Quiz #1 Flashcards

1
Q

What is the main function of restriction nucleases in bacteria, and how are they protected from them?

A

Restriction nucleases cut DNA at specific sites.

Bacterial DNA has modifications, often methylations, that restriction nucleases don’t recognize.

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2
Q

Why are enzymes that produce staggered cuts more advantageous over enzymes that produce blunt ends in making recombinant DNA molecules?

A

It is easier to join DNA with staggered cuts, or “sticky ends” since there are ss, unpaired bases

(Can draw picture)

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3
Q

What reagent allows the visualization of DNA on an agarose gel? And, if you want to visualize and separate very large DNA molecules (>15 Kbs), what technique do you use?

A

EtBr

Pulsed-field gel electrophoresis

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4
Q

There are two ways (using two different enzymes) to label purified DNA. Name the two enzymes. Which one makes for a stronger probe? Why?

A

Using DNA polymerase to copy DNA in the presence of nucleotides labeled with 32P or chemically. Makes for a stronger probe bc each nt is labeled

Using polynucleotide kinase to label the 5’ end of each DNA chain with 32P

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5
Q

Northern and Southern blots detect what, respectively? Southern can detect what types of mutations?

A

Northern blots detect RNA
Southern blots detect DNA

Deletion or insertion

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6
Q

Three common features of an artificially constructed plasmid vectors are? Which one allows for maintaining of the cell?

A
  1. Initiation site
  2. Resistance to antibiotic - maintaining the cell
  3. Polylinker
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7
Q

What is the main difference between genomic and cDNA library in terms of specificity? Why is that?

A

Genomic libraries contain the entire genome (introns and exons), making it less specific
cDNA libraries only contain the DNA that is complementary to the mRNA (only exons), making it more specific

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8
Q

What three things do you need in a PCR reaction, in addition to a DNA polymerase?

A
  1. Primer
  2. Nucleotides
  3. Template strand
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9
Q

What is the advantage of taq polymerase, but what is the problem with taq polymerase?

A

The advantage of taq polymerase is that it can avoid being deactivated at high temperatures

The disadvantage of taq polymerase is that it lacks 3’ to 5’ exonuclease proofreading activity, resulting in low replication fidelity

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10
Q

What is the main drawback with using primary culture cells?

A

Primary culture cells die off eventually due to shortening of telomeres

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11
Q

Immortalized cancer cells typically have a mutated version of this gene, which allows for cells to bypass the cell cycle checkpoints. This is important for normal maintenance of cells, i.e. cell division, apoptosis (cell death), etc. What is this gene called?

A

Oncogenes are the mutated version of protooncogenes

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12
Q

You want to isolate mitochondria from your cells. How do you go about doing this?

A

Column chromatography. Different spin speeds isolate different elements. A medium spin speed would isolate mitochondria in a pellet.

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13
Q

How does gel-filtration chromatography work?

A

Based on size. The smaller molecules get trapped inside of the beads and the larger ones travel quickly to the bottom

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14
Q

Give me an example of a recognition tag you can use to make fusion protein.

A

GST

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15
Q

Concerning SDS-PAGE, the addition of SDS serves what purpose?

A

SDS is a detergent that denatures the protein, making it linear. It also gives the protein a negative charge.

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16
Q

Concerning SDS-PAGE, the addition of a reducing agent serves what purpose? Also, name me a reducing agent.

A

The reducing agent is beta-mercaptoethanol and it breaks disulfide bonds

17
Q

You want to identify protein or proteins that interact with Protein Z. There are many ways, but name one, and describe how this technique works.

A

Affinity chromatography. It works by using the enzyme-substrate interaction. Proteins that interact with Protein Z will be retained in the matrix.

18
Q

A plasmid was digested with Bam HI. On the agarose gel electrophoresis, you observe three bands 200, 4000, and 700 bp. How many BamHI sites are present in this plasmid? And, what is the size of the plasmid?

A

Three BamHI sites are present. The size of the plasmid is 200 bp + 400 bp + 700 bp = 1300 bp

19
Q

Proteins that bind to DNA to control gene expression are called _______ factors.

A

transcription

20
Q

A procedure for digestion of DNA with a restriction enzyme includes a final incubation stop of 5 minutes at 95C. What is the purpose of this final step?

A

Inactivate enzymes

FINISH

21
Q

The cells in an individual animal contain nearly identical genomes. In an experiment, a tissue composed of multiple cell types is fixed and subjected to in situ hybridization with a DNA probe to a particular gene. To your surprise, the hybridization signal is much stronger in some cells than in others. Explain this result.

A

The gene is expressed differently in different cell types (got 1 out of 2 points)

FINISH