Assignment task:
Please give Respond to EACH STUDENT answer (below). Make sure your Respond is at least 100 words in length with new information and written in your own words and providing thoughtful commentary.
Provide more information or explanation about student topics. DO NOT REPEAT what student already said.
Provide Questions with your respond.
Provide References to support your feedback/comments/respond.
Student 1:
DNA microarrays are used to primarily study gene expression but can also be used to identify single nucleotide polymorphisms (SNP) and pathogens (Trevino et al., 2007). DNA microarrays are created by synthesizing the complement of a target sequence through reverse transcriptase and undergoing PCR amplification. These amplified sequences have probes that are then attached to them that allow for visualization on glass, and the probes are then excited and analyzed by computer software (Trevino et al., 2007).
cDNA microarrays are not the only source of microarrays (Trevino et al., 2007). Microarrays can also be created by using small, single-stranded oligonucleotides. This methodology can provide less errors when compared to cDNA microarrays. For example, cDNA microarrays can have uneven melting temperatures due to size and GC concentration differences, as well as cross-hybridization. The oligonucleotide method can avoid these issues as it maximizes the specificity of its target (Trevino et al., 2007).
CD Genomics is a company that offers cDNA microarray services for research purposes (CD Genomics, 2024a). They note that the use of cDNA compared to oligonucleotides allows for more successful hybridization as the cDNA is longer. CD Genomics performs its microarray services by creating a cDNA probe that attaches to a glass microarray chip. The RNA is added to the chip, allowing for binding to the probes and therefore the presence of fluorescence through laser excitation (CD Genomics, 2024a). CD Genomics also offers oligonucleotide microarray services (CD Genomics, 2024b). The oligonucleotides are shorter than cDNA and the method therefore has high specificity and low cross-hybridization. Instead of being labeled with fluorophores, the oligonucleotides are labeled with biotin and are hybridized to a chip, which allows for less variability (CD Genomics, 2024b). Similar to last week's discussion, researchers must choose the methodology of microarray analysis that will best benefit their experiment - there is not a one-size-fits-all approach.
Student 2:
TakaRa Bio-Home (partnered with Clontech Laboratories) offers Subtractive Hybridization Kits to research teams wishing to amplify differentially expressed genes (TakaRa Bio-Home, n.d.). They offer a multitude of kits with a wide array of methods to retrieve one's desired target DNA. Some kits they offer is a Bacterial Genome Subtraction Kit which is marketed for comparing entire bacterial genomes. In as little as a few days, they claim that researchers can build a library of sequences that will accurately depict genes present in one bacterial strain and absent in another. Figure 1 depicts the summarized workflow demonstrated in this kit's user manual. Another kit they offer is their PCR-Select Differential Screening Kit. This kit identifies differentially expressed clones in a subtracted library. The kit's strength is to confirm differential expressions between genomes quickly. TakaRa is explicitly for research use only and it's website specifically states that it is not for diagnostic procedures unless the product is specifically noted to do so (TakaRa Bio-Home, n.d.).
Subtractive Hybridization is well-known for being a tedious technical method in the face of others, such as cDNA microarray (Goetz, 2003). Though, TakaRa kits claim that their kits provide a straightforward method with only few steps required due to their PCR-Select method. In this method, subtraction occurs in one round and selective amplification of differentially expressed genes instead of by physical separation. The kit also allows subtraction to occur with as little as 2 µg of RNA and quotes a 3-4 day complete process range. I believe the weakness of these kits is the method itself, SH, depending on your intended application.
Since the method itself is where the flaws lay (or the lack of efficiency), I believe its main competitor is the use of different, more efficient, methods such as cDNA microarrays. This method provides a highly sensitive quantitative and qualitative gene expression level detection (CD Genomics, n.d.). CD Genomics offers a variety of cDNA Microarray services that I believe potentially compete with Clontech SH Kits.