Set 1
Part A.
1. BLAST - This algorithm compares the various pieces of primary biological sequence information, such as the nucleotides of DNA sequences or the amino acid sequences of different proteins, in order to identify the library sequences that bear similarities with the query sequence above a certain threshold.
2. ORF Finder – This tool is used when searching for open reading frames (ORFs) in the DNA sequence that the user enters where the information provided includes the protein translation and the range of each open reading frame.
3. Compute pl/Mw tool – This tool computes the isoelectric point (pl) and Molecular weight (Mw) for the sequences that the user entered or for a list of UniProt Knowledgebase entries.
4. Restriction Mapper – This tool is used to map the sites of restriction endonucleases or restriction enzymes in DNA sequences.
5. Entrez – This is a retrieval and search tool that NCBI (National Center for Biotechnology Information) developed to enable a user to search more than one NCBI database through the use of just one query where the results can include a combination of various types of data.
Part B.
Check Size Using SDS Page
The BLAST algorithm can be used when checking the molecular size. While the SDS page can be used for separating proteins based on their electrophoretic mobility, BLAST can be used to make comparisons of the proteins’ sequence information.
ORF Finder
Sequence the plasmid to confirm correct ligation
The ORF Finder can be used for identifying the α peptide’s reading frame. The disruption of this reading frame by the deletion of vector sequences or by the insertion of a foreign DNA fragment prevents the α complementation from occurring, which would make the bacterial colonies give off a white or light blue color.
Compute pl/Mw tool
Sequence the plasmid to confirm correct ligation
The tool can be used for getting the vector-insert ratio, which requires the measurement of the molecular weight.
Restriction Mapper
After being given the cDNA of the gene BRCA1 in a cloning vector
After the gene is obtained, the Restriction Mapper can be used for determining the restriction sites, which will be used for designing the PCR primers in order to amplify the coding sequence. In addition, if the desired DNA fragment’s PCR amplification is neither possible nor desirable, the tool can be used when employing the restriction enzyme digestion of the target DNA.
Design PCR primers to amplify the coding sequence
Restriction digest the PCR product and ligate it into a restriction digested expression vector.
The tool can be used when a need for cutting the vector using a restriction enzyme is determined before the vector’s insertion into the organism.
Transform into bacteria
The tool can be used to perform a restriction enzyme digestion, which in turn can be used for identifying the recombinant vector. This verifies whether the correct insertion was made or not.
Entrez
Design PCR primers to amplify the coding sequence
This tool can be used for searching the NCBI database for the sequencing information of whole or partial genomes, which will aid in the design of primers for the amplification of the DNA fragment directly from the genomic DNA of the desired organism.
Set 2
Part A.
1. BLAST – This is an algorithm that enables the comparison of a database or a library of sequences to a query sequence such that the library sequences resembling the query sequence above a particular threshold is identified.
2. ORF Finder – This is used when you want to find ORFs (open reading frames) in the DNA sequence that you enter, particularly the range of each open reading frame and its protein translation.
3. Compute pl/Mw tool – This tool is used for the calculation of the estimated isoelectric point (pl) and Molecular weight (Mw) of an AA sequence that the user entered or of a specific Swiss-Prot/TrEMBL entry.
4. Restriction Mapper – This tool is used for mapping the sites for restriction enzymes, which are then used to obtain structural information on a piece of DNA.
5. Entrez – This is a tool that can be used for searching multiple NCBI (National Center for Biotechnology Information) databases with a single query. The search results include various types of data such as macromolecular structures, protein sequences, and nucleotide sequences.
Part B.
1. BLAST
This algorithm can be used when checking the size of the molecule using the SDS page. It can be used for comparing the biological sequence information of the proteins.
2. ORF Finder
- When the plasmid is sequenced to conform to the correct ligation, the reading frame
of the alpha peptide is identified using the ORF Finder. It is used to determine if the reading frame is disrupted, which results in the bacterial colonies appearing light blue or white.
3. Compute pl/Mw tool
After the DNA is prepared for ligation, this tool can be used for measuring the molecule’s weight in order to obtain the molar ratio of insert to vector.
4. Restriction Mapper
This tool can be used after the researcher is provided with the cDNA of the gene
BRCA1 in a cloning vector in order to determine the restriction sites. This information is needed when the PCR primers are designed and can also be used if there’s a need to employ the DNA’s restriction enzyme digestion.
Similarly, this tool can be used when the PCR product is restriction digested and ligated into a restriction digested expression vector. In particular, it can be used if the vector needs to be cut before being inserted into the organism. Moreover, once the vector is transformed into bacteria then the restriction mapper can be used for performing a restriction enzyme digestion.
5. Entrez
This tool can be used in the stage where the PCR primers are designed (with restriction sites on each) in order to amplify the coding sequence. In particular, the Entrez tool can be used for quickly and easily amplifying the desired sequences. With the Entrez tool, the researcher will be able to search the NCBI database, which will in turn enable the researcher to design primers that will amplify their DNA fragment of interest directly from the desired organism’s genomic DNA. The database will be able to provide the researcher with information on the sequencing of partial or whole genomes of organisms across all biological kingdoms.
