Chapter 20, Problem 25EQ

Summary Introduction

To analyze:

In the figure below, it shows the evolution of the globin gene family. Though all the genes show homology, there is the difference in sequence similarity depending on the time of divergence. The more recent the divergence, the higher the sequence similarity.

Asan example- the sequence similarity between genes $\alpha 1$ and $\alpha 2$ is higher than in $\alpha 1$ and $\zeta$ genes. The degree of similarity can be checked using the southern blotting technique, where different conditions or variables specifically distinguish higher complementary probe and DNA with lower complementary probe and DNA.

The variables or conditions in southern blot are- temperature and ionic concentration. High stringency refers to very high temperature and low ionic salt concentration; the hybridization shows nearly a perfect complementarity of the probe with the DNA. Low stringency refers to low temperature and high ionic salt concentration; the hybridization does not show a perfect complementarity of the probe with the DNA. In lower stringency, the homologous genes can be detected.

In a southern blot, if a sample of human genome DNA and probe used to match with the $\text{β}$ globin gene, arrange the following in high stringency to low stringency on a blot.

$\left({\text{Mb, α}}_{\text{1}}{\text{, β, γ}}_{\text{A}}\text{,δ,}\text{ε }\right)$.

Introduction:

Southern blotting is a technique used to detect the target gene sequence within a mixture of several genes. This method was introduced by Edwin Southern in $1975$.

The goal of southern blotting is to detect if a sample of the genome contains a complementarity with the specific probe.

In this technique, first, the genomic DNA is isolated and followed by digestion of Restriction enzymes that results in the production of several fragments. These fragments are separated on gel electrophoresis according to their masses. Later, the DNA is blotted onto a nylon membrane; this ensures that the DNA is bound to the membrane. This membrane is incubated and labeled; probes are introduced with unlabeled genomic DNA in a solution.

If there is complementarity with the labeled probe and unlabeled DNA, then it will show a band on the gel.

The major variables in southern blotting are temperature and the ionic strength of hybridization.

High stringency refers to very high temperature and low ionic salt concentration; the hybridization shows nearly a perfect complementarity of the probe with the DNA.

Low stringency refers to low temperature and high ionic salt concentration; the hybridization does not show a perfect complementarity of the probe with the DNA.