TAG TGC TGG GCG GTA AGG ACA CGG ACC TGG AAG
GAG CGC GCG CGA GGG AGG GAG GCT GGG AGT CAG AAT CGG GAA AGG GAG GTG CGG GGC
GGC GAG GGA GCG AAG GAG GAG AGG AT TAA TGA TAG CGA GCG GCG CTG GCG GGG GTG
CGT AGT GGG TGG AGA AAG CCG CTA GAG CAA ATT TGG GGC CGG ACC AGG CAG CAC TCG
GCT TTT AAC CTG GGC AGT GAA GGC GGG GGA AAG AGC AAA ATG AAG GGG TGG TAG AAT
TAA TAA AAA TTA ATA TAT AAA

Problem 2: Will you need to add any additional nucleotides to the 5’ or 3’ ends of any of the primers in
problem 1? Explain why or why not.
Problem 3: Next, you want to insert the entire CFTR coding region (abridged sequence shown below)
downstream of GFP. The MCS downstream of GFP is shown.

a) Identify the start and stop codons.

b) Design forward and reverse primers to amplify the entire gene and insert the PCR product
downstream of GFP using BspEI and HindIII.

Hint: Primers will be 20 nucleotides long, have a Tm of 55-60C, and less than 60% GC content.
Remember to maintain the CFTR reading frame. There is a codon chart on the next page.

CFTR Sequence:

CGA GAG ACC ATG CAG AGG TCG CCT CTA AAA AAG GCC AGC GTT GTC TCC AAA …. TTA CCT
CTG CCT CAG AGA ACA AGG ATG AAT TAA GCC ATT TTT TAA

Problem 4: Using the same MCS and CFTR sequence as shown above to insert CFTR downstream of GFP:

a) Design forward and reverse primers to amplify the entire gene and insert the PCR product
downstream of GFP using XhoI and HindIII.
Hint: you can use the same annealing sequence as above

Problem 5: Will you need to add any additional nucleotides to any of the primers in problem 4? Explain
why or why not.
Problem 6: You decide that an N-terminal GFP is ineffective and want to insert the entire CFTR coding
region (abridged sequence shown below) so that you have a C-terminal GFP. The MCS upstream of GFP
is shown. Start and stop codons are bold.

a) Design forward and reverse primers to amplify the entire gene and insert the PCR product
upstream of GFP using HindIII and BamHI.
Hint: Remember to maintain the open reading frame.

CFTR Sequence:

CGA GAG ACC ATG CAG AGG TCG CCT CTA AAA AAG GCC AGC GTT GTC TCC AAA …. TTA CCT
CTG CCT CAG AGA ACA AGG ATG AAT TAA GCC ATT TTT TAA