Molecular Biology Quiz (Lectures 11 & 12)

A Southern Blot is used to detect Restriction Fragment Length Polymorphisms (RFLPs) and analyze genetic variation. It involves transferring DNA fragments from a gel to a solid support, such as a nitrocellulose membrane, and then hybridizing the DNA with a labeled probe that is complementary to the target sequence. This technique allows for the identification and analysis of genetic variations, such as deletions, insertions, or mutations, within the DNA fragments.

Southern Blots are a laboratory technique used to study DNA. This technique involves the separation of DNA fragments by size using gel electrophoresis, followed by transfer of the DNA fragments to a solid support (such as a membrane). The transferred DNA fragments are then hybridized with labeled DNA probes to detect specific sequences. Therefore, Southern Blots are specifically used to study DNA and not RNA or proteins.

Northern Blots are used to study RNA only. This technique involves the separation of RNA molecules by gel electrophoresis, followed by transfer onto a membrane and hybridization with a labeled RNA probe. It allows researchers to determine the size and abundance of specific RNA molecules in a sample. Northern Blots are not used to study DNA or proteins, as other techniques like Southern Blots and Western Blots are specifically designed for those purposes.

Improvements in sequencing methods have led to a decrease in the cost of sequencing larger molecules and an increase in the speed of sequencing. This means that it is now more affordable to sequence larger molecules, making it more accessible for researchers to study complex genomes or conduct large-scale sequencing projects. Additionally, the increase in sequencing speed allows for faster processing of samples, reducing the time required to obtain sequencing results. These advancements in sequencing methods have significantly improved the efficiency and affordability of genetic sequencing.

The correct order for steps to be taken in a Southern Blot is: III, VI, II, V, I, IV. First, DNA is digested with a restriction enzyme (III). Then, the DNA fragments are size fractionated in an agarose gel and stained with EtBr to visualize all DNA (VI). Next, the dsDNA in the gel is denatured using NaOH (II). The ssDNA is then transferred from the gel to a membrane (V). Specific fragments are detected by hybridizing a DNA probe to the DNA on the membrane (I). Finally, the hybridized bands are visualized using a P-imager or film (IV).

Sanger sequencing is a method used to determine the sequence of DNA. It involves synthesizing a new strand of DNA in vitro, using a DNA template and a primer. The method includes the use of dideoxy nucleotides (ddNTP) that lack a 3' hydroxyl group, which terminates DNA synthesis. Each tube used in Sanger sequencing contains a known dideoxy nucleotide (ddATP, ddGTP, ddTTP, or ddCTP), along with regular nucleotides (dATP, dGTP, dTTP, and dCTP). The termination of DNA synthesis at different points allows the determination of the sequence of the original DNA template.

The major difference between northern and Southern blots, besides the specific substance being studied, is that northern blots do not require restriction endonucleases. However, in both northern and Southern blots, the substance being studied must be denatured in heated formaldehyde instead of SDS.

The Sanger method of sequencing DNA involves the use of dideoxy nucleotides, which are modified versions of the normal nucleotides. These dideoxy nucleotides lack a 3' hydroxyl group, preventing the addition of further nucleotides during DNA synthesis. This results in the termination of the DNA chain at specific positions, allowing for the determination of the sequence. Therefore, the correct answer is "dideoxy, chain termination."

The correct answer is "2nd Generation" Sequencing includes "454" Pyrosequencing and Illumina.,"3rd Generation" Sequencing includes single molecule sequencing. This is because "2nd Generation" sequencing methods refer to the technologies that came after the first-generation Sanger sequencing, and both "454" Pyrosequencing and Illumina are examples of these technologies. On the other hand, "3rd Generation" sequencing methods are the latest advancements in sequencing technology, and single molecule sequencing is one of the key features of these methods.

Cycle sequencing is an improvement on the Sanger method because it utilizes a cyclic reaction to amplify and sequence DNA. This method allows for the automation of the sequencing process, making it more efficient and less labor-intensive. It does not specifically include the "454" and "illumina" methods, but they are examples of sequencing technologies that utilize cycle sequencing principles.

454 Pyrosequencing is a DNA sequencing method that is based on the release of the pyrophosphate (P-P) molecule during the sequencing reaction. This method generates relatively long "reads" of approximately 400 base pairs (bp), allowing for the sequencing of longer DNA fragments. Additionally, it is capable of sequencing approximately 500 million base pairs (Mbp) of DNA per day, making it a high-throughput sequencing technique.

Reporter Genes are "Synthetic" and used to study the function of regulatory sequences such as promoters, RNA stability, and translation regulation. They include the "regulatory sequence" from the gene of interest. Additionally, they add a coding region for a specific "Reporter Protein" that is the same as the experiment being conducted. This allows researchers to track and measure the activity of the regulatory sequences in the gene of interest.

The differences between Automated Sequencing and Sanger Sequencing include the use of fluorescent dyes instead of (^35)S for labeling DNA, the presence of dyes on Dideoxynucleotides (ddNTPs) instead of deoxynucleotides (dNTPs), the electrophoresis of DNA in polyacrylamide gel instead of agarose gel, the detection of DNA by exciting the dye with a laser, and the use of one lane and four colors, one for each nucleotide, for detection.