Question

1. Briefly describe the purpose/role of): Transformation of bacteria with ligation reactions. What specifically did this step allow for and how is the step important to the overall sequencing analysis?

2. Does a 15 nucleotide deletion alter the protein sequence?

Answer 1

1. Transformation is a process in which bacteria takes up the recombinant DNA from ouside environment and then makes multiple copies of it. This is called cloning. Ligation refers to the process of joining our 'insert' (desired gene) with that of vector genetic material. The enzyme used for ligation is T4 DNA ligase. It links the 5' phosphate and 3' OH groups with the help of ATP and Mg2+ ions. These are present in the ligation mixture. Ligation results into the formation of phosphodiester bonds. It is an important step as it results into the formation of 'recombinant DNA'. This rDNA would be taken up by the host which will produce multiple copies of it, necessary for cloning and for sequencing.

2. Yes, it wil affect the protein sequence. A deletion mutation causes the change in frame of reading the codon tripletes. Thus, they are called framsehift mutations. Though, in some cases when the number of nucleotides are in multiple of three, silent mutations may occur. But if large deletions like that of 15 nucleotides occur, it may cause loss of an entire gene. Thus, affecting the protein structure and function adversely.