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 should be:

Step 1: Cloning Step 2: Restriction Mapping Step 3: Gel Electrophoresis Step 4: DNA Sequence Analysis  gel electrophoresis and dna analysis needs to elaborated on.

Answer 1

PCR with primers to regions of ribosomal RNA genes that are conserved across a wide range of bacteria to amplify the corresponding sequences from the bacterium of interest could be used. If it appeared to be a single band in gel electrophoresis, i.e., the PCR product is sufficiently specific, and was in enough quantity, then the DNA sequence could be determined directly. If the product is not specific, the the PCR might need to be optimised, perhaps by adjusting the concentration of magnesium ions, increasing the annealing temperature or using touchdown PCR. If very little product is obtained in the PCR, then cloning it might be needed before sequencing.

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.