Question

based on the given graph how would these be answered?

sa sequence from a sheep PCR product. The template used was genomic DNA and the forward and se primers were designed to amplify partofthe priongene, which encodes the causative agentot scrapie. is black;Cisblue; Ais green: Tisred. The forward primerwasusedasasequencing primer. TheNattheten ow means that the computer can't decide which nucleotide is at that position in the sequence because there are two peaks (red=T and blue-C) that are the same height. The computer called the nucleotide A at the ghtarrow, but it was wrong because there are two peaks at the same position of nearly the same height (green=A and black=G). GIENCE TAG AG GIC C TIC TITAATITIS GCAA TIG AC 28A Which strand of DNA would resultfrom sequencing with the forward primerifthe top strand is the sense strand (translatable, same as mRNA sequence)and the bottom strand is the non-sense strand? Which strand of DNA wouldresultfrom sequencing with the reverse primerifthe top strand is the sense strand and the bottom strand is the non-sense strand? AB. Codons are indicated by vertical marks. What are the amino acids encoded by the codons at the: First arrow (left). Secondarrow(right) How would you label the: X-axis: y-axis 92The arrowheads point to positions where there are two peaks. If the ratio of their height on the y-axis is 1:1, how is this correlated with the genomic DNA template used and the prion gene alleles?

Answer 1

During a PCR reaction, The template DNA separates and the primers anneal to each of the strands to amplify the region of interest. The primers always anneal to the 3' end of the template and the Taq DNA polymerase starts adding nucleotides in the 5'>3' direction (shown in the picture below). The forward primer anneals to the 3' end of the 3' > 5' template strand (non-sense strand / bottom strand) whereas the reverse primer anneals to the 3' end of the 5' > 3' template strand (sense strand/ top strand).

$image?w=601&h=305&rev=141&ac=1&parent=1n$

a. Since the forward primer would anneal to the bottom, non-sense strand, the resulting strand from sequencing would be the top, sense strand.

b. Since the reverse primer would anneal to the top, sense strand, the resulting strand from sequencing would be the bottom, non-sense strand.

c.

GENCIJAG AG GIC CITTAA TETTO GCAA GAG

The amino acids for each triplet code demarcated above can be decihered using the codon table. The triplet code can be used by looking for the first letter in the left of the table, second letter in the middle and the third letter in the right of the codon table.

The codon at the first arrow is GNC which could be either GTC or GCC. GTC codes for Valine whereas GCC codes for Alanine.

The codon at the second arrow could be CAT or CGT which code for Histidine or Arginine respectively.

d. The graph represents the fluoresence signal intensities of different nucleotides of the DNA sequence. The X-axis could be labeled as "nucleotide" and the Y-axis "amplitude" .