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MCDB 2150 Lecture Notes - Lecture 15: Dna Microarray, Fluorescence Microscope, Microscope Slide

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tealhedgehog29
3 Mar 2017
School
CU-Boulder
Department
Molecular Cell & Developmental Biology
Course
MCDB 2150
Professor
Jennifer Knight

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Document Summary

To study gene expression, mrna can be prepared from a particular type of cell such as a muscle cell or from cells that have been subjected to an environmental agent, such as a hormone. This mrna that is expressed from such cells is used to make cdna. cdna is made in a test tube by an enzyme called reverse transcriptase. Identify genes that are expressed in a cell specific manner. Identify genes that are expressed or repressed under one set of conditions. Unit 2 study guide: tumor profiling identify gene expression pattern in a tumor can be used to sub-classify tumors that are morphologically the same, detect genetic variation. Mutant alleles may not hybridize: distinguish between closely related species, prepare cdna probe. In the disease sample, there is a mutation in this gene. In tumor cells, the same gene is upregulated. The spot on the merged micro array would be red: rna sequencing (rna-seq)

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Related Questions

1. You have cloned a human gene along with its promoter and ribosome-binding site in a bacterial cloning vector and then introduced the recombinant plasmid into a bacterial host cell that supports the replication and segregation of the cloning vector. Unfortunately, even though you know the whole human gene sequence is present (by DNA hybridization); you cannot get expression of the gene. Which of the following is NOT a reason why there is no expression?

A. The vector cannot replicate

B. The human promoter is not recognized by the bacterial host cell

C. The ribosome-binding site is not recognized by the bacterial host cell

D. The human gene lacks introns, so it is not transcribed by the host bacterium

2. Restriction endonucleases are:

A. sequence-specific

B. able to recognize any 4-, 6-, or 8-base pair sequence

C. always able to produce single stranded tails

D. only able to cleave bacterial DNA

3. Which of the following is NOT a required characteristic of cloning vectors?

A. Genes for replication of vector DNA and segregation to progeny cells

B. Genes for selection of cells carrying the vector (e.g., antibiotic-resistance)

C. DNA sequence for the introduction of foreign DNA where the integrated DNA does not disable the vector

D. None of the above

4. A poly (T) primer is used to produce cDNA from mammalian mRNA. Which of the following would be used to produce cDNA from bacterial mRNA?

A. A poly (T) primer

B. A random DNA sequence primer

C. A poly (A) primer

D. A poly (GC) primer

1. What reaction is catalyzed by reverse transcriptase and used in the laboratory to generate cDNA?

a. transcription of RNA to DNA

b. transcription of DNA to RNA

c. conversion of ribonucleotides into deoxyribonucleotides

d. translation of RNA to DNA

e. conversion of a ribonucleotide to a DNA double helix

2. In eukaryotic cells, _________determine the amount of mRNA arising from a particular gene.

a. transcriptional-level controls

b. RNA processing-level controls

c. translational-level controls

d. replication-level controls

e. b and c

3. In eukaryotic cells, _________determine how a primary transcript is converted to mature mRNA

a. transcriptional-level controls

b. RNA processing-level controls

c. translational-level controls

d. replication-level controls

e. a and d

4. In eukaryotic cells, _________determine when a particular mRNA is actually used in protein synthesis.

a. transcriptional-level controls

b. RNA processing-level controls

c. translational-level controls

d. replication-level controls

e. None of the above because protein synthesis takes place immediately

5. Under what circumstances do cells selectively activate and suppress their genes?

a. at different stages of embryonic development

b. in different tissues

c. exposure to different types of stimuli

d. No such circumstances exist in nature—cells express all their genes throughout life.

e. a, b and c

6. Which statement about the control of gene expression is FALSE?

a. A single gene may be controlled by different transcription factors.

b. A single transcription factor may be necessary for the expression of different genes.

c. Each cell type has a characteristic pattern of gene transcription, which is determined by the particular complement of transcription factors contained in that cell.

d. Expression of protein genes is primarily controlled in nucleoli

e. Control of gene transcription is complex and influenced by various circumstances.

7. What is thought to maintain X chromosome inactivation in females?

a. DNA acetylation

b. DNA methylation

c. RNA inactivation

d. Histone methylation

e. None of the above because both X chromosomes are active in females and males.

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)?

A)

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?

A)

3 Gm

B)

300 Kb

C)

3 Mb

D)

3 Gb

E)

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