(U) DNA Technologies
Describe each of the 5 steps of a Southern Blot:(1) Cut(2) Gel Electrophoresis(3) Filter Paper(4) Probe(5) X-ray
(1) Cut - Restriction Enzymes are used to cut the DNA into smaller pieces.(2) Gel Electrophoresis - DNA fragments are placed in wells and will travel from one end of the gel to the other, getting separated based on their size (note that the Khan Academy video incorrectly states that the fragments are separated based on size and charge, but they will only be separated based size since the mass-to-charge ratio is equal between fragments).(3) Filter Paper - Filter paper is place on the gel, and the fragments will transfer over to the filter paper. (4) Probe - A radio-labelled single-stranded DNA fragment is added to the filter paper. It will hybridize to the gene of interest.(5) X-ray - In order to visualize the radio label, the filter paper is exposed to an x-ray, which will only show a band for the gene of interest.
Describe the four steps of DNA Cloning:(1) Cut(2) Paste(3) Transform(4) Amplify
(1) Cut - Restriction Enzymes are used to cut the gene of interest out of a greater body of DNA.(2) Paste - That gene is then inserted into a plasmid. (3) Transform - The plasmid is placed in a solution with bacteria. Heat is applied, which causes the bacteria to take in the plasmid. This uptake process is termed transformation." (note that Khan Academy incorrectly uses the term "transformation" to describe the Paste step.(4) Amplify - The bacteria are grown on a plate
Describe what happens during each step of PCR and why the temperature is set to what it is during each step:(1) Denaturation (96° C)(2) Annealing (55° C)(3) Extension (72° C)
(1) Denaturation - The double stranded DNA that you are interested in studying is denatured as the temperature is increased to 96° C. (2) Annealing - As the temperature is decreased to 55° C, the primer anneals to the end of the region that you want to copy on each strand of DNA.(3) Extension - DNA Polymerase (which is most active at a temperature of 72° C) begins extending the DNA from the primer.
Describe the steps of DNA Sequencing: (1) PCR(2) PCR with ddNTPs (3) Gel Electrophoresis(4) Analysis
(1) PCR - PCR is used to amplify the gene of interest.(2) PCR with ddNTPs - The sample is separated into four different containers, one containing regular nucleotides plus radio-labelled ddGTP, another container with ddCTP, another with ddTTP, and another with ddATP. PCR is then continued in each of the four containers.(3) Gel Electrophoresis - The sample is place in a well and the DNA strands travel across the gel, becoming separated by size.(4) Analysis - The DNA strands are analyzed in order from shortest to longest fragment.
What is the preferred gel for DNA electrophoresis called?(A) Agarose gel(B) Polysaccharide gel(C) Sucrose gel(D) Polyacrylamide gel
(A) Agarose gelAgarose gel is the preferred gel for DNA electrophoresis.
Put the following steps of a Southern Blot in the correct order from first to last:I. Gel ElectrophoresisII. CutIII. ProbeIV. Filter PaperV. X-ray(A) II, I, IV, III, V(B) II, I, IV, V, III(C) I, III, V, IV, II(D) II, IV, I, III, V
(A) II, I, IV, III, VThe steps of a Southern Blot are as follows:(1) Cut(2) Gel Electrophoresis(3) Filter Paper(4) Probe(5) X-ray
CRB Microarrays are used to compare the transcription of specific genes between different cells. How do you preserve these transcription profiles before using a microarray?(A) Convert the mRNA into tRNA to increase stability(B) Convert the mRNA into cDNA to increase stability(C) Convert the mRNA into dsDNA to increase stability(D) Add stabilization solution to preserve the mRNA
(B) Convert the mRNA into cDNA to increase stabilityBecause RNA is prone to degradation, scientists will convert the mRNA into a complementary strand of DNA (cDNA) to increase the transcription profile's stability.
What separation method(s) can be used to isolate a protein on the basis of the isoelectric point?I. Isoelectric focusingII. Ion-exchange chromatographyIII. SDS-PAGE(A) I Only(B) I and II Only(C) II and III Only(D) I, II, and III
(B) I and II OnlyIsoelectric focusing and ion-exchange chromatography both separate proteins based on charge, which can indirectly separate proteins based on their pI.
Put the steps of DNA Sequencing in the correct order: I. Gel ElectrophoresisII. PCR with ddNTPsIII. AnalysisIV. PCR(A) II > IV > I > III(B) IV > II > I > III(C) I > III > I > IV(D) III > I > IV > I
(B) IV > II > I > IIIThe steps of DNA Sequencing are as follows: (1) PCR(2) PCR with ddNTPs (3) Gel Electrophoresis(4) Analysis
CRB Which of the following would happen if you restriction digested a vector at exactly two Restriction Sites?(A) 2 fragments form(B) The Vector is linearized and 2 fragments form(C) The vector is linearized as 1 fragment.(D) None of the above.
(B) The Vector is linearized and 2 fragments form
After the first cycle of PCR, you have 2 double-stranded DNA fragments. After the second cycle, you have 4. And after the third cycle, you have 8. How many will you have after the 20th cycle?(A) 248,642(B) 486,922(C) 1,048,576(D) 1,854,392
(C) 1,048,5762^20 = 1,048,576. You will have 1,048,576 double-stranded DNA fragments after the 20th cycle of PCR. To know how many fragments of DNA after any cycle simply calculate 2 raised to the cycle's number (2^n).*Need help with MCAT math? Become an MCAT math wizard using my High-speed Math Mastery Course @ https://mcatselfprep.com/course/andrews-high-speed-math-mastery-course/
Put the following steps of DNA Cloning in order from first to last:I. UptakeII. CutIII. AmplifyIV. Paste(A) I > III > II > IV(B) III > I > II > IV(C) II > IV > I > III(D) II > IV > III > I
(C) II > IV > I > IIIThe steps of DNA Cloning are as follows: (1) Cut(2) Paste(3) Uptake(4) Amplify
Place the steps of a polymerase chain reaction (PCR) in the correct order:I. Annealing II. Extension III. Denaturation(A) III > II > I(B) II > III > I(C) III > I > II(D) I > II > III
(C) III > I > IIThe polymerase chain reaction (PCR) happens subsequently in the following order: (1) Denaturation(2) Annealing(3) Extension
cDNA is generated using which of the following enzymes?(A) Ligase(B) Complimentary Synthase(C) Reverse Transcriptase(D) RNA Replicase
(C) Reverse TranscriptasecDNA is generated using the enzyme Reverse Transcriptase.
CRB Which of the following would happen if you restriction digested a vector at exactly one Restriction Site?(A) 2 fragments form(B) The Vector is linearized and 2 fragments form(C) The vector is linearized as 1 fragment.(D) None of the above.
(C) The vector is linearized as 1 fragment.Think of this like making one cut in the band of a wedding ring. It would no longer be an intact circle shape, but all of the metal would still be connected!
Ethidium bromide is a common example of a(n):(A) reducing agent(B) biological agent(C) intercalating agent(D) oxidizing agent
(C) intercalating agentEthidium bromide is a common example of an intercalating agent.
CRB Which of the following statements best describes how most sequences are inserted into cloning vectors?(A) Both the insert and the cloning vectors are digested with restriction enzymes, providing Blunt Ends, and then the two are ligated together.(B) Both the insert and the cloning vectors are digested with restriction enzymes, providing Blunt Ends. To prevent those cleaved sequences from recombining with their smaller fragments, the restriction digest products undergo Gel Electrophoresis, and the largest band is extracted from the gel. Then, the insert and vector are ligated together.(C) Both the insert and the cloning vectors are digested with restriction enzymes, providing Sticky Ends, and then the two are ligated together.(D) Both the insert and the cloning vectors are digested with restriction enzymes, providing Sticky Ends. To prevent those cleaved sequences from recombining with their smaller fragments, the restriction digest products undergo Gel Electrophoresis, and the largest band is extracted from the gel. Then, the insert and vector are ligated together.
(D) Both the insert and the cloning vectors are digested with restriction enzymes, providing Sticky Ends. To prevent those cleaved sequences from recombining with their smaller fragments, the restriction digest products undergo Gel Electrophoresis, and the largest band is extracted from the gel. Then, the insert and vector are ligated together.
CRB Which of the following statements about cDNA libraries are true?I. cDNA Libraries will lack all introns typically removed by alternative splicing.II. cDNA Libraries can vary based on the type of cell the RNA was taken from.III. cDNA Libraries isolated from one cell/cell type can be called Expression Libraries.(A) I only(B) I and II only(C) II and III only(D) I, II and III
(D) I, II and IIIEach of the following statements are true:I. cDNA Libraries will lack all introns typically removed by alternative splicing.II. cDNA Libraries can vary based on the type of cell the RNA was taken from.III. cDNA Libraries isolated from one cell/cell type can be called Expression Libraries.
DNA Cloning requires the use of which of the following enzymes?I. DNA PolymeraseII. DNA LigaseIII. Restriction Enzymes(A) I Only(B) II Only(C) I and II Only(D) II and III Only
(D) II and III OnlyDNA Cloning requires the use of DNA Ligase and Restriction Enzymes.
DNA Cloning is done _______________ while PCR is done ________________.(A) in vitro, in vitro(B) in vitro, in vivo(C) in vivo, in vivo(D) in vivo, in vitro
(D) in vivo, in vitroDNA Cloning is done in vivo (in a living thing such as a bacteria) while PCR is done in vitro (in a test tube).
During PCR, which side does the DNA primer bond to? (3' or 5')
3'. And the the DNA Polymerase adds bases from 5' to 3' on the new strand (3' to 5' on the base strand
In Gel Electrophoresis, what is the DNA ladder and why is it used?
A DNA ladder is a standard with known sizes of molecules. When performing gel electrophoresis, the unknown sample is compared against the DNA ladder to determine the size of the different sequences in the sample.
What is a restriction map?
A map of a gene showing where restriction enzymes cut
What is the purpose of a microarray?
A microarray compares the mRNA contents of two cells. This allows us to understand how the expression of certain genes differs between the two cells.
Why is adding an intercalating agent (such as ethidium bromide) an important step in the process of running gel electrophoresis?
Adding an intercalating agent (such as ethidium bromide) allows us to physically see the DNA sample. The agent works by placing itself between the nucleotide base pairs of the DNA and becomes fluorescent when you apply UV light to it. This allows us to visualize the DNA and thus determine how far it traveled in the gel.
What is the difference between the two most common electrophoresis types: agarose gel electrophoresis vs. sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis?
Agarose gel electrophoresis is used for larger DNA fragments and the mobility of DNA fragments in this gel depends on both size and charge. This agarose gel is derived from seaweed.Sodium dodecyl sulfate (SDS)-polyacrylamide gel is used for smaller macromolecules (either small DNA or small proteins). Mobility through this gel strictly depends on size alone, since SDS disrupts all noncovalent interactions, including causing the protein to unfold. The polyacrylamide gel itself is a synthetic polymer.
Once DNA or cDNA is inserted into a cloning vector, how is that genetic material amplified"?"
Amplification of the DNA/cDNA is accomplished through inserting the cloning vector into a bacteria that can replicate that genetic material over and over again. From there, we can sequence the gene of interest.
CRB Draw out both Sticky and Blunt ends. Which do you expect to ligate more easily?
Because the Sticky Ends share exposed, matching base pairs, these should ligate more easily.
In Gel Electrophoresis, why do larger molecules with more SDS bonded (more negative charge) travel less than smaller molecules?
Because the gel only has small pores, which allows small molecules to easily travel through the gel, while the large molecules will get trapped.
Explain how microarray experiments depend on hybridization.
Cellular contents are injected into each well. If the well has a probe strand targeting the cDNA for a gene the cell was expressing, that cDNA and the probe will hybridize (bind). Then, if you wash out any unbound contents in the well, that cDNA will remain in the well, showing that gene is expressed in the original cell.
What is the purpose of a Southern Blot?
Conducting a Southern Blot allows the researcher to determine if a specific gene is present within a larger body of DNA.
What is the purpose of DNA Cloning?
DNA Cloning allows us to generate many copies of the same gene. Researchers can then do research using the many copies generated.
If cDNA is single-stranded, how can it be inserted to a cloning vector that makes up a cDNA library?
DNA Polymerase sythesizes the complimentary strand, making the cDNA double-stranded and able to be inserted into a cloning vector for a cDNA library.
What is a DNA ladder and why do we need one when running a DNA gel electrophoresis?
DNA ladder is a set of standardized DNA molecules of known size that are used to determine the size of an unknown DNA sample molecule run on a gel during electrophoresis.
What is a DNA library and what is its purpose?
DNA library is a collection of the total genomic DNA from a single organism that have been cloned into vectors. With access to such a library, researchers can identify and isolate the DNA fragments that interest them for further study.
Why is DNA Polymerase from Thermus aquaticus (Taq, a bacteria that lives in the hot springs of Yellowstone National Park) used instead of DNA Polymerase from a human or bacteria?
DNA polymerase from Thermus aquaticus (Taq, a bacteria that lives in the hot springs of Yellowstone National Park) is very heat resistant, meaning that it will not denature at the high temperature required to denature the DNA strands (96° C). If human or bacteria DNA Polymerase was used, the enzyme would denature and not work.
How do dideoxynucleotides (ddNTPs) differ from typical deoxynucleotides (dNTPs) in terms of structure and function?
Dideoxynucleotides (ddNTPs) lack 3' OH group typical of regular deoxynucleotides (dNTPs). Because they lack this OH group, ddNTPs cause DNA polymerization to stop since the phosphate head of the downstream nucleotide can't bind to an H group.
A microarray is a piece of plastic with many tiny wells. What makes each well different from each other?
Each well contains probe strands for a specific cDNA. For instance, cell 1 might contain the mRNA that codes for protein A, cell 2 might contain the mRNA that codes for protein B, etc., and there would be a different well with probe strands targeting the cDNA for one specific protein.
True or False? Each band that you see during DNA gel electrophoresis represents a single DNA strand.
False. Each band represents tons of DNA strands of the same length. If there was only one DNA strand, it would be impossible to see it.
CRB True or false? If a PCR product is meant to be inserted into a vector, the Primers should be designed with restriction sites inside the desired sequence.
False. If a PCR product is meant to be inserted into a vector, the Primers should be designed with restriction sites flanking the desired sequence. These same restriction sites should also be present in the vector of interest.
CRB True or false? Transgenic Mice expressing the phenotype of the Recombinant Vector in all of their cells must have had parents with the Recombinant Vector in all of their cells too.
False. Transgenic Mice expressing the phenotype of the Recombinant Vector in all of their cells must have had parents with the Recombinant Vector in their Germ Cells, or the Recombinant Vector was added to embryonic stem cells.
Why do we transfer the DNA fragments to a filter paper as opposed to leaving them on the gel?
Filter paper is more sturdy than the gel.
What is gene therapy?
Gene therapy is inserting a working gene into a patient using viral vectors. This is used when the patient has a gene that isn't being expressed.
DNA libraries consist of either genomic DNA or cDNA (complementary DNA). What is the difference between genomic libraries and cDNA libraries?
Genomic DNA libraries contain larger fragments of double-stranded DNA and include both exons (coding regions) and introns (noncoding regions).cDNA libraries only contain an organism's expressed genetic information in the form of double-stranded cDNA.
Which DNA library (genomic library or cDNA library) has promoter and enhancer sequences present?
Genomic libraries have promoter and enhancer sequences present, but not necessarily in the same clone.cDNA libraries do not have these regions present since they would not be found within the exons of the gene.
CRB There are four directional types of Blots in general. Match the type of blot with the information it provides.I. SouthernII. NorthernIII. EasternIV. Western(A) Post-Translational Protein modifications(B) DNA strands present(C) Protein levels (D) mRNA levels
I. Southern - (B) DNA strands presentII. Northern - (D) mRNA levelsIII. Eastern - (A) Post-Translational Protein modificationsIV. Western - (C) Protein levelsI like to think of these as a bizarro-compass rose. Instead of North South East West"
A certain insurance company requires DNA testing before you are allowed to enroll in their program. Why might this be unethical?
If they discovered that you have a predisposition to a certain illness, they might charge you higher rates. This would be seen as discriminatory.
How would these steps differ if you wanted to clone cDNA?
Instead of using restriction enzymes to isolate DNA in step 1, you would use Reverse Transcriptase to generate cDNA templates of the cell's mRNA. Then you would make that cDNA double-stranded via DNA Polymerase. This double-stranded DNA can then be pasted into the plasmid and the process continues as normal from step 2 onward.
What does Ethidium Bromide do in gel electrophoresis?
It makes the DNA sample fluoresce blue under UV light. This allows you to better see the sample.
Why is it not possible to infer mRNA sequence from a protein sequence while it is possible to infer mRNA sequence from DNA?
It not possible to infer mRNA sequence from a protein sequence because multiple codons code for the same amino acid. For instance, if a protein contains the amino acid Alanine, you wouldn't know whether the mRNA sequence that coded for that amino acid was GCU, GCA, GCG, or GCC. With DNA/mRNA however, cytosine in the mRNA strand is always the result of guanine in the DNA template strand.
CRB Many experiments talk about Knockout Genes". What is the mechanism behind preventing the typical phenotype caused by that gene?"
Knockout Genes, or Knockout Animal Models for a gene, have a gene that has been intentionally deleted or interfered with so that it will not produce the proper mRNA or protein product.
If larger DNA strands have a larger overall negative charge, why don't they migrate farther?
Large DNA strands do have a higher overall charge, but they also have a higher overall mass. The mass-to-charge ratio is the same for any DNA strand. Thus, the sole factor that differentiates a big strand from a small strand is that big strands will experience more drag as they try to move through the agarose gel.
Why do the plasmids used in DNA Cloning contain an antibiotic resistance gene?
Many bacteria in the petri dish will not take in the plasmid. We want these bacteria to die so that we know we only have bacteria on our plate that contain the gene of interest. To kill off all bacteria without a plasmid, we add an antibiotic. Only bacteria that contain the plasmid will survive since the plasmid contains an antibiotic resistance gene that makes them immune to that specific antibiotic.
What is the purpose of the Polymerase Chain Reaction (PCR)? How does it work?
Polymerase Chain Reaction is used to make more copies of DNA. A DNA strand is treated to separate the strands. After this, a specific primer bonds to a specific region of DNA that you want. Free nucleotides then bind onto the strand after the primer. This results in two strands of DNA based on the original strand.
Compare Reverse Genetics with Forward Genetics.
Reverse Genetics seeks to understand the phenotype via the alteration and/or composition of the genotype (as in knock out experiments). Forward Genetics, on the other hand, seeks to understand the genotype simply by observing the natural phenotype (as in observing bean plants and their shapes and sizes.Note: Khan Academy's use of the term Reverse Genetics is not the most accurate. See this link for more information on this topic: https://en.wikipedia.org/wiki/Reverse_genetics
What does SDS do in gel electrophoresis?
SDS breaks non-covalent interactions (like hydrogen bonds) and denatures the DNA SDS also bonds to every other side chain group in the DNA strand, and adds negative charges from its phosphate group. This ensures that all DNA moves towards the anode (positive end) instead of towards the cathode.
You conduct Isoelectric Focusing on three proteins with the same pKa but differing molecular weights of 250 Daltons, 100 Daltons, and 25 Daltons. Which will travel the furthest in the gel?
Since each protein has the same pKa, each protein will travel the same distance on the gel. In Isoelectric Focusing, a protein stops moving when the pH of the gel is equal to its isoelectric point, which would be the same for each of these protein.
Which DNA will travel the farthest during gel electrophoresis? A DNA with 6,000 base pairs or DNA with 3,500 base pairs?
The DNA with 3,500 base pairs will travel farther during gel electrophoresis because the gel acts like a sieve, allowing small particles to travel through more easily while retaining large particles; consequently, shorter DNA will travel farther through the gel.
Working on a research project, you run a gel electrophoresis on some DNA samples. Subsequently, you find out from your principal investigator that your DNA samples have been damaged during the gel electrophoresis. You find out that the buffer solution in which you bathed the DNA was acidic. Why did this damage your DNA and ruin your gel electrophoresis?
The acidic solution lilkely affected the charge on the DNA due to the protonation of phosphate groups. This affected the rate at which the DNA migrated towards the positive electrode.
In what direction do the anions and cations move towards in an electrical field?
The anions always move toward the anode while the cations always move toward the cathode in an electrical field.
What are the relative benefits of agarose (polyacrylamide gel) compared to sodium dodecyl sulfate (SDS)-polyacrylamide gel?
The benefits of agarose gel (polyacrylamide gel) are that it allows a complete protein to be recovered after an analysis since SDS is a denaturant and can damage DNA or denature proteins, and secondly the agarose gel (polyacrylamide gel) determines the globular size of proteins.
What are the relative benefits of sodium dodecyl sulfate (SDS)-polyacrylamide gel compared to agarose (polyacrylamide gel)?
The benefits of sodium dodecyl sulfate (SDS)-polyacrylamide gel is that it can be used to eliminate charge as a factor and focus specifically on molecule's size.
What is hybridization?
The joining of complementary base pair sequences, can be DNA-DNA or DNA-RNA
In gel electrophoresis, which direction do the molecules travel? (anode/cathode) What is the charge at this side?
The molecules move towards the anode, which has a positive charge.
You just robbed a bank, and a piece of your hair was found at the scene of the crime. GASP! You are taken into custody as a suspect, and your DNA is tested against the DNA found in the hair originally discovered. How might DNA technology be used by the police to prove that you are guilty?
The police may use PCR to create many copies of two different regions of your genome known to contain a variable number of repeats (known as Short Tandem Repeats (STRs)). They would then run that DNA using gel electrophoresis to measure the size of the DNA (the more repeats, the less far it will migrate). If the tested fragments from hair DNA migrate to the same locations as the tested fragments from your DNA, they'd pin you as the criminal!
What is the purpose of gel electrophoresis?
The purpose of Gel Electrophoresis is to figure out the size and charge of macromolecules such as DNA, RNA and proteins.
What is the purpose of Polymerase Chain Reaction (PCR)?
The purpose of PCR is to generate tons and tons of copies of a single fragment of DNA that you are interested in. This is essential for researchers since it would be very hard to work with just a single copy of DNA since they are so tiny and fragile. Having many copies allows you to better test and visualize the DNA you are interested in.
In a particular DNA sequencing experiment, ddGTP was labelled yellow, ddCTP was labelled blue, ddTTP was labelled red, and ddATP was labelled green. What is the sequence of the new DNA strand from 5' to 3' if the gel displayed the following from top (closest to where samples were loaded) to bottom (furthest from where samples were loaded):RedYellowRedRed
The shortest fragments are located at the bottom of the gel, and DNA is synthesized in the 5' to 3' direction. Thus, the new sequence of the DNA is Red, Red, Yellow, Red: 5'-TTGT-3'
CRB What would happen if a researcher skipped the Gel Electrophoresis in between the restriction digest and ligating?
The smaller, digested fragments would be able to re-bond with their original molecules, decreasing the efficiency of the ligation.
Restriction enzymes (restriction endonucleases) work by recognizing what types of sequences?
The special traits in how restriction enzymes work are by recognizing palindromic sequences (meaning that the 5' to 3' sequence of one strand is identical to the 5' to 3' sequence of the other strand in antiparallel orientation) and by producing offset cuts yielding sticky ends on the fragments.
A farmer is frustrated because the canna virus is destroying his crops. How might this farmer use DNA technology to save the day?
This farmer could inject his plant's seeds with a plasmid that contains a canna virus resistance gene. The next generation would be resistant to the virus!
Anna is 20-weeks pregnant and has her fetus's DNA tested for a variety of diseases. It is discovered that her fetus has a gene that will predispose the baby to a serious illness later in life. DNA technology such as CRISPR could potentially be used to correct this defect. Why might it be unethical to do so?
This might be unethical because by altering the genome, a new mutation may be created that is worse. Also, we don't know the long-term affects of altering genomes like this, so it is extremely risky.
T/F: restriction enzymes are used to cut palindromic DNA sequences to make bacterial vectors for gene cloning.
True
CRB True or false? Expression Libraries and Recombinant Vectors could be used for constructing Transgenic Animals to further study the affected genes.
True. Expression Libraries and Recombinant Vectors could be used for constructing Transgenic Animals to further study the affected genes.
CRB True or false? The Vector that has been ligated with the cDNA insert can be called a Recombinant Vector.
True. The Vector that has been ligated with the cDNA insert can be called a Recombinant Vector.
CRB True or false? This method of using chain-terminating ddNTPs during DNA Polymerase experiments is often called Sanger Sequencing.
True. This method of using chain-terminating ddNTPs during DNA Polymerase experiments is often called Sanger Sequencing.
How is cDNA generated?
We can isolate mRNA from a cell and use the enzyme reverse transcriptase to generate single-stranded cDNA using the mRNA as a template. This cDNA represents the original template strand's exons.
In a certain microarray experiment, the cDNA in the regular cell is labelled with a green fluorescent tag while the cDNA in the cancer cell is labelled with a red fluorescent tag. When red and green are mixed, they produce a yellow color. What can you conclude regarding the following genes based on the results of the microarray:- Gene A's well has no color. - Gene B's well has a red color.- Gene C's well has a yellow color.- Gene D's well has a green color.
You can conclude the following:- Gene A's well has no color. This gene is not expressed in either the normal or cancerous cell.- Gene B's well has a red color. This gene is expressed primarily in the cancerous cell. Perhaps this gene's protein is contributing to the cancer.- Gene C's well has a yellow color. This gene is expressed equally well in both the normal and cancerous cell.- Gene D's well has a green color. This gene is primarily expressed in the normal cell. Perhaps the lack of this gene's protein is contributing to the cancer.
Originally, vaccines entailed injecting people with a virus in the hopes that they would develop immunity against that virus. The problem with this is that the vaccine might actually give you the disease. How was DNA technology used to overcome this obstacle?
You could extract the mRNA that produces the virus's capsid (outer layer). Then you could produce cDNA using reverse transcriptase, and insert that cDNA into a cloning host such as e Coli. Then extract the protein and inject it into humans. This would allow the human's immune system to build immunity against that virus without the risk of getting the disease.
You are interested in understanding the role of a certain protein in the progression of lung cancer. How might you use a Knock Out Experiment to further your understanding?
You could knock out the gene that produces the protein you are interested in. Then you can observe what happens in this lung cancer cell as opposed to a lung cancer cell that still has the gene present. The differences in function between the two cells can be attributed to the lack of the certain protein.
If you are a physician who wants to produce tons of insulin or human growth hormone for your patients, how might you use DNA technology to accomplish this?
You could use DNA cloning to produce tons of e Coli that contain either the insulin or human growth hormone gene. Then you can harvest the proteins from these e Coli.
On which side of the gel electrophoresis (negative electrode or positive electrode) would you place samples of DNA fragment?
You would place samples of DNA fragments on the same side of the gel as the negative electrode. This way, DNA (with its negatively-charged phosphate backbone) will migrate across the gel towards the positive electrode.
what is a chimera (in terms of mice).
a Chimera mouse is a mouse with two groups of stem cells. One with transgenes and one without (and they have patchy fur). These Chimeras can be bred to produce mice that are heterozygous for the transgene and mice that are homozygous for the transgene
Why is beta-mercaptoethanol used in gel electrohporesis?
beta-mercaptoethanol is used to break disulfide bonds
Which DNA library (genomic library or cDNA library) has genes that can be expressed in a cloning host to produce functioning proteins?
cDNA libraries have genes that can be expressed in a cloning host to produce functioning proteins. This is due to the fact that the DNA is pre-spliced" (introns are already removed). Genomic libraries cannot be used to produce functioning proteins since the host (typically bacteria) that proteins are produced in do not contain spliceosomes
What is the difference between the content of DNA libraries and cDNA libraries?
cDNA libraries only contain information on the coding regions (exons) of the DNA. DNA libraries contain the entire genome
Explain Polymerase Chain Reaction (PCR)
denature DNA using heat in a solution with free nucleotides, cool to allow reannealing and allow a DNA primer to bond, then allow the DNA polymerase to extend the DNA primer to make it a complete double strand. This makes a bunch of copies of DNA without using bacteria
What are transgenes and how are they used in mice and knockout mice?
knockout mice are mice who's DNA was altered by removing a gene before birth. Effects of this are then studied as the mice develop. Transgenes are genes that were instead introduced in mice, and the effects of these are then studied.
In the equation for gel electrophoresis: V = E*q/f What do each of the variables mean?
v is the velocity of the molecule E is the strength of the electric field q is the molecule's charge f is the coefficient of friction This equation is used to find the velocity of the molecule in terms of the strength of the electric field, the molecule's charge, and the coefficient of friction.
