Question

Below is a portion of DNA sequence, introns are shown in bold. Determine which strand contains a start codon and could serve as the template for synthesis of an mRNA molecule.

5’ ATCGCGCTCAGCTAGTACCGATCAGCAGTCAGCAGTCAGCATCGGT 3’ (top) 3’ TAGCGCGAGTCGATCATGGCTAGTCGTCAGTCGTCAGTCGTAGCCA 5’ (bottom)

a. Which strand will serve as the template strand for transcription?

b. Based on the template strand you have chosen, write the mature mRNA sequence on your answer sheet in the 5’ to 3’ direction. Be sure to label 5’ and 3’ ends of the mRNA and any additional elements found in mature mRNA.

c. Based on your mRNA sequence, draw a line between each codon in part b, above, and write the sequence of the polypeptide that can be translated from the N-terminus to C-terminus. Be careful of the codons you choose and be sure to label the N- and C- termini of the sequence you write.

d. If Cas9 used an 8 bp long guide RNA sequence, which of the following sequences would be best knock out this gene assuming that Cas9 cuts the DNA down the middle of the guide RNA sequence? Explain your choice and why the other guide RNA is not ideal. Include in your answer a description of why cutting by Cas9 will result in a knockout of the gene where no functional protein being produced.

Guide RNA #1: 5’ACCGAUCA 3’ Guide RNA #2: 5’ATCGCGCT 3

Answer 1

DNA sequence given -

5’ ATCGCGCTCAGCTAGTACCGATCAGCAGTCAGCAGTCAGCATCGGT 3’ (top)

3’TAGCGCGAGTCGATCATGGCTAGTCGTCAGTCGTCAGTCGTAGCCA 5’ (bottom)

a. Answer- 5’ ATCGCGCTCAGCTAGTACCGATCAGCAGTCAGCAGTCAGCATCGGT 3’ (top) - Coding strand
3’ TAGCGCGAGTCGATCATGGCTAGTCGTCAGTCGTCAGTCGTAGCCA 5’ (bottom) -Template strand

b.Template starnd will be used to obtain the mRNA-

3’ TAGCGCGAGTCGATCATGGCTAGTCGTCAGTCGTCAGTCGTAGCCA 5’ (bottom) -Template strand

The Adenine base will change to the Uracil in mRNA.

First it will form the pre mRNA -

  5' AUCGCGCUCAGCUAGUACCGAUCAGCAGUCAGCAGUCAGCAUCGGU 3'- pre- mRNA

The intron region (shown in purple) will be spliced ,only flanking exon join to form the mature mRNA.

5' AUCGCGCUCAGCUAGCAGUCAGCAUCGGU 3' - Mature mRNA .

C. 5' AUC GCG CUC AGC UAG CAG UCA GCA UCG GU 3' - Mature mRNA .

Mature mRNA will code for the amino acid (polypeptide sequence).

N' terminal Ile- Ala-Leu-Ser-stop-Gln-Ser-Ala-Ser C' terminal.

D. Guide RNA #1: 5’ACCGAUCA 3’

Guide RNA #1: 5’ACCGAUCA 3’ will bind to the intron region and get spliced off, thus would be best for knock out this gene assuming that Cas9 cuts the DNA down the middle of the guide RNA sequence.

5' AUCGCGCUCAGCUAGUACCGAUCAGCAGUCAGCAGUCAGCAUCGGU 3'- pre- mRNA

Guide RNA #2: 5’ATCGCGCT 3 -binds to exon region,thus will not be helpful for the knockdown.

5’ ATCGCGCTCAGCTAGTACCGATCAGCAGTCAGCAGTCAGCATCGGT 3’ (top)