AN ASSIGNMENT ON MOLECULAR BIOLOGY

(MCB 305)

BY

ARIYO BISOLA OMOLARA

MCB/19/2637

TO BE SUBMITTED TO THE DEPARTMENT OF MICROBIOLOGY

FEDERAL UNIVERSITY OF TECHNOLOGY, AKURE

LECTURER IN CHARGE: PROF. A.K ONIFADE

Question1: Stella is working on helicobacter pylori, she wants to know the full length of the
organism genome at the same time study some virulent genes and know other genes.
Which of the DNA sequencing Techniques will give this details.

Answer:

For Stella's research on Helicobacter pylori, she would benefit from using whole-genome
sequencing (WGS). WGS provides the full-length sequence of the organism's genome, allowing
her to study virulent genes as well as other genes present in the genome. This technique provides
comprehensive information about the organism's genetic makeup, enabling detailed analysis and
identification of specific genes of interest.

Question 2: Outline the steps involved in the above sequencing Techniques.

Answer:

Here is an outline of the steps involved in whole-genome sequencing (WGS):

1. Sample Collection: Obtain a sample of Helicobacter pylori containing its genetic

material (DNA). This can be collected from various sources such as culture plates,
clinical samples, or environmental samples.
2. DNA Extraction: Isolate the DNA from the collected sample using techniques such as
phenol-chloroform extraction, silica-based methods, or commercial DNA extraction kits.
This step ensures that only the DNA of interest is extracted for sequencing.
3. Library Preparation: Fragment the extracted DNA into smaller, manageable pieces.
Adapters containing known sequences are ligated to the ends of these fragments. This
process helps in amplifying and sequencing the DNA fragments.
4. Sequencing: Perform the actual sequencing of the DNA fragments. There are different
sequencing platforms available such as Illumina, PacBio, and Oxford Nanopore, each
with its own sequencing chemistry and workflow. Sequencing generates millions of short
reads (sequences) that cover the entire genome.
5. Data Analysis: Process the raw sequencing data to reconstruct the original genome
sequence. This involves several steps such as quality control, read alignment, assembly
(for de novo sequencing), and variant calling (to identify genetic variations).
Bioinformatics tools and software are used extensively in this stage to analyze the vast
amount of sequencing data.
6. Annotation: Annotate the assembled genome to identify genes, regulatory elements, and
other features. This step involves comparing the sequenced genome to known databases
of genes and proteins to assign putative functions to the identified sequences.
7. Functional Analysis: Analyze the annotated genome to understand the biological
functions and pathways encoded by the genes. This may involve further bioinformatics
analysis, comparative genomics, and experimental validation of gene functions.
 8. Interpretation: Interpret the results in the context of Stella's research objectives. Identify
virulent genes, drug targets, or other genomic features relevant to Helicobacter pylori
pathogenesis or biology.
9. Reporting: Compile the findings into a comprehensive report, including the sequenced
genome, identified genes, genetic variations, and any other relevant information. This
report serves as the basis for further research or applications in fields such as medicine,
microbiology, or biotechnology.

By following these steps, Stella can obtain the full-length sequence of the Helicobacter pylori
genome and study both virulent and other genes of interest.

Question 3:

What is the advantage over other DNA sequencing Techniques.

Answers:

Whole-genome sequencing (WGS) offers several advantages over other DNA sequencing
techniques:

1. Comprehensive Coverage: WGS provides a complete view of the entire genome of an

organism, including both coding and non-coding regions. This allows for the detection of
all types of genetic variations, from single nucleotide polymorphisms (SNPs) to large
structural variations.
2. High Resolution: WGS generates high-resolution data, with the ability to detect genetic
variations at the single-base level. This makes it suitable for studying subtle differences
between closely related strains or populations.
3. Discovery of Novel Genes and Features: WGS enables the discovery of novel genes,
regulatory elements, and other genomic features that may be missed by targeted
sequencing approaches. This can lead to new insights into the biology, pathogenesis, and
evolution of the organism being studied.
4. Flexibility and Versatility: WGS is a versatile technique that can be applied to a wide
range of organisms, from bacteria to humans. It can also be used for various applications,
including microbial genomics, cancer genomics, evolutionary biology, and personalized
medicine.
5. Cost-Effectiveness: While WGS was initially more expensive compared to targeted
sequencing approaches, advances in sequencing technology and decreasing costs have
made it increasingly cost-effective, especially for large-scale projects or when multiple
analyses are required.
6. Long-term Data Utility: WGS generates data that can be stored and reanalyzed over
time as new research questions arise or as new computational methods become available.
 This long-term data utility adds value to WGS datasets and facilitates ongoing research
efforts.

Overall, the comprehensive nature, high resolution, versatility, and long-term data utility of
whole-genome sequencing make it a powerful tool for studying the genetic makeup of organisms
and addressing a wide range of research questions.

Question 4: Patience was advised by her supervisor to use metagenomic technique in

carrying her project on the biodiversity of a given soil sample, Discuss the steps involved in
achieving this project.

Answer:

Here are the steps involved in conducting a metagenomic study to explore the biodiversity of a
soil sample:

1. Sample Collection: Patience should collect a representative soil sample from the study
area. The sample should be collected using appropriate techniques to ensure that it
captures the diversity of microorganisms present in the soil.
2. DNA Extraction: Once the soil sample is collected, Patience needs to extract the total
DNA from the sample. This step involves breaking open the cells of microorganisms
present in the soil and isolating their genomic DNA. Various DNA extraction methods
are available, including chemical-based methods and commercial DNA extraction kits.
3. Library Preparation: Next, Patience needs to prepare a metagenomic DNA library. This
involves fragmenting the extracted DNA into smaller pieces and attaching sequencing
adapters to the ends of the fragments. The DNA fragments are then amplified using
polymerase chain reaction (PCR) to generate a library suitable for sequencing.
4. Sequencing: The prepared metagenomic DNA library is sequenced using high-
throughput sequencing technologies such as Illumina sequencing or Oxford Nanopore
sequencing. The choice of sequencing platform depends on factors such as sequencing
depth, read length, and cost.
5. Data Preprocessing: After sequencing, the raw sequence data undergoes preprocessing
to remove low-quality reads, adapter sequences, and other artifacts. This step helps
improve the quality of the data before downstream analysis.
6. Taxonomic Profiling: Patience can perform taxonomic profiling to identify the microbial
species present in the soil sample. This involves comparing the sequenced DNA
fragments to reference databases of known microbial genomes using bioinformatics tools
such as BLAST or Kraken. Taxonomic profiling provides information about the diversity
and abundance of different microbial taxa in the soil sample.
7. Functional Annotation: In addition to taxonomic profiling, Patience can annotate the
functional potential of the microbial community present in the soil sample. This involves
 predicting the functions of genes based on their sequence similarity to known genes and
metabolic pathways. Functional annotation provides insights into the ecological roles and
metabolic capabilities of the microbial community.
8. Statistical Analysis: Patience can perform statistical analysis to compare the microbial
composition and functional potential of the soil sample with other samples or
environmental conditions. This may involve methods such as alpha diversity analysis,
beta diversity analysis, and differential abundance testing.
9. Interpretation and Visualization: Finally, Patience interprets the results of the
metagenomic analysis in the context of the research objectives and scientific literature.
Visualization tools such as heatmaps, bar charts, and network diagrams can be used to
present the results in a clear and informative manner.

By following these steps, Patience can gain valuable insights into the biodiversity of the soil
sample and the functional potential of its microbial community using metagenomic techniques.

Question 5: With representative examples, Discuss the role of nucleic acid replication in
molecular biology

Answers:

Nucleic acid replication plays a crucial role in molecular biology by ensuring the faithful
transmission of genetic information from one generation to the next. Here are some
representative examples of the role of nucleic acid replication:

1. DNA Replication in Cell Division: DNA replication is essential for cell division in both
prokaryotic and eukaryotic organisms. During cell division, DNA is replicated to ensure
that each daughter cell receives a complete set of genetic information. For example, in
eukaryotic cells, DNA replication occurs during the S phase of the cell cycle before
mitosis or meiosis.
2. DNA Repair Mechanisms: Nucleic acid replication also plays a role in DNA repair
mechanisms. Cells have evolved sophisticated repair systems to correct errors that occur
during DNA replication or to repair damage caused by environmental factors such as UV
radiation or chemical mutagens. For example, base excision repair and nucleotide
excision repair are two common mechanisms used by cells to repair damaged DNA.
3. PCR (Polymerase Chain Reaction): PCR is a widely used technique in molecular
biology that relies on nucleic acid replication. PCR amplifies a specific region of DNA
through repeated cycles of denaturation, annealing, and extension using DNA polymerase
enzymes. PCR is used for various applications, including DNA sequencing, genotyping,
gene expression analysis, and molecular cloning.
4. DNA Sequencing: Nucleic acid replication is fundamental to DNA sequencing
techniques. For example, Sanger sequencing relies on DNA replication to generate a
 series of DNA fragments of varying lengths, each terminating with a labeled nucleotide.
These fragments are then separated by size to determine the sequence of the original
DNA molecule. Next-generation sequencing (NGS) techniques also rely on nucleic acid
replication to generate sequence data from millions of DNA fragments in parallel.
5. Gene Expression Regulation: Nucleic acid replication is involved in the regulation of
gene expression. For example, DNA replication origins play a role in determining the
timing and efficiency of DNA replication during the cell cycle. Additionally, epigenetic
modifications such as DNA methylation can influence DNA replication and gene
expression patterns.
6. Viral Replication: Many viruses rely on nucleic acid replication to replicate their
genomes and produce progeny virions. For example, RNA viruses such as influenza virus
and retroviruses replicate their RNA genomes using viral RNA-dependent RNA
polymerases or reverse transcriptases, respectively. DNA viruses such as herpesviruses
and adenoviruses replicate their DNA genomes using host cell DNA replication
machinery.

Overall, nucleic acid replication is a fundamental process in molecular biology with diverse roles
ranging from cell division and DNA repair to PCR, DNA sequencing, gene expression
regulation, and viral replication. Its importance cannot be overstated in understanding the
mechanisms underlying genetic inheritance and the functioning of living organisms.