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BIOLOGY 305 Lecture Notes - Lecture 12: Sticky And Blunt Ends, Methylation, Dna Microarray

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purplebeaver856
21 Jul 2016
School
U of M
Department
Biology
Course
BIOLOGY 305
Professor
Marcus Ammerlaan

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Document Summary

In the 1970s, we gained the ability to manipulate genes. They cut somewhere in the middle of the molecule where a specific set of bases occur. They are all palindromes due to the anti-parallel relationship. These restriction enzymes most of the time bind as dimers (so you get one protein binding to the top and bottom strand) Each cut the phosphodiester bond in one place. Different enzymes not only bind to different 4-6 sets of bases but they cut in different places. That can mean that you sometimes get an overhang of 4 bases or cut right in the middle (thus no overhang) Bacteria have these enzymes to chop up virus dna. Different species will have different restriction enzymes recognizing different particular sets of 4-6 bases. A given bacteria might have 2-4 of these enzymes. The idea is that once the bacteria phage injects it"s dna, the green dots (these are the enzymes) chop up the pieces of viral dna.

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Related Questions

1) Which of the following is FALSE about plasmid vectors ued in recombinant DNA research?
A) Usually carrry an antibiotic resistance gene
B) Clonin sites are always located between genes, that is, outside coding sequences
C) May hae a multiple clonin gsite which can be cut by several restriction enzymes
D) May be expression vectors

2) Which of the following is FLASE about Sanger Dideoxy DNA sequencing protocols?
A) Different specfiic chemical reactions break the DNA at each base.
B) Fluorescent molecules can be used to detect the DNA
C) The Template is single stranded DNA
D) Many steps can be automated

3) A southern blot transfers ___ to a Nitrocellulose membrane.
A) Protein
B) RNA
C) DNA
D) Lipid

4) Which of the following is true about cDNA?
A) It is made from DNA template
B) It is isolated from bacteria
C) Reverse transcriptase is used to synthesize cDNA
D) It has introns

5) The restriction endonucleases used in recombinant DNA work:
A) Always produce blunt ends (No unpaired bases)
B) Recognize sequences 4-6 bp long
C) Cut the DNA outisde the recognition sequence
D) All of the above

S17

THANK YOU VERY MUCH!!!

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

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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

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Plasmids are put into bacterial cells by

restriction enzymes
DNA ligase
binding of cohesive stickyends
transformation

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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

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

Restriction enzyme
DNA Ligase
Reverse transcriptase
DNA polymerase
Helicase

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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.

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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.

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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.

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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.

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

True
False

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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

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In your

1. In addition to identifying the genetic material, the experiments of Avery, MacLeod, and McCarty with different strains of Streptococcus pneumoniae demonstrated that
DNA is a double helix.
DNA may be taken up by bacterial cells and be active.
DNA is present in bacteriophage T2.
eukaryotic cells may be genetically transformed.
DNA binds to the cell wall of the bacterium.
2. In order to show that DNA in cell extracts is responsible for genetic transformation in Streptococcus pneumoniae, important corroborating evidence should indicate that _______ also destroy transforming activity.
temperatures high enough to kill cells
enzymes that hydrolyze proteins
enzymes that hydrolyze DNA
enzymes that hydrolyze polysaccharides
enzymes that hydrolyze RNA
3. Based on what you have learned about the experiments conducted by Griffith and Avery and colleagues with bacteria, which of the following would result in transformation of living R cells?
Pure RNA from S cells
Cell-membrane extracts from S cells
A mixture of pure RNA and protein from S cells
Cell-free extracts from S cells
Pure protein from S cells
4. A-T base pairs in a DNA double helix
form three hydrogen bonds with each other.
are more heat-stable than G-C base pairs.
are of a substantially different length than G-C base pairs.
are not accessible to DNA binding proteins.
are chemically distinct from G-C base pairs.
5. If 23 percent of the bases in a sample of double-stranded DNA are adenine, what percentage of the bases are uracil?
27
50
0
73
23
6. The uniform diameter of the DNA structure provides evidence for
antiparallel DNA strands.
a right-handed helix.
a double-stranded helix.
3′ end phosphorylation.
purine–pyrimidine pairing.
7. If a sequence of one strand of DNA is 5′-TGACTATC-3′, what is the complementary strand?
5′-ACTGATAG-3′
3′-TGACTATC-5′
3′-ACTGATAG-5′
3′-ACTGATAG-3′
5′-TGACTATC-3′
8. What structural aspect of the DNA facilitates dissociation of the two DNA strands for replication?
3′ end phosphorylation
Purine–pyrimidine pairing
Antiparallel DNA strands
Covalent bonds between sugars and phosphate groups
Hydrogen bonding between paired bases
9. If the Meselson–Stahl density gradient experiment had resulted in two bands of DNA molecules after only one round of replication, one containing only 15N and the second only 14N, this result would have indicated that replication was
unidirectional.
dispersive.
conservative.
bidirectional.

semiconservative.
10. The nucleoside analogue acyclovir, which is used to treat herpes simplex virus (HSV) infections, lacks a 3′ hydroxyl group (–OH). Predict what will happen if the host cell DNA polymerase incorporates a molecule of acyclovir into an elongating strand of HSV DNA.
The replication complex will no longer bind to origins of replication.
The DNA polymerase will only incorporate acyclovir molecules.
Acyclovir will bind single-strand binding proteins.
DNA polymerase will not be able to link a successive nucleotide.
Acyclovir will block the activity of the DNA helicase.
11. Which of the following does not demonstrate the stability of the DNA double helix?
Single-strand binding proteins are required to keep the strands apart.
Paired bases form hydrogen bonds.
High temperatures are required to denature it.
DNA synthesis requires energy.
DNA helicases require ATP.
12. What effect would a primase inhibitor have on DNA replication?
DNA polymerase would not be able to add bases to the DNA.
Primase would be blocked from joining the DNA replication complex.
Primase would not be able to provide primers for DNA polymerases.
DNA polymerase would not be able to use DNA as a template.
There would be no effect on DNA replication.
13. To replicate their DNA in a timely manner, most eukaryotic chromosomes
normally have uncoiled DNA.
have multiple origins of replication.
contain linear molecules of single-stranded DNA.
have two replication forks that move in the same direction.
are composed entirely of DNA.
14. Which statement about DNA replication is false?
Okazaki fragments are synthesized as parts of the leading strand.
Error rates for DNA replication are reduced by proofreading of the DNA polymerase.
The sliding clamp increases the rate of DNA synthesis.
Replication forks represent areas of active DNA synthesis on the chromosomes.
Ligases and polymerases function in the vicinity of replication forks.
15. In many eukaryotes, there are repetitive sequences called telomeres at the ends of chromosomes. After successive rounds of DNA replication, the _______ strand becomes shorter. In some cells, an enzyme called _______ repairs the shortened strand.
leading; telomerase
lagging; telomerase
lagging; DNA polymerase I
leading; DNA polymerase I
leading; replicase
16. A researcher studies normal human fibroblast cells. They can be maintained in culture but die off after about 30 cell generations. Unexpectedly, a colony of cells continues to survive and divide past 30 generations. Which scenario is most likely true for these cells?
DNA polymerase is constantly active.
Primase is able to extend the telomeres.
The mismatch repair system is extra efficient.
The telomerase gene is being expressed.
The cells do not need the ends of their chromosomes.
17. If DNA polymerase III introduces an incorrect nucleotide, what is the first corrective action made by the DNA repair system?
The replication complex excises the incorrect nucleotide.
The excision repair proteins remove the incorrect nucleotide.
The mismatch repair system scans for base-pairing mismatches.
There is no correction, because nucleotide errors by DNA polymerases are not repaired.
DNA ligase connects the correct nucleotide.
18. Choose the correct order of the following four events in the excision repair of DNA:
(1) Base-paired DNA is made complementary to the template.
(2) Damaged bases are recognized.
(3) DNA ligase seals the new strand to existing DNA.
(4) Part of a single strand is excised.
1, 2, 3, 4
2, 1, 3, 4
3, 4, 2, 1
2, 4, 1, 3
4, 2, 3, 1
19. Six complete cycles of PCR should result in a _______-fold increase in the amount of DNA.
64
32
12
128
6
20. When double-stranded DNA is heated to temperatures above 90°C, it denatures. Denaturation is a process that
breaks covalent bonds.
hydrolyzes DNA.
separates double-stranded DNA into two single strands.
causes new hydrogen bonds to form.
irreversibly destroys the double helical structure.