Question

You are studying a bacterium that grows in a particular ecological niche. You cannot culture it in the laboratory, but you can isolate small quantities of cells that microscopic analysis indicates are not contaminated with other bacteria. How will you obtain the ribosomal RNA gene sequence data to study the taxonomy of the bacterium? •How will you determine the complete genome sequence of the bacterium?

The steps performed for determining the complete genomics sequence of the bacterium include- Step 1: Cloning Step 2: Restriction Mapping Step 3: Gel Electrophoresis Step 4: DNA Sequence Analysis

Gel electrophoresis- The DNA fragments after Restriction digestion are separated by electrophoresis, a process that involves application of an electric field to cause the DNA fragments to migrate into an agarose gel. The gel is then stained with a methylene blue stain to visualize the DNA bands and may be photographed. Although methylene blue dye is not as sensitive as ethidium bromide it may be used to stain the higher quantities of DNA that are used in this experiment. The movement of the fragments will always be towards the positive electrode because DNA is a negatively charged molecule. The fragments move through the gel at a rate that is determined by their size and shape, with the smallest moving the fastest.

DNA Sequence Analysis- This method uses dideoxynucleotide triphosphates(ddNTPs) whichwhich an H on the 3’ carbon of the ribose sugar instead of the normal OH found in deoxynucleotide triphosphates (dNTPs). Dideoxynucleotides are chain terminators. In a synthesis reaction, if a dideoxynucleotide is added instead of the normal deoxynucleotide, the synthesis stops at that point because the 3’OH necessary for thetaddition of the next nucleotide is absent. The sequencing gel is able to resolve fragments that differ in size from each other by only one base.

**I need explain exactly gel ectrphoresis and DNA sequence analysis will help find the bacteria genome

Answer 1

The type of bacteria considered here is a non-culturable bacteria. So these type of studies are called Metagenomic studies, where we try to study the microbial flora of an ecological niche which are not culturable in the laboratory.

For studying the taxonomic charecteristics of this bacterium we need to go for meta - genome isolation (isolation of DNA directly from the environmental sample) from the specific ecologial niche. This Meta-DNA will contain DNA of all the species present there.

After the DNA isolation, we need to go for Density Gradient Gel Eletrophoresis, where DNA gets seperated not only because of size, but also because of its specific GC content. The Density is of a DNA denaturing chemical i.e Formaldehyde. This will show bands according to the number of species present because each species of bacteria will have its won specific GC content and denature according to it.

Now from the small quantities of cells that can be isolated, can be sent for sequencing, and we will know the GC content of its genome. Now we have to match the GC content of the sequenced data and the GC content present in the Gel. After this we can determine our bacteria and cut it out and sent for sequencing. But before sequencing, we need to amplify it with 16s rRNA primer (which is a universal primer for prokaryotes).

The DNA sequnce can be analyzed and from bioinformatic softwares we can estimate its taxonomical charecteristics.