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BISC300 Chapter Notes - Chapter 17: Polyacrylamide Gel Electrophoresis, Southern Blot, Recombinant Dna

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erinbear648
27 Nov 2016
School
University of Delaware
Department
Biological Sciences
Course
BISC300
Professor
Cooper Carlton

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Document Summary

17. 1 key discoveries led to development of recombinant dna technology: restriction enzymes. Recombinant dna is dna with a new nucleotide sequence. Such dna is formed by joining fragments from two or more different sources restriction enzymes or restriction endonucleases, recognize and cleave specific sequences about four to eight base pairs long. Restriction enzymes identify specific dna sequences called recognition sites; each restriction enzyme has its own recognition site. These enzymes can be used to prepare dna fragments containing specific genes or portions of genes. Sticky ends-- an end of a dna double helix at which a few unpaired nucleotides of one strand extend beyond the other: genetic cloning and cdna synthesis. Sticky ends of fragments to anneal that is, to base pair with one another and then covalently joined the fragments with the enzyme dna ligase. Plasmid vectors into which foreign dna fragments had been inserted during gene cloning had been developed.

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Related Questions

View/perform/read ALL THREE of the following prior to answeringthe questions.

http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120078/micro10.swf::Stepsin Cloning a Gene (Links to an external site.)

http://www.discoverbiotech.com/wiki/-/wiki/Main/Applications ofCloning (Links to an external site.)

http://www.wiley.com/college/boyer/0470003790/animations/cloning/cloning.htm(Links to an external site.)

Skip to question text.

From the list below, which of the following is the most logicalsequence of steps for splicing foreign DNA into a plasmid andinserting the plasmid into a bacterium?
I. Transformbacteria with recombinant DNA molecule
II. Cutthe plasmid DNA using restriction enzymes
III. Extractplasmid DNA from bacterial cells
IV. Hydrogen-bondthe plasmid DNA to nonplasmid DNA fragments
V. Useligase to seal plasmid DNA to nonplasmid DNA

IV, V, I, II, III
III, II, IV, V, I
III, IV, V, I, II
II, III, V, IV, I
I, II, IV, III, V

Flag this Question

Plasmids (or vectors) are important in biotechnology becausethey are

a vehicle for the insertion of recombinant DNA intobacteria.
surfaces for respiratory processes in bacteria.
recognition sites on recombinant DNA strands.
surfaces for protein synthesis in eukaryotic recombinants.
proviruses incorporated into the host DNA

Flag this Question

Plasmids are put into bacterial cells by

restriction enzymes
DNA ligase
binding of cohesive sticky ends
transformation

Flag this Question

Restriction enzymes usually

cut donor DNA evenly so smooth edges result
cut donor DNA but do not affect plasmids
make staggered cuts at specific sequences in DNA in both donorDNA and plasmid
are used in ligating plasmids into bacterial host cells
more than one of the above

Flag this Question

After combining DNA fragments in a cloning experiment, ___ isused to covalently join the DNA segments.

Restriction enzyme
DNA Ligase
Reverse transcriptase
DNA polymerase
Helicase

Flag this Question

It is theoretically possible for a gene from any organism tofunction in any other organism. Why is this possible?

All organisms have ribosomes.
All organisms have the same genetic code.
All organisms are made up of cells.
All organisms have similar nuclei.
All organisms have transfer RNA.

Flag this Question

Skip to question text.

Assume that you are trying to insert a gene into a plasmid andsomeone gives you a DNA sample cut with restriction enzyme X. Thegene you wish to insert from the given sample has sites on bothends for cutting by restriction enzyme Y. You have a plasmid with asingle site for Y, but not for X. Your strategy should be to

cut the plasmid with restriction enzyme X and insert thefragments cut with Y into the plasmid.
cut the plasmid with enzyme X and then insert the gene into theplasmid.
cut the DNA again with restriction enzyme Y and insert thesefragments into the plasmid cut with the same enzyme.
cut the plasmid twice with restriction enzyme Y and ligate thetwo fragments onto the ends of the human DNA fragments cut withrestriction enzyme X.
insert the fragments cut with X directly into the plasmidwithout cutting the plasmid.

Flag this Question

Which of the following is/are false in regard to expressionplasmids (also called expression vectors)?

They are used to make proteins using a cloned gene.
They contain a promotor.
They are the first plasmid type used to clone a gene.
They contain a terminator.
More than one of the above is false.

Flag this Question

What is NOT a potential problem(s) associated with usingbacteria containing a cloned eukaryotic gene (e.g. a human gene) toproduce a functional protein?

If the eukaryotic gene contains introns the bacteria will notremove them and the resulting amino acid sequence will be differentthat that made by a eukaryote.
The bacteria may not fold the protein correctly.
The bacteria may degrade the protein.
All of the above are potential problems.

Flag this Question

Cloning allows for production of proteins in much larger amountsthan occurs in the cells from which the gene is isolated.

True
False

Flag this Question

Question 111 pts

Gene cloning is used to do all of the following except

Make insulin
Making genetically identical animals (e.g. Dolly thesheep)
Make vaccines
Perform Gene Therapy
Making genetically engineered plants

Flag this Question

In your

View/perform/read ALL THREE of the following prior to answeringthe questions.

http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120078/micro10.swf::Stepsin Cloning a Gene (Links to an external site.)

http://www.discoverbiotech.com/wiki/-/wiki/Main/Applications ofCloning (Links to an external site.)

http://www.wiley.com/college/boyer/0470003790/animations/cloning/cloning.htm(Links to an external site.)

Skip to question text.

From the list below, which of the following is the most logicalsequence of steps for splicing foreign DNA into a plasmid andinserting the plasmid into a bacterium?
I. Transformbacteria with recombinant DNA molecule
II. Cutthe plasmid DNA using restriction enzymes
III. Extractplasmid DNA from bacterial cells
IV. Hydrogen-bondthe plasmid DNA to nonplasmid DNA fragments
V. Useligase to seal plasmid DNA to nonplasmid DNA

IV, V, I, II, III
III, II, IV, V, I
III, IV, V, I, II
II, III, V, IV, I
I, II, IV, III, V

Flag this Question

Plasmids (or vectors) are important in biotechnology becausethey are

a vehicle for the insertion ofrecombinant DNA into bacteria.
surfaces for respiratoryprocesses in bacteria.
recognition sites onrecombinant DNA strands.
surfaces for protein synthesisin eukaryotic recombinants.
proviruses incorporated intothe host DNA

Flag this Question

Plasmids are put into bacterial cells by

restriction enzymes
DNA ligase
binding of cohesive stickyends
transformation

Flag this Question

Restriction enzymes usually

cut donor DNA evenly so smoothedges result
cut donor DNA but do notaffect plasmids
make staggered cuts atspecific sequences in DNA in both donor DNA and plasmid
are used in ligating plasmidsinto bacterial host cells
more than one of theabove

Flag this Question

After combining DNA fragments in a cloning experiment, ___ isused to covalently join the DNA segments.

Restriction enzyme
DNA Ligase
Reverse transcriptase
DNA polymerase
Helicase

Flag this Question

It is theoretically possible for a gene from any organism tofunction in any other organism. Why is this possible?

All organisms haveribosomes.
All organisms have the samegenetic code.
All organisms are made up ofcells.
All organisms have similarnuclei.
All organisms have transferRNA.

Flag this Question

Skip to question text.

Assume that you are trying to insert a gene into a plasmid andsomeone gives you a DNA sample cut with restriction enzyme X. Thegene you wish to insert from the given sample has sites on bothends for cutting by restriction enzyme Y. You have a plasmid with asingle site for Y, but not for X. Your strategy should be to

cut the plasmid withrestriction enzyme X and insert the fragments cut with Y into theplasmid.
cut the plasmid with enzyme Xand then insert the gene into the plasmid.
cut the DNA again withrestriction enzyme Y and insert these fragments into the plasmidcut with the same enzyme.
cut the plasmid twice withrestriction enzyme Y and ligate the two fragments onto the ends ofthe human DNA fragments cut with restriction enzyme X.
insert the fragments cut withX directly into the plasmid without cutting the plasmid.

Flag this Question

Which of the following is/are false in regard to expressionplasmids (also called expression vectors)?

They are used to make proteinsusing a cloned gene.
They contain a promotor.
They are the first plasmidtype used to clone a gene.
They contain aterminator.
More than one of the above isfalse.

Flag this Question

What is NOT a potential problem(s) associated with usingbacteria containing a cloned eukaryotic gene (e.g. a human gene) toproduce a functional protein?

If the eukaryotic genecontains introns the bacteria will not remove them and theresulting amino acid sequence will be different that that made by aeukaryote.
The bacteria may not fold theprotein correctly.
The bacteria may degrade theprotein.
All of the above are potentialproblems.

Flag this Question

Cloning allows for production of proteins in much larger amountsthan occurs in the cells from which the gene is isolated.

True
False

Flag this Question

Question 111 pts

Gene cloning is used to do all of the following except

Make insulin
Making genetically identicalanimals (e.g. Dolly the sheep)
Make vaccines
Perform Gene Therapy
Making genetically engineeredplants

Flag this Question

In your

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