Bioinformatics Module 2 - Assignment 3
Question 1.1.1: BioEdit Restriction Site Analysis for Accession Number
FM995514.1
To determine the restriction sites for the sequence associated with the accession
number FM995514.1, I utilized the BioEdit software. The following steps were taken:
1. I opened BioEdit and loaded the sequence from the FM995514.1.fasta file, which
was downloaded from NCBI.
2. After loading the sequence, I attempted to use BioEdit's restriction analysis tool.
However, due to some tool limitations in my setup, I transitioned to using the
online tool NEBcutter.
The following restriction sites were identified using NEBcutter for the sequence of
FM995514.1.
NEBcutter restriction site analysis screenshot:
Question 1.1.2: NEBcutter Restriction Site Analysis for FM995514.1
Using the NEBcutter tool, I input the FM995514.1 sequence to analyze restriction
enzyme sites. The software generated a restriction map highlighting the positions of
various enzyme cut sites within the sequence.
Question 1.1.3: Enzyme Name and Function from NCBI
According to the accession number FM995514.1, the correlated enzyme is alcohol
dehydrogenase originated from bacterium Arthrobacter sp. JEK-2009 that inhabits the
Sydney tar ponds.
Enzyme Function: Alcohol dehydrogenase as one of the enzymes the plays a crucial
role in the metabolism of alcohol in that it transforms alcohols (ethanol) into aldehydes
(acetaldehyde) and ketones. It has profound biochemical significance in the catabolism
of alcohol in virtually all living entities ranging from bacteria, yeasts right up to human
beings. In bacteria, this enzyme is responsible for the breakdown of alcohols in order
get energy.
Question 1.1.4: Restriction Sites for BcgI, TaqII, and MboII in BioEdit
For the enzymes BcgI, TaqII, and MboII, I used NEBcutter to find the restriction sites
within the sequence of FM995514.1. The resulting restriction sites specific to these
enzymes are as follows:
Question 1.1.5: Translation of the Sequence to Protein Using BioEdit (Frame 1)
I used BioEdit to translate the nucleotide sequence of FM995514.1 to its corresponding
protein sequence using Frame 1. The following protein sequence was generated:
Question 2.1: PCR Primer Design Parameters
Designing primers for PCR involves several key considerations:
1. Primer Length: Primers are typically 18-25 nucleotides long to ensure specific
binding.
2. Melting Temperature (Tm): The Tm of the primers should ideally be between
50-60°C to ensure stable binding during the PCR process.
3. GC Content: Primers should have a GC content between 40-60%, as this
ensures the primer's stability when binding to the target sequence.
4. Specificity: Primers should be specific to the target DNA region and avoid
forming secondary structures like dimers.
Question 2.2: Methods to Find Primers for Gene Amplification
To find primers that can amplify a specific gene, you can use the following methods:
1. Primer3 Software: A widely used tool to design primers based on a given
sequence.
2. BLAST (Basic Local Alignment Search Tool): Use BLAST to compare your
sequence to known sequences in the database and find primers used in related
research.
3. Commercial Databases: You can use commercial primer databases, such as
PrimerBank, to find primers designed for genes similar to your target.
Question 2.3: Alcohol Dehydrogenase Gene Primer Pair
A research project focusing on the amplification of the alcohol dehydrogenase gene can
use primers from published research. After reviewing research articles, the following
primer pair was selected for amplifying the gene:
Question 2.4: Primer Design for AJ427225.1 Using Primer3
Using the Primer3 software, primers were designed for the nucleotide sequence
associated with accession number AJ427225.1. The first set of primers that can be
used for PCR amplification are as follows:
Conclusion:
This assignment involved various tools and methodologies to analyze nucleotide
sequences, design primers, and interpret enzyme functions. By using software such as
BioEdit and NEBcutter, important molecular biology techniques were applied to a real-
world sequence, providing insights into its structure and function.
