Below are 120 consecutive base pairs of DNA that include only the beginning of t

Question

Below are 120 consecutive base pairs of DNA that include only the beginning of the sequence of gene X. The underlined and italicized sequence (from position 15-40) represents the promoter for gene X. The underlined and italicized sequence (from position 61-69) encodes the gene X ribosome binding (RBS) site. Transcription begins at and includes the T/A base pair at position 50 (bold). Answer A, B, C, D and E A) What are the first 3 nucleotides of the mRNA from gene X? Label the 5' and 3' ends of the sequence. B) What are the first 3 amino acids encoded by gene X? Label the N and C termini of the sequence. C) Design a forward primer that could be used as part of a primer pair to amplify this entire sequence. Label the 5' and 3' ends of the primer in your answer. D) Calculate the Tm of the primer. Show your working. E) You have found cells with a mutation of gene X. The mutant cells have deletion of three base pairs (5' CTA 3') that includes the C/G base pair at position 80 (shown in bold). If the mRNA from this mutant gene can be translated, would you expect the protein to be longer, shorter, or the same as that produced from the normal gene X? If longer or shorter, indicate by how many amino acids.

Explanation / Answer

First 4 questions answered

A) Since It is given that Transcription starts with T-A base pair at position 50, so the first 3 nucleotides of m-RNA will be 5'- UAG- 3'. we know in transcription the m -RNA grows in 5' to 3' direction and it contains the same information as the Coding strand except Thymine (T) is replaced by Uracil (U).

B) The translation will start from base 76-78 where it finds an initiation codon (i.e; AUG) next to Ribosome Binding site(RBS) . In the given sequence the first 3 codon will be AUG- ACU- AGC, starting from base 76 to 84. So the first 3 amino acid will be N terminal-Met-Threonine- Serine-C terminal.

C) The expected forward primar would be 5'-GGCTACTAATAAACTTGAC-3'. We need to amplify the entire sequence. So we had to choose the forward primer wchich will start from very begining of the sequence. The primer sequence is 19 bp long with 37% GC content.

D) For sequences longer than 13 nucleotides, the equation used for Tm calculation is

     Tm= 64.9 +41*(yG+zC-16.4)/(wA+xT+yG+zC). where w,x,y,z are the number of the bases A,T,G,C in the sequence, respectively. Assuming that assume that the annealing occurs under the standard conditions of 50 nM primer, 50 mM Na+, and pH 7.0.

for the above primer sequence the Tm is 44.6

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