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gene 3200 test 5 > Test 5 > Flashcards

Test 5 Flashcards

1
Q

What is in situ hybridization?

A

Detects positions of target mRNA, non-destructive.

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2
Q

What is northern blotting?

A

Measures size of mRNA after gel electrophoresis, destructive.

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3
Q

What is RNA sequencing?

A

A method of analyzing large sets of RNAs that involves making cDNAs and sequencing them, destructive.

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4
Q

What is immunofluorescence?

A

Detects position of target proteins, non-destructive.

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5
Q

What is western blotting?

A

Measures size of proteins after gel electrophoresis, destructive.

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6
Q

What is proteomics?

A

Study of the structure and function of proteins in the human body (proteome), destructive.

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7
Q

What is primary (direct) immunofluorescence?

A

One antibody chemically linked to a fluorophore, then attached to epitope on antigen.

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8
Q

What is secondary (indirect) immunofluorescence?

A

Primary antibody binds to target protein (epitope), secondary antibody binds to fluorophore, then binds to primary.

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9
Q

Best molecules for gel electrophoresis?

A

Smaller, negatively-charged, and tightly condensed globular molecules migrate faster.

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10
Q

What does it mean if no signal is detected in northern blot?

A

Gene is not expressed OR RNA sample is degraded. Use control probes to detect other genes that would also be present.

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11
Q

What is quantitative PCR?

A

Measures mRNA level of the same gene between samples.

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12
Q

What do PCR reactions need?

A
  • dsDNA template
  • Mixture of all four dNTPs
  • Stable DNA polymerase
  • Two primers for the opposite strands
  • Aqueous buffer solution
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13
Q

What happens in PCR amplification phase 1?

A

Sample heated and template denatures into single strands.

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14
Q

What happens in PCR amplification phase 2?

A

Temperature reduced and primer hybridize to their complementary sequence.

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15
Q

What happens in PCR amplification phase 3?

A

Temperature increased for DNA synthesis (longer template takes longer).

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16
Q

What are the 4 major transformations?

A
  • PCR and cloning
  • Recombinant DNA
  • DNA sequence identification
  • Genome targeting (CRISPR)
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17
Q

What is the right primer (reverse)?

A

Upper strand.

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18
Q

What is the left primer (forward)?

A

Lower strand.

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19
Q

What do restriction enzymes do?

A

Recognize a specific sequence of bases anywhere within the DNA.

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20
Q

Which direction does DNA move in gel electrophoresis?

A

Towards positive charge, as it is negatively charged.

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21
Q

What are germline mutations?

A

Source of all genetic variation, occurs in gametes and is transmitted to progeny.

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22
Q

What are somatic mutations?

A

Occurs in somatic cells, mutation inherited by daughter cells.

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23
Q

What are gene mutations?

A

Small alterations that affect a single gene or locus.

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24
Q

What are chromosome mutations?

A

Large alterations that affect chromosome structure or number.

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25
Q

Where do mutations occur?

A

Anywhere.

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26
Q

What are the consequences of mutations?

A

Either no functional consequences or can affect gene function/expression.

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27
Q

What are mutations in protein coding regions?

A
  • Can affect amino acid sequence
  • Substitutions: missense, nonsense or silent
  • Insertion/deletion: frameshift or in-frame
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28
Q

What are mutations in noncoding regions?

A

Can affect rate of transcription or post-transcriptional processes.

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29
Q

What is a transition point substitution?

A

Pyrimidine to pyrimidine or purine to purine (C <–> T or A <–> G), more frequent.

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30
Q

What is a transversion point substitution?

A

Purine to pyrimidine or pyrimidine to purine (A <–> C or T; G <–> C or T).

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31
Q

What is a missense mutation?

A

A mutation that changes one amino acid.

32
Q

What is a nonsense mutation?

A

Changes a normal codon into a stop codon.

33
Q

What is a silent mutation?

A

A mutation that changes a single nucleotide, but does not change the amino acid created.

34
Q

What is a conservative mutation?

A

The new amino acid is of the same type as the original (chemical properties are similar), usually no effect on protein function.

35
Q

What is a nonconservative mutation?

A

Chemical properties of mutant amino acid are different from original amino acid, effects protein function.

36
Q

What is a frame-shift mutation?

A

Insertion or deletion of a nucleotide in the DNA sequence.

37
Q

What is an in-frame mutation?

A

Insertion or deletion of a trinucleotide set that does not change the reading frame of the codon.

38
Q

What are loss of function mutations?

A

Reduced/abolished gene activity, recessive inheritance.

39
Q

What is a null mutation?

A

Complete loss of function.

40
Q

What is a hypomorphic mutation?

A

Reduced gene function.

41
Q

What is haploinsufficiency?

A

When one gene copy is not sufficient for organism to survive.

42
Q

What is a gain of function mutation?

A

A mutation that confers new or enhanced activity on a protein, dominant.

43
Q

What is a hypermorphic mutation?

A

Generates more gene product or the same amount of a more efficient gene product.

44
Q

What are neomorphic mutations?

A

Generate gene product with new function or that is expressed at at the wrong time or place.

45
Q

What is a forward mutation?

A

Wild type to mutant form (A to a), more frequent.

46
Q

What is a reverse mutation?

A

Mutant to wild type (a to A).

47
Q

What is CRISPR/Cas9?

A

Bacteria protects bacterial genomes from invading DNAs.

48
Q

What is adaptation CRISPR?

A

Cas complex integrates small pieces of viral DNA into CRISPR array of host cell.

49
Q

What is expression CRISPR?

A

CRISPR array transcribed into long pre-crRNA and processed by Cas proteins. crRNA will partially base pair with tracrRNA and form effector complex with Cas.

50
Q

What is interference CRISPR?

A

Needs PAM; complementary base-pairing between crRNAs and viral DNA; Cas protein cleaves viral DNA.

51
Q

What do the effector complex and PAM do?

A

Unwind nearby DNA.

52
Q

What is the DNA endonuclease?

A

Cas9.

53
Q

What is the protospacer adjacent motif?

A

NGG sequence in DNA, not on sgRNA.

54
Q

What is nonhomologous end joining?

A

Error-prone DNA repair pathway for DSBs, creates random indels.

55
Q

What is homology-directed repair?

A

Repairs DSBs by replacing damaged DNA with another DNA molecule by homologous recombination.

56
Q

What is Duchenne muscular dystrophy?

A

X-linked recessive; usually in boys.

57
Q

What is a model organism?

A

Easy to raise, reproduce in lab, rapid life cycle, numerous offspring, inbred populations.

58
Q

What is convergent evolution?

A

Different organisms independently evolve similar traits.

59
Q

What is forward genetics?

A

Driven by phenotype to genotype, identify the gene mutation that causes the mutant phenotype, germline.

60
Q

How to do forward genetics?

A

Linkage mapping with positional cloning or DNA sequencing.

61
Q

What is reverse genetics?

A

Genotype to phenotype, disrupt the gene sequence to identify functions.

62
Q

How to do reverse genetics?

A

Knockouts, RNAi, CRISPR.

63
Q

What are genetic screens for forward genetics?

A

Gain or loss of function mutations, in vivo mutagenesis, loss of biochemical pathways (microbes).

64
Q

What is knockdown?

A

synthesized siRNAs silence gene expression, temporary effect.

siRNAs are complementary to mRNA of gene

65
Q

What is a disadvantage of knockdown?

A

Can’t explain which gene is underlying the phenotype.

66
Q

What is a knockout?

A

Removing/inactivating a gene from the gene sequence

67
Q

What is a conventional knockout?

A

Target gene is knocked out in all tissues at all times.

68
Q

What is a conditional knockout?

A

A gene that can be selectively deactivated in specific tissues when this happens.

69
Q

What are global knockout limitations?

A

Hard if gene is in multiple places, deleting a gene in one place can affect it in another, hard to determine cause of phenotype.

70
Q

What happens when both loxP are on the top strand in the same direction?

A

DNA between them is removed.

71
Q

What happens when one loxP is on the top and one on the bottom in opposite direction?

A

DNA between will be inverted.

72
Q

What are transgenes?

A

A gene from one organism is put into another at random locations.

73
Q

What is the goal of transgenes?

A

Gain info about function of gene products and identify regulatory sequences.

74
Q

What are transgene limitations?

A

Variability, random insertions, codon bias, post-translational mods.

75
Q

What is whole genome shotgun sequencing?

A

Randomly breaking up genome into small DNA fragments, sequence individually, then reassemble.

76
Q

What is annotation?

A

Identify the location of genes and functional sequence in genome.

gene 3200 test 5 (2 decks)

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