Help with Genetic Cloning, Splicing, and Sequencing - GRE Subject Test: Biochemistry, Cell, and Molecular Biology

The correct answer is the ampicillin the nutrient agar plates expired. The plasmid expresses beta-lactamase, an enzyme that degrades ampicillin. Successfully transformed bacteria will be able to grow in the presence of ampicillin, but untransformed bacteria should not grow. Since the bacteria in our control transformation do not have ampicillin resistance conferred by beta-lactamase, we would expect that no growth would be observed on any nutrient agar plates with ampicillin. However, we observe uncontrolled growth on these plates, indicating that the ampicillin has expired and is no longer a viable selectable marker for transformed bacteria.

The annealing temperature is the optimal temperature at which the primers bind the template DNA sequence. This temperature takes into account several factors: the number of basepairs in the primers, the relative guanine-cytosine content, and their melting point.

None of the answer choices are correct. In order to successfully clone a bacterial gene into a mammalian expression vector, the following must be completed: First, the bacterial gene should be PCR amplified from bacterial cDNA, which contains only exons. Second, the vector backbone should be digested with restriction enzymes to linearize the vector so that the bacterial gene can be inserted at a specifc locus (multiple cloning site). Third, the amplified bacterial gene and the digested vector backbone are ligated together to create the desired expression construct.

The correct answer is cDNA. Given that this gene most likely has several introns, complementary DNA (cDNA) should be used as a template since it only contains exons. When subcloning a gene into an expression vector, only coding sequence should be present because the transcribed RNA will not be processed like it is in host cells.

Plasmids are circular pieces of DNA that are resistant to exonucleic degradation (exonucleases cut from DNA ends). Bacteria harbor native plasmids that replicate independently from their genome and express genes that often confer a survival advantage. Scientists often clone genes into plasmids and express them in a variety of host cells.

Frederick Sanger invented the chain termination method of DNA sequencing in 1977, which has been widely used since its invention for short DNA sequences. The method is know as Sanger Sequencing. Leroy Hood was responsible for automating this method, Kary Mullis was the inventor of the polymerase chain reaction to create copies of DNA, E. M. Southern lent his name to another DNA study method called Southerns, and Craig Venter was responsible for other later DNA sequencing methods.

Polymerase chain reaction requires two, not one, synthesized primer molecules to correctly amplify a DNA molecule into new copies. One primer attaches to each end of your desired DNA segment, and amplification occurs from these sites by polymerase molecules. The other options are all correct facts about PCR.

Cosmids could handle DNA insert sizes of 25-50kb. BAC's could handle insert sizes of 100-300kb, and YAC's even more. These are still used for tricky genome projects due to this large insert size, like antifreeze protein genes in Arctic fish. Plasmids can generally only handle insert sizes of 5-10kb.

These were all important advances in improvement of the Sanger technique that increased DNA sequence output. Modification of polymerases allowed quicker thermal cycling reactions and incorporation of manufactured bases with fluorescent tags. Four different fluorescent tags for each base allowed for easy identification of different bases. Lastly, capillary chromatography allowed for huge parallelization of DNA sequencing.

All of these answers are correct. Next generation sequencing substantially increased DNA sequence generation speed, and makes it much easier to assemble full genomes. Earlier, Sanger sequencing requires that you know a primer sequence for each fragment you'd like to sequence, but its read lengths are still longer than next generation sequencing methods, which rely on creation of many relatively short sequences.

Directional cloning is the process by which two restriction enzymes cut the plasmid and the subject DNA, creating a situation in which the plasmid cannot recircularize because the only viable combination of linking is plasmid-subject DNA-plasmid.

Cloning with the Lambda phage involves all of these basic steps. The subject DNA is annealed to sticky ends inside the phage DNA. A plate of bacteria is infected with the phage, which actually does the replication of your DNA. The phages are specifically engineered to only insert DNA into the bacteria for replication if it actually incorporated your DNA fragment.

The correct answer is second next-generation sequencing requires amplification of DNA, but third next-generation sequencing can sequence single molecules. This major advance in sequencing technology allows a researcher to only sequence the DNA present in a given cell, for example, with third generation sequencing. This reduces amplicon bias that can occur when amplifying DNA to prepare it for second generation sequencing, generating truly representative sequencing.

The correct answer is TAS amplifies conserved yet divergent genes, such as rRNA genes, to identify species. Many species do not have sequenced genomes. Metagenomic DNA isolation is performed regularly to survey microbial species in a given sample and can be sequenced by next-generation sequencing. However, identifying species based on whole genome sequencing is very difficult because we have not sequenced the genomes for most organisms. Furthermore, computationally aligning whole genomes to reference genomes is very tedious and time consuming. rRNA genes have highly conserved regions that allow researchers to design primers to anneal in almost every species. However, the highly divergent portions of rRNA genes allow for specie identification. TAS amplifies and sequences specific genes, such as rRNA genes, for specie identification studies.

The correct answer is polymerase cycling assembly. In this reaction, multiple template sequences are included into a reaction with primers to the 5' most and 3' most sequence of the desired final product. The template sequences must also have regions of homology overlap with each other such that upon denaturing and annealing they hybridize to form a larger fragment. Upon hybridization, the primers will promote polymerase amplification of one large product.

The correct answer is the start codon should be 5' to the biochemical tag and the stop codon should be 3' to the protein of interest. When designing fusion proteins with N-terminal biochemical tags, it is important to remove the native start codon for the protein of interest to prevent initiation of transcription at multiple sites. A start codon is required immediately 5' to the biochemical tag to include the tag in the expression of the fusion protein. Only one stop codon is required at the end of the fusion protein sequence (3').

The correct answer is calcium chloride surrounds the bacterial membrane and attracts negatively charged DNA. Plasmid DNA is introduced to calcium chloride incubated bacteria and are mixed at for up to one hour. Then, a heat shock to causes the plasma membrane to loosen, allowing the plasmid DNA to enter the cells. A recovery period in nutrient broth at promotes plasma membrane recovery and initiation of bacterial replication.

The correct answer is that there is no control over the orientation of the insertion into the plasmid backbone. Since there are no single stranded base pair overhangs that normally occur with restriction enzyme digestion in a blunt cutter digestion, the insertion (with blunt ends) will ligate with the plasmid backbone (with blunt ends) in both orientations randomly. This creates a grave difficulty in analyzing clones to make sure the gene insertion is in the correct orientation.

The correct answer is that the transformed bacteria need time to transcribe and translate the antibiotic resistant gene on the plasmid. Bacteria used in transformations do not inherently contain antibiotic resistance genes. The genes that confer antibiotic resistance for bacteria often come from exogenous plasmids introduced by techniques such as transformations. There is a lag in expression of these plasmids, and as such, the transformed bacteria will not be immediately antibiotic resistant following introduction of the plasmid.