Question

2) Additional experiments confirmed that the difference in sequence between the expected genomic sequence and the experimentally determined cDNA sequence is neither due to a replication error nor to a DNA sequencing mistake. One of your coworkers decided to perform 5 independent experiments. In each experiment, the gene was first transcribed and then the transcript was purified and reverse- transcribed to produce double stranded cDNA copies of the gene. Finally, one copy of synthesized cDNA from each independent experiment was sequenced (5 samples sequenced in total). The difference between the cDNA and genomic DNA sequences described in part 1 was found in the 5 sequences. Explain why this result suggested that a transcription error was unlikely responsible for the sequence difference.

Answer 1

Any genomic sequence of any gene consists of exons and introns. Exon are sequences that codes for protein whereas introns are sequences present in between exons, that don't code for protein. When mRNA is transcribed from genomic DNA, both Exon and intron sequences are transcribed as hnRNA. hnRNA is then undergone 5' capping, 3' polyadenylation and splicing. Splicing allows to remove intronic sequences from the Exon sequences and allows exon sequences to join to one another and finally forms mRNA. This mRNA is translated into protein.

When cDNA is synthesized from mRNA, only exonic sequences come. No intronic sequences are present in the cDNA.

Thus, any genomic DNA sequence will contain both exon and intron sequences but cDNA sequence only contains exons.

Thus there is a difference in the sequence and this sequence difference is not dur to transcriptional error.

Some times alternative splicing occurs during maturation of hnRNA into mRNA. During this alternative splicing, some exonic sequences may also be skipped and are not included in the mRNA. Thus, alternative spliced mRNA sequence may be differed from other spliced mRNA sequence.