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BIOMG3300 > Unit 9 > Flashcards

Unit 9 Flashcards

1
Q

Describe the five general procedures in DNA cloning.

A
  1. Obtain DNA segment to be cloned
  2. Select the cloning vector
  3. Join the two DNA fragments w/ DNA ligase to form recombinant DNA
  4. Move recombinant DNA to host organism
  5. Select/Identify the cells w/ your target DNA
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2
Q

Are restriction enzymes made by eukaryotes or prokaryotes?

A

Prokaryotes

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3
Q

What is the function of restriction enzymes in vivo?

A

To recognize + cleave foreign DNA (ex: viral DNA)

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4
Q

Discuss the restriction-modification system.

A
  • Restriction endonucleases cleave DNA at specific sequences
  • Sequences within the organism’s own DNA that match those recognized by restriction enzymes are protected by methylation.
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5
Q

What is a restriction endonuclease that generates sticky ends and one that generates blunt ends?

A

Sticky End Enzymes = BamHI, ClaI, EcoRI, HindIII, NotI, PstI, Tth111I

Blunt End Enzymes = EcoRV, HaeIII, PvuII

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6
Q

What are blunt and sticky ends?

A

Sticky ends: Two overhanging DNA ends w/ unpaired nucleotides as a result of staggered cleavage by a restriction endonuclease

Blunt ends: Two DNA ends w/ no unpaired bases – result of straight-across cleavage by a restriction endonuclease

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7
Q

What is a plasmid?

A

A circular DNA molecule that replicates separately from the organism’s chromosome

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8
Q

What is transformation?

A

The introduction of exogenous DNA into a cell, causing the cell to acquire a new phenotype

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9
Q

Why do plasmids contain a selectable marker gene?

A
  • The selectable marker gene can be used to select for cells that contain the desired gene.
  • Selectable marker either permits the growth of the cell (positive selection) or kills the cell (negative selection) under a defined set of conditions.
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10
Q

Why do plasmids contain an origin of replication (ori)?

A
  • The origin of replication is a sequence to which cellular enzymes can bind to initiate replication.
  • Necessary for the propagation of the plasmid.
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11
Q

Why do plasmids contain unique recognition sequences for restriction endonucleases?

A
  • The recognition sites provide areas where the DNA can be cleaved for the insertion of foreign DNA.
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12
Q

How are plasmids used to clone DNA?

A
  • The plasmid is cleaved by restriction enzymes
  • DNA fragments to be cloned are ligated (joined) to the cleaved plasmid by DNA ligase
  • Cells incorporate the plasmid + are transformed
  • Cells are grown under certain conditions to determine which ones took up the plasmid
  • The plasmid is replicated within the cell.
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13
Q

What is an expression vector?

A

A cloning vector w/ the transcriptional + translational signals needed for the regulated expression of a cloned gene.

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14
Q

Why do you need an expression vector to express a eukaryotic gene in a prokaryote?

A

Eukaryotic genes lack the DNA sequence elements necessary for expression in prokaryotic cells (promoter for RNA polymerase binding, ribosome-binding sites) +

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15
Q

What is the importance of the origin of replication (ori)?

A

DNA sequence to where replication of the plasmid is initiated.

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16
Q

What is the importance of the selectable genetic marker?

A

Gene used to identify which cells incorporated the plasmid (ex: antibiotic resistance gene).

17
Q

What is the importance of the polylinker?

A

Contains restriction sites where, once cleaved, the desired gene can be inserted into the plasmid.

18
Q

What is the importance of the promoter?

A

DNA sequence to direct RNA polymerase binding for mRNA synthesis.

19
Q

What is the importance of the operator (O)?

A

DNA sequence to which a repressor protein can bind, regulating RNA synthesis.

20
Q

What is the importance of the ribosome-binding site?

A

Provides sequence signals for efficient translation of mRNA synthesized from the gene.

21
Q

Why is it necessary to have a promoter in the expression vector when using a cDNA inserts?

A

So that genes inserted at restriction sites have regulated expression. Most eukaryotic genes lack the instructions for RNA polymerase that they need to work in prokarya.

22
Q

Why is it desirable to have a regulatable promoter in an expression vector?

A
  • Rate of expression controlled by replacing the gene’s own promoter + regulatory sequences w/ more efficient + convenient versions leading to high expression of the cloned gene

o High efficiency leads to overproduction of the cloned gene’s protein product– at high concentrations of cloned gene, foreign proteins can kill the cell

o If promoter is able to be regulated, expression of the cloned gene can be regulated.

23
Q

Define site-directed mutagenesis.

A
  • A set of methods used to create specific alterations in the sequence of a gene
  • Alter DNA sequence of cloned gene to change amino acid sequence of resulting protein (way to study protein structure + function)
24
Q

Distinguish the synthetic insert method from oligonucleotide directed mutagenesis.

A
  • Site-directed mutagenesis: cut out DNA + insert synthetic DNA w/ altered nucleotide sequence
  • Oligonucleotide directed mutagenesis:

o Two complementary oligonucleotide sequences act as primers for synthesis of mutant plasmid

25
Q

Describe the steps of oligonucleotide-directed mutagenesis.

A

(1) Denature plasmid + anneal oligonucleotide primers with mutation
(2) Use DNA polymerase to extend primers
(3) Digest parental plasmid with methylation-specific nuclease + anneal newly synthesized strands
(4) Transform dsDNA into cells. Cell repairs nicks in the DNA.

26
Q

Describe how DNA primers and probes of a specific sequence are synthesized.

A

(1) Nucleoside attached to solid silica support at 3’ hydroxyl + 5’ hydroxyl protected by DMT
(2) Protecting group is removed
(3) Next nucleotide is added
(4) Phosphite linkage is oxidized to form phosphotriester linkage
- Steps #2-#4 repeated until all residues are added
(5) Protecting group removed from bases
(6) Cyanoethyl groups removed from phosphates
(7) Chain is cleaved from silica support

27
Q

Describe a procedure used to tag proteins thereby facilitating their purification using affinity chromatography techniques.

A
  • Fuse gene of target protein w/ the gene of a protein/peptide that binds a ligand with high affinity + specificity
  • ## Altering gene of target protein to express a fusion protein which can be purified by affinity chromatography
28
Q

What are transposons? How much of the human genome do they represent?

A
  • Mobile genetic elements that can self-replicate

- 45% of human genome

29
Q

How many protein coding genes are in the human genome? What fraction of the genome encodes protein

A
  • 20,000 protein coding genes

- 1.5% of genome encodes proteins

30
Q

What fraction of the human genome consists of genes?

A
  • 30%

- Genes include protein coding sequences + introns

31
Q

Ion semiconductor sequencing

A
  • A method of DNA sequencing where the addition of nucleotides is detected by measuring the release of protons
32
Q

Single-molecule real-time (SMRT) sequencing

A
  • DNA sequencing technology in which nucleotide additions are detected as flashes of fluorescent-colored light
33
Q

Describe next-generation reversible terminator sequencing.

A
  • DNA sequencing technology in which nucleotide additions are detected + scored by the color of fluorescence displayed when a labeled nucleotide is added
  • Blocking groups on each o fluorescently-labeled nucleotide prevent multiple nucleotides from being added in a single cycle
    ♣ Add blocked, fluorescently-labeled nucleotides
    ♣ Correct nucleotide is added – color is observed + recorded
    ♣ Remove labels + blocking groups, wash, add blocked, fluorescently-labeled nucleotides
    ♣ Correct nucleotide is added – color is observed + recorded
    ♣ Repeat.
34
Q

Describe pyrosequencing.

A

o DNA sequencing technology
o Using flashes of light to detect the addition of complementary nucleotides to a DNA template (to be sequenced)
o When a nucleotide is incorporated, pyrophosphate is released and pyrophosphate is used to generate ATP
o When ATP is generated, luciferase reacts w/ luciferin to produce light

35
Q

What is radioactivity vs. fluorescence?

A

o Radioactivity: Emission of particles by some atoms when their unstable nuclei disintegrate
o Fluorescence: Emission of light by excited molecules as they revert to ground state

36
Q

What are micro-RNAs?

A

a class of small RNA molecules involved in gene silencing by inhibiting translation and/or promotion of degradation of particular mRNAs

37
Q

Explain how RNAi can be used as a gene silencing method.

A

o RNAi (RNA interference) - siRNAs bind to mRNA – prevent translation or degrade targeted mRNA

38
Q

Discuss the Sanger method for DNA sequencing.

A

♣ Use DNA polymerases + primers to synthesize DNA strand complementary to the strand under analysis
♣ Each added deoxynucleotide is complementary (through base pairing) to a base in template strand
♣ Sequence obtained = newly synthesized complementary strand
♣ Dideoxynucleoside triphosphates (ddNTPs) are used to interrupt DNA synthesis
• ddNTPS lack the 3’ –OH necessary for strand elongation – so synthesis is terminated prematurely

BIOMG3300 (1 decks)

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