AP Biology : Biotechnology Review Questions
How is CRISPR useful in cancer therapy?
A possible application of the CRISPR/Cas9 system to cancer therapy is related to the regulation of endogenous gene expression. As mentioned above, catalytically inactive dCas9 can be recruited by gRNAs to specific target DNA sites, and when fused to transcriptional activation or inhibition domains, can be exploited to activate or repress specific target genes.
Explain what happens during Annealing, and the temperature at which Annealing occurs.
Annealing - when the temperature is lowered to enable the DNA primers to attach to the template DNA.The annealing temperature (typically between 48-72°C) is related to the melting temperature (Tm) of the primers and must be determined for each primer pair used in PCR.
How is CRISPR used as a biotechnology tool?
Cas9 enzymes together with CRISPR sequences form the basis of a technology known as CRISPR-Cas9 that can be used to edit genes within organisms.
Describe the process of cloning an organism.
Cloning is the process of taking genetic information from one living thing and creating identical copies of it. The copied material is called a clone.
What role does electric charge play in the distribution of DNA bands?
DNA is negatively charged, therefore, when an electric current is applied to the gel, DNA will migrate towards the positively charged electrode. Shorter strands of DNA move more quickly through the gel than longer strands resulting in the fragments being arranged in order of size.
Explain what happens during Denaturation, and the temperature at which Denaturation occurs.
Denaturing - when the double-stranded template DNA is heated to separate it into two single strands. The reaction temperature is increased to 95 °C, which melts (disrupts the hydrogen bonds between complementary bases) all dsDNA into single-stranded DNA (ssDNA).
Pick one real-world application that uses PCR and gel electrophoresis and specifically explain how each process is used in that application.
Disease Diagnosis- The process in which a scientist can determine what disease you may have is by first grabbing sample from patients, then isolate the germ and also isolate the DNA from the bacteria. PCR (Polymerase chain reaction) the DNA with priers specific for the gene specific to this disease and then after the PCR use gel electrophoresis of the amplified data. After the gel electrophoresis stain the gel and observe the gel through the UV transilluminator and if the band of the amplified DNA matches the band of your control DNA then the patient has the disease.
How does gel electrophoresis work? What properties of the DNA does it utilize?
Gel electrophoresis is a technique used to separate DNA fragments according to their size. ... DNA fragments are negatively charged, so they move towards the positive electrode. Because all DNA fragments have the same amount of charge per mass, small fragments move through the gel faster than large ones.
What is the purpose of CRISPR in nature?
It allows researchers to easily alter DNA sequences and modify gene function. Its many potential applications include correcting genetic defects, treating and preventing the spread of diseases and improving crops.
Where will the largest fragments be found? How is the size of a particular fragment determined?'
Larger fragments will be found at the top. The reason for this is there is a certain pore size depending on the concentration of agarose/acrylamide in the gel.
Explain the purpose of the Polymerase Chain Reaction. Why is it useful?
Polymerase chain reaction (PCR) is a method widely used in molecular biology to rapidly make millions to billions of copies of a specific DNA sample allowing scientists to take a very small sample of DNA and amplify it to a large enough amount to study in detail.
Explain the function of primers.
Primers serve as the starting point for DNA synthesis. The polymerase enzyme can only add DNA bases to a double strand of DNA.
Compare and contrast therapeutic cloning with reproductive cloning.
Reproductive cloning involves creating an animal that is genetically identical to a donor animal through somatic cell nuclear transfer. ... In therapeutic cloning, an embryo is created in a similar way, but the resulting "cloned" cells remain in a dish in the lab; they are not implanted into a female's uterus.
Explain the relationship between single nucleotide polymorphisms ("SNPs") and restriction fragment length polymorphisms ("RFLPs")? How are they caused and why do they matter?
SNPs are DNA sequence variations that occur when a single nucleotide in the genome is altered. In order for an alteration to be considered an SNP, it has to be present in at least 1% of the population. RFLPs is one of the first techniques used to study the genetic variation of a population and it is based on the restriction enzymes that cut DNA on specific sites. The sequences produced by these cuts, are then studied. They matter because they are used to detect differences in the DNA of a population and study the genetic variation.
What is the purpose of a microarray? Give an example of a real-world application of microarray analysis (you probably need to use the internet).
Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome. Examples include sequencing by hybridization, resequencing, mutation detection, assessment of gene copy number, comparative genome hybridization, drug discovery, expression analysis, and immunoassay (protein microarrays).
What is the purpose of a DNA "library"? How can specific genes be retrieved from a DNA library?
So that researchers can identify and isolate the DNA fragments that interest them for further study. DNA libraries can be used to isolate a specific gene of interest, as they generally include at least one fragment that contains the gene.
Explain the significance of "sticky ends" and why they were given that name.
Sticky ends want to bond to create base pairs and thus a new molecule with the same DNA.
Explain the function of Taq polymerase.
Taq polymerase is an enzyme that copies DNA.
Explain what happens during Elongation, and the temperature at which Elongation occurs.
The elongation of the primers completes one PCR cycle. The primer molecules are elongated by the action of DNA polymerase. Since the Taq polymerase, which is usually added to the PCR, works the best at around 72 degrees centigrade, the temperature of the test tube is raised (Scheme - Elongation).
Where will the smallest fragments of DNA be found on a gel after it runs?
The smallest DNA fragments will be found near the bottom of the gel.
Why is it necessary to utilize probes for labeling particular DNA sequences? How is this process accomplished?
The use of dyes, fluorescent tags or radioactive labels enables the DNA on the gel to be seen after they have been separated. They will appear as bands on the gel.By comparing the bands of the DNA samples with those from the DNA marker, you can work out the approximate length of the DNA fragments in the samples.
Explain the function of thermal cycler.
Thermal cyclers, are instruments used to amplify DNA and RNA samples by the polymerase chain reaction. The thermocycler raises and lowers the temperature of the samples in a holding block in discrete, pre-programmed steps, allowing for denaturation and reannealing of samples with various reagents.
How do restriction enzymes work?
They cut DNA at sites that have a specific nucleotide sequence.
What is Cas-9 and how does it work?
This acts as a pair of 'molecular scissors' that can cut the two strands of DNA at a specific location in the genome so that bits of DNA can then be added or removed.
Describe the function of a vector
a DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and/or expressed. A vector containing foreign DNA is termed recombinant DNA.
Give examples of things used as vectors.
plasmid, cosmid, Lambda phages
