Question

10. How would you try to figure out the function of a novel gene you cloned from a bacterium?

Answer 1

For determine the function f a novel gene the following steps are to be done:

1. Gene sequencing

After isolation of gene you want to know the sequence of gene. Gene sequencing is the process of determining the nucleic acid sequence in the order of nucleotides in DNA or RNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine. (A, G, C and T or U). The methods for gene sequencing are like; Sanger’s method (for short nucleotides and it’s a basic method), Maxim-Gilbert sequencing (basic method), Shotgun sequencing etc. NGS or Next Generation sequencing is one of the best method for sequencing the gene.

2. Finding the homologs

In this step you can check the homologs of the novel gene using a software BLAST (Basic Local Alignment Search Tool). It enables to compare a subject protein or nucleotide sequence with a library or database of sequences, and identify library sequences that resemble the gene sequence above a certain threshold. Using BLAST we can identify the homologs gene and the function of novel gene because the novel gene will show the same function as the homologs gene found from the BLAST

If the gene is completely novel means there is no homologs for the gene we can use another methods for determining the function the gene they are:

• You can find the function by expression study by examining when and where the gene is expressed in the cell or in the whole body of organism. Gene expression can be accomplished by replacing coding region by a reporter gene. Usually the genes used as reporter genes are that induce visually identifiable characteristics usually involve fluorescent and luminescent proteins (Eg: luc, rfp, gfp, lacZ etc.). Expression of the reporter gene can be monitored by tracking the fluorescence or enzymatic activity of protein transcribed from the gene.
• Once you know the sequence you can use functional predication algorithms such as InterProScan that attempt to identify functional proteins domains