BIOL 2100 Chapter Notes - Chapter 20: Polymerase Chain Reaction, Reverse Transcriptase, Dna Microarray

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limesardine98

9 Dec 2016

School

University of Virginia

Department

Biology

Course

BIOL 2100

Professor

Kittlesen David

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Document Summary

20. 2 biologists use dna technology to study gene expression. Analyzing gene expression: studying expression of single genes. We can detect mrna by nucleic acid hybridization with molecules of complementary sequence (which is called nucleic acid probe) Cloned gene used as template and complementary probe made. Solution with probe molecules applied to observed embryos, and probes hy(cid:271)(cid:396)idize with (cid:272)o(cid:373)ple(cid:373)e(cid:374)ta(cid:396)y se(cid:395)ue(cid:374)(cid:272)es o(cid:374) (cid:373)rna"s. Reverse transcriptase polymerase chain reaction - turns mrna into double stranded dna. Enzyme reverse transcriptase used to make reverse transcript of each mrna molecule. Short complementary strand of thymines (complementary to poly-a tail) acts as primer. After degradation of mrna, second strand made by dna polymerase. Resulting dna called complementary dna (cdna: studing the expression of interacting groups of genes. Systems approach studying the expression of large groups of genes. Tiny amounts of a large number of single-stranded dna fragments representing different genes fixed to a glass slide in a grid.

Related Questions

1)What enzyme is used to attach different colors of stained nucleotides to create a DNA molecule along the mRNA?

Select one:

a. reverse transcriptase

b. fluorescent primer

c. restriction enzyme

d. radioactive probe

The process in which two complementary strands of DNA from different sources pair with one another is called hybridization.

Select one:

True

False

3) Explain the significance of a red microarray spot revealed by the microarray scanner.

Select one:

a. this gene is not expressed in either type of cell

b. this gene is expressed in cancer cells

c. this gene is expressed in healthy cells and cancer cells

d. this gene is expressed in healthy cells

4)What is the function of primers in PCR?

Select one:

a. raw material for new DNA

b. attach new nucleotides to DNA strand

c. attach to either side of target DNA

d. denature, or separate, DNA

5)What is the function of DNA polymerase in PCR?

Select one:

a. raw material for new DNA

b. attaches to either side of target DNA

c. attaches new nucleotides to DNA strand

d. denatures, or separates, DNA

Question 1

1 pts

Cystic fibrosis is caused by nonsense and missense mutations in the CFTR gene, which encodes for a chloride channel. You are studying cystic fibrosis patients to determine what mutation they possess in the CFTR gene. The difference between the mutant and wild type CFTR genes can be uncovered by examining the CFTR:

	DNA
	mRNA
	protein
	tRNA

Question 2

1 pts

You decide to identify the CFTR mutation by analyzing the genomic DNA of your patients compared to healthy individuals. You specifically are looking to see whether a specific 3' gene truncation has occurred in the patients. You will determine this using hybridization techniques with samples from healthy and CF patients. Which of the following will allow you to accomplish this?

	Using an RNA probe complementary to the region not removed by the truncation.
	Using an RNA probe complementary to the region removed by the truncation.
	Using an DNA probe complementary to the region not removed by the truncation.
	Using an DNA probe complementary to the region removed by the truncation.

Question 3

1 pts

You would like to ensure that this experiment (to determine whether patients have a specific CFTR gene truncation using hybridization) is properly controlled. Which of the following samples must you test?

	The genomic DNA of a healthy individual who does not have cystic fibrosis.
	The genome of a CFTR patient known to have the specific truncation you are trying to identify.
	The genome of a CFTR patient with a missense mutation but full length gene.
	The genome of a healthy individual married to a CFTR patient with the specific truncation you are trying to identify.
	The genome of a patient with muscular dystrophy, which can be due to a trucation in the dystrophin gene.

Question 4

1 pts

To conduct the hybridization experiment, you are trying to decide between using a DNA or RNA probe. Which would be ideal to use and why?

	As both are composed of nucleic acids, using either would result in identical results.
	An RNA probe because RNA has uracil bases.
	An RNA probe because it could also be used in a translation experiment.
	A DNA probe because it is more stable than RNA.
	A DNA probe because RNA cannot bind to DNA.

Question 5

1 pts

Which of the following will lower the Tm of a given DNA strand?

	Increasing the percentage of GC base pairs.
	Raising the pH of the solution from neutral to basic.
	Decreasing the buffer concentration from 50mM NaCl to 5mM NaCl.
	None of the above.

Question 6

1 pts

One step of the Hershey/Chase experiment involved blending the virus/cell mixture before centrifugation and probing the pellet for radioactivity. Why was the blending step necessary?

	To collect the bacteria at the bottom of the tube.
	To break open the bacteria to release the genome.
	To separate the bacteria from the bacteriophages.
	To be able to detect the radioactivity.

Question 7

1 pts

Imagine Hershey/Chase had used an RNA virus (genome composed of RNA) instead of a DNA virus in their experiment. Would radioactivity still have been found in the pellet?

	No, because only DNA can be labeled with radioactivity.
	No, because the RNA genome would not enter the bacteria upon infection.
	No, because while DNA and RNA nucleotides are similar, they are not identical.
	Yes, because DNA and RNA nucleotides are similar.
	Yes, because genome in any form (DNA, RNA, protein) would be labeled similarly.

Question 8

1 pts

Griffith and Avery's transformation experiments allowed us to identify that DNA is our genetic information. Which of the following scenarios would result in bacterial cells that are capable of killing mice upon injection?

	Heat killed non-virulent bacteria is added to a live virulent bacteria strain.
	Heat killed virulent bacteria is added to a heat killed non-virulent bacteria strain.
	A heat killed virulent bacteria that is treated with a nuclease, is then added to a non-virulent bacteria strain.
	Heat killed mouse cells are added to a non-virulent bacteria.

Question 9

1 pts

The human genome consists mostly of non-coding DNA. Which of the following are benefits of this?

	Random DNA mutations generally won't affect RNA and protein function.
	It is faster to duplicate the genome when these are present.
	The existence of introns can lead to multiple variations of proteins encoded by a single gene.
	It is unlikely transposons would exist in the genome if there was too much protein coding DNA.

Question 10

1 pts

Andrew Murray's sister, Andrea, is adding to her brother's work on chromosomes. She is using cells that are unable to synthesize adenine (âade) and histidine (âhis). The plasmid she is currently working with consists of an origin of replication and the Ade gene.

Following her transformation of the plasmid into her yeast, what media will the cells be plated on to select for cells that have picked up the plasmid?

	Media containing histidine
	Media containing adenine
	Media lacking adenine
	Media lacking histidine

tealcockroach755

Which of the following did Erwin Chargaff observe?

A)	Base composition changes as the organism ages.

B)	Base composition of DNA does not vary from one species toanother.

C)	In all species the number of adenines equals the number ofguanines.

D)	In all species A + T = G + C.

E)	DNAs isolated from different tissues of the same species havethe same base composition.

The melting temperature (Tm) of a DNA duplex is:

A)	the temperature at which double-stranded nucleic acids degradeinto free nucleotides.

B)	the temperature at which double-stranded DNA is toosingle-stranded to ever renature back to double strands.

C)	the temperature at which double-stranded DNA is completelymelted.

D)	the temperature at which double-stranded DNA has become 50%single-stranded.

E)	the temperature at which double-stranded DNA begins to melt.

Given your knowledge and the descriptions of each of thesemethods, which of the following does NOT rely on the ability ofnucleic acids to hybridize with each other?

A)	Southern blotting for the detection of specific DNA fragmentsthat have been separated by gel electrophoresis.

B)	Colony blot hybridization to identify a specific sequence from acollection of sequences that have been inserted into bacteria.

C)	Northern blotting for the detection of specific RNA fragmentsthat have been separated by gel electrophoresis.

D)	A DNA oligonucleotide microarray incubated with probes made fromcDNA.

E)	Western blotting for the detection of specific proteins thathave been separated by gel electrophoresis.

Which of the following is a possible reason why DNA uses thymineinstead of uracil?

A)	Cytidine is deaminated to uracil so it would be difficult forany repair mechanism to differentiate between uracil that belongsin the sequence and uracil that resulted from deamination.

B)	Adenine is deaminated to uracil so it would be difficult for anyrepair mechanism to differentiate between uracil that belongs inthe sequence and uracil that resulted from deamination.

C)	When uracil is bound to a deoxyribose it will become morereactive, forming covalent bonds with other bases.

D)	None of the above.

Which of the following is an enzyme used in cloning to breakcovalent bonds?

A)	DNA ligase

B)	Restriction endonuclease

C)	Reverse transcriptase

D)	DNA polymerase I

E)	Alkaline phosphatase

Which of the following could you use to remove the 5' phosphatesafter cleavage of a plasmid with a single type of restrictionenzyme to ensure that the plasmid would not simply fuse backtogether upon addition of ligase?

A)	Reverse transcriptase

B)	Alkaline phosphatase

C)	DNA ligase

D)	DNA polymerase I

E)	Restriction endonuclease

Which of the following would you use to create a cDNA librarythat you would not use for a genomic library?

A)	Alkaline phosphatase

B)	Reverse transcriptase

C)	Restriction endonuclease

D)	DNA ligase

E)	DNA polymerase I

A plasmid vector typically has which of the followingfeatures?

A)	An origin of replication

B)	A selectable marker to ensure that the only bacteria growingcontain the intact or recombinant vector.

C)	Unique restriction sites for insertion of DNA.

D)	Small size to facilitate entry into the cell and biochemicalmanipulation of the DNA.

E)	All of the above are features of plasmid vectors.

Which of the following is FALSE of genomic or cDNAlibraries?

A)	Genes represented in cDNA libraries include control sequencesthat direct transcription.

B)	cDNA libraries contain cDNAs made from mRNA.

C)	Genomic libraries contain fragments of genomic DNA that aregenerated by partial digests with restriction enzymes.

D)	Genomic libraries usually contain large fragments and are likelyto be constructed in YAC or BAC vectors.

E)	cDNA libraries contain sequences from expressed genes only.

Which of the following is NOT a typical component of apolymerase chain reaction (PCR)?

dNTPs

B)	DNA containing the sequences to be amplified

C)	DNA ligase to connect the fragments together

D)	Primers complementary to each end of the sequence to beamplified

E)	Heat stable Taq polymerase

How can a fusion protein be formed?

A)	Site-directed mutagenesis where a fragment is replaced with asynthetic sequence containing the altered DNA sequence.

B)	Two proteins can recombine in the bacterial cell to form a newhybrid protein.

C)	Portions of two different genes are ligated together to create anew hybrid gene. When expressed the gene product is a fusion of thetwo gene products.

D)	Using oligonucleotide-directed mutagenesis to introduce specificmutations that cause the expressed protein to fuse to otherproteins.

E)	A portion of the gene can be removed to form a shorter gene andtherefore a shorter protein.

Why is green fluorescent protein (GFP) so useful in visualizingfusion proteins in eukaryotes?

A)	The GFP protein recognizes and binds to fusion proteins allowingthe location of the fusion protein to be determined.

B)	The reaction is only dependent on the presence of GFP and oneother cofactor.

C)	It is easily cloned and expressed in bacterial cells.

D)	The presence of just a few molecules of GFP fusion proteins canbe sufficient for observing the proteins microscopically allowingthe study of its location and movements in the cell.

E)	It is derived from fire flies, which are easy to cultivate inthe lab.

You wish to determine the cellular location of a protein butunfortunately it is not particularly antigenic (can t develop astrong antibody response). How could you solve this problem?

A)	Create a fusion protein with an epitope tag that can bevisualized by incubation with fluorescently tagged antibody to theepitope.

B)	Create a fusion with green fluorescent protein (GFP).

C)	Express the protein as a fusion with biotin. The addition of GFPwill cause fluorescence.

D)	A and B might work.

E)	A, B, and C might work.

F)	None of the above will work.

A positive result for the yeast two-hybrid analysis means thefollowing:

A)	The proteins that you are studying can be made into fusionproteins, but do NOT interact with each other within the nucleus ofyeast.

B)	A fusion protein containing the Gal4p activation domain, hasbound to RNA polymerase resulting increase transcription ofnumerous genes, including the reporter gene, and formation ofcolonies.

C)	Two fusion proteins, one containing the Gal4p binding sitedomain and one containing the Gal4p activation domain, haveinteracted through their Gal4p domains resulting in transcriptionof the reporter gene.

D)	Two fusion proteins interact with each other and because of theGal4p binding site domain and the Gal4p activation domain containedin each fusion protein, the reporter genes are activated and thecells die resulting in no colonies.

E)	The colonies growing are expressing the two fusion proteins, butthe two proteins have not interacted with each other.

Which of the following is the approximate size of the humangenome?

3 Gm

300 Kb

3 Mb

3 Gb

3 Mm

Which of the following is correct about the structure orlocation of genes?

Question 22 options:

A)	Prokaryotic genes typically possess their own regulatorysequences that only control expression of a single gene.

B)	Eukaryotic genes often encode introns that are spliced outbefore translation.

C)	Eukaryotic genes are mostly located at the telomeres and thecentromeres of chromosomes.

D)	Eukaryotic genes are typically arranged in operons.

E)	Prokaryotic genes often encode exons that are spliced out beforetranslation

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BIOL 2100 Chapter Notes - Chapter 20: Polymerase Chain Reaction, Reverse Transcriptase, Dna Microarray

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