Question

A portion of DNA sequence is

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

Assume GTACCGATCAGCAGTCA and CATGGCTAGTCGTCAGT are introns

1) Which strand will be the template strand for the transcription?

2) Write down the mature messenger RNA sequence, label the 5' and 3' ends, and additional elements found in mature messenger RNA.

3) Based on the mRNA sequence, draw a line between each codon in question 2) and write the sequence for the polypeptide that can be translated from the N-terminus to C-terminus.

4) If Cas9 used an eight bp long guide RNA sequence, which of the following two sequences would be, assuming that Cas9 cuts the DNA down the middle of the guide RNA sequence?

#1: 5’ACCGAUCA 3’ #2: 5’ATCGCGCT 3’

Answer 1

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

Coloured region shows the intron.

1)  5’ ATCGCGCTCAGCTAGTACCGATCAGCAGTCAGCAGTCAGCATCGGT 3’ (top) - Coding strand
3’ TAGCGCGAGTCGATCATGGCTAGTCGTCAGTCGTCAGTCGTAGCCA 5’ (bottom) -Template strand

2) mRNA will be transcribed from template strand -

3’ TAGCGCGAGTCGATCATGGCTAGTCGTCAGTCGTCAGTCGTAGCCA 5’ (bottom) -Template strand

5' AUCGCGCUCAGCUAGUACCGAUCAGCAGUCAGCAGUCAGCAUCGGU 3'- pre- mRNA

Exon Intron Exon     

Intron region will be removed by the splicing and exons combine to form the mature mRNA -

5' AUCGCGCUCAGCUAGCAGUCAGCAUCGGU 3' - Mature mRNA .

3) 5' AUC GCG CUC AGC UAG CAG UCA GCA UCG GU 3'

N terminus - Ile- Ala-Leu-Ser-stop-Gln-Ser-Ala-Ser. (Polypeptide sequence).

4) #1 5’ACCGAUCA 3’ would be correct answer , assuming that Cas9 cuts the DNA down the middle of the guide RNA sequence, because it binds in the intron middle region.

5' AUCGCGCUCAGCUAGUACCGAUCAGCAGUCAGCAGUCAGCAUCGGU 3'- pre- mRNA

#2: 5’ATCGCGCT 3’ binds in exon region -

5’ ATCGCGCTCAGCTAGTACCGATCAGCAGTCAGCAGTCAGCATCGGT 3’ (top) - Coding strand