Chapter 12, Problem 1RQ

Summary Introduction

To review:

The important considerations while designing a strategy to select mutants.

Introduction:

The technique used for identification and selection of individuals having a specific mutant phenotype in a mutagenized population is known as mutagenesis screening or genetic screening. The study of mutant population can offer vital information about the role of different genes. It also provides important information about the molecular events in a particular biological process or pathway.

Explanation of Solution

The important points to remember for designing a mutant selection strategy are as follows:

1. The strategy used should select for mutant bacterial cells by culturing them in a specific condition. In case of loss of function mutation, a master plate of mutagenized cells should be prepared. The cell from this master plate should be replica plated onto a culture plate requiring the wildtype gene function.

2. The selection strategy should test for the presence or absence of the wildtype gene function in the population of mutants. It should be clear from the beginning of the experiment about the functional consequences of the specific mutation in a cell. The study of Escherichia coli O157:H7 genes required for the growth and survival at pH (potential of hydrogen) 2.0 is done through the screening of mutant bacterial cells that are unable to grow at pH 2.

3. The mutant cells should possess a selectable marker like antibiotic resistance gene. This eases the differentiation between wildtype and mutant cells.

4. The identified mutant cells should be further selected using specific DNA (deoxyribonucleic acid) probes. In case of transposon mediated insertional mutation of target genes, a pair of primers is used to amplify the junction between the insertionally inactivated gene and transposon. The sequencing of the amplified product using transposon specific primer determines the insertion site and the gene into which the insertion has taken place.