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BIOL 200 Study Guide - Final Guide: Agarose Gel Electrophoresis, Tata-Binding Protein, Transfer Rna

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pinkgoldfish355
22 Jun 2016
School
McGill University
Department
Biology
Course
BIOL 200
Professor
Richard Roy

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Document Summary

You run the mrna which is coated with poly a tail through a matrix which is bound by poly t oligonucleotides. Use reverse transcriptase to convert the mrna back to cdna. Use probes to study speci c areas of the dna (complementarity) Use rt + pcr to amplify the dna, rather than cloning will make tons of cdna in a couple of hours. Make insulin through bacteria inject the insulin plasmid into a bacterium and collect the product. Lac operon promoter will drive expression of lac z gene uses galactose as an energy source. If you put the gene of interest downstream of the lac gene, when you introduce lac promoter to galactose, you make your protein. Must add a poly a tail to any dna and use viral vectors to add it. Use drugs to kill the cells that don"t except the vector (with antibiotic resistance) Used to tag genes of interest to discover location.

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Related Questions

A student written question: New Belgium Brewery creates its own yeast strains for brewing beer. Some yeast strains secret a toxic protein, which is not detectable until after the brewing process. Therefore the brewery must find a way to detect the yeast that secretes this toxic protein. Because they collaborate with Dr. Wolkow, a yeast-whiz, the brewery decides to hire you to work with Dr. Wolkow to make a cDNA expression library from the toxin-secreting strain. You decide to use a bacterial expression vector since it is easy to work with.

1. What components must the expression vector contain?

a. promoter, translation start sequence, cloned cDNA sequence, DNA polymerase

b. DNA ligase, translation start signal, cloned cDNA sequence, polyA tail

c. Shine-Dalgarno sequence, promoter, cloned cDNA sequence, antibody

d. promoter, Shine-Dalgarno sequence, cloned cDNA sequence, transcription termination sequence

2. Suppose the radioactive 3 antibody did not attach to any of the protein products. What can you conclude from this?

a. the toxic protein product is produced by all the yeast strains

b. the toxic protein product is produced by one or more of the yeast strains

c. the toxic protein product is not produced by any of the yeast

d. the yeast DNA was destroyed when the cells were lysed

3. Why would the brewer want to specifically use an expression vector rather than another type of vector?

a. It is specifically for eukaryotic DNA

b. It is used to produce the protein encoded by a cloned gene

c. It is only used for cloning cDNA sequences

d. They want to produce mRNA encoded by a cloned gene

4. This type of blot, where protein is transferred onto the membrane and is then hybridized to a tagged-antibody is a blot.

a. Southern

b. Northern

c. western

d. southwestern

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

ANSWER THE FOLLOWING QUESTIONS(COULD BE MORE THAN ONEANSWER)

19) Describe the roles of M13 genes and their proteins:

a) Genes 1, 4 and 11 produces proteins required for assemblingthe the phage.

b)Genes 3.6,7,8 and 9 produces capsid proteins.

c)Gene 2 nicks the circular phage ssDNA to make it doublestranded by DNA polymerase

d)Gene 5 produces single strand binding proteins and gene 10protein makes M13 SSDNA circular.

e)Protein five bound single stranded circular M13 moved to theE. coli membranne and is released out coated will proteins 3, 6, 7,8 and 9 .

2) What is a phagemid and how does it work?

a)it is a recombinant DNA with a filamentous phage genome and aplasmid genome.

b)it carries two origins of replication: eg., oir f1 and oriColE1

c)May carry a gene for selection: e.g. Ampr gene.

d)May carry a marker gene; e.g, lacZ gene with promoter andregulator

e)May contain MCS, as is pUC18.

3)For storing functional gene for future applications you willneed the following:

a)A vector DNA

b) A cloning site in the vector matching with the ends of a geneto be cloned

c)A host organism capable to hold the recombined vector.

d)A marker gene present in the recombinant vector

e)A functional gene of interest.

24)identify which of the following is true for thebacteriophage lambda?

a)It has two major body parts, the head and the tail.

b)The genome is approximately 49 thousand bases long.

c) It has a linear dsDNA with single stranded 12 bases long costerminals at both

d) The single stranded lambda DNA cos terminals appear to be asfollows: (a) GGGCGGCGACCT and (b)CCCGCCGCTGGA

e)it can accept 7-10 kb long foreign DNA replacing itsnon-essential b2 region.

29) For developing a recombinant lambda DNA with a gene ofinterest and expressing the gene in a host you have to take thefollowing steps:

a)Remove the b2 region from lambda DNA using a restrictionenzyme

b)digest the gene of interest, outside its stop and startcodons, with the same restriction enzyme.

c)Recombine the lambda DNA digest with the digested gene ofinterest carrying complementary overhangs with ligase.

d)Form lambda particles by adding head proteins, tail proteinsand packaging enzynes to the recombined lambda DNA.

e)Add the packaged lambda particles to Agrobacetrium tumefaciensand incubate them in culture.

f)Add the packaged lambda particles to laboratory strains ofEscherichia coli and incubate them in culture.

32)The amino acids coded by our genes differ by thefollowing:

a)Polarity

b) Acidity

c)Basicity

d)Aroma

e)Hydrophobicity

38)What is an M13 phage and what are its specialty?

a)They are rod shaped Escherichia coli bacteriophage.

b)ts genome is made of a single stranded circular DNA.

c)Its DNA is 6400 nucloetides long coding for elevenproteins.

d)After infecting a bacterial cell it starts replicating in 10minutes producing 1,000 phages in an hour

e) soon its kills the host Escherichia coli and gets out to theenvironment.

40)You have a gene with BamH cut sites out side its start andthe stop codons. You can use the following procedures to prepare aclone of the gene in pBR322 and select the transformed E.Coli cellscarrying pBR322, except:

a) Digest both the gene and the plasmid pR322 with BamH1

b) Ligate the fragments using T4 DNA ligase.

c)Transform E coli with the reconstructed plasmid

d)Select the cells transformed cells grow in Lb-agar-ampicilinplates

e)Select the cells transformed cells grow inLB-agar-tetracycline plates