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Studying > Molecular Genetics | Final Exam > Flashcards

Molecular Genetics | Final Exam Flashcards

1
Q

What is Dideoxy (chain termination sequencing) useful for?

A

(AKA Sanger sequencing or cycle sequencing)

Useful for isolating ans sequencing small sections of the genome

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2
Q

How does sanger sequencing differ from Next gen?

A

Sanger is useful for specific sequences, Next gen is a whole genome sequecing technique

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3
Q

What are the general steps of Sanger sequencing?

A

DNA primer + fluorescently labeled ddNTPs, stops sequence at evey nucleotide

Analysis involves looking at the color of each size of sequenced piece of DNA and building final sequence

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4
Q

What are the three types of DNA polymerase used in DNA sequencing?

A

Klenow polymerase
Sequenase
Taq polymerase

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5
Q

What are the properties of Klenow polymerase?

A

Originated by E. coli
No 5’ to 3’ exonuclease activity
low processivity (~250bp)
5’ –> 3’ polymerase
3’ –> 5’ exonuclease (proofreading)

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6
Q

What are the properties of Sequenase?

A

Origin = T7 phage
Greater processivity than klenow (>250bp)

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7
Q

What are the properties of taq polymerase?

A

Origin = Thermus aquaticus
Required for cycle sequencing

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8
Q

How are nucleotides tracked in Chain Termination Sequencing?

A

Fluorescently labeled ddNTPS

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9
Q

What is the preferred method of DNA separation in Chain Termination Seq?

A

Capillary Electrophoresis

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10
Q

How does automated sequencing increase the rate of sequencing?

A

Hundreds of capillary tubes run simultaneously

Electrical current used to excite fluorophores, recorded by computer

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11
Q

Define adapters (NGS)

A

Contains a sequence complementary to oligos bound to flow cell, a unique identfiable sequence, and a sequence complementary to the primer.

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12
Q

Define Next gen Sequencing

A

A massively parallel method of sequencing entire genomes or smaller segments and genes

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13
Q

Define tagmentation

A

Tn5 transposase with adapters binds to the gene and breaks it apart.

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14
Q

Define barcode sequence

A

A sequence that is used to identify a target molecule

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15
Q

Define consensus sequence

A

The final sequence of the target gene, built from the combination of the fragmented genes

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16
Q

Define contig

A

A series of overlapping DNA sequences used to create the consensus sequence

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17
Q

Define read depth

A

Number of reads that cover an individual site

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18
Q

Define emulsion PCR

A

Beads of DNA are suspended in water, which is then put in oil to prevent contamination

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19
Q

Define third gen sequencing

A

Uses one strand of DNA.

Two types:
1. Nanopore
2. SMRT

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20
Q

What are two methods of fragmenting genomic DNA for NGS library prep

A

Tagmentation and Sonication

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21
Q

What are the basic steps of Illumina Sequencing?

A
  1. Break up the DNA
  2. Add adapter sequences
  3. PCR amplify
  4. Inclusion of fluorescently labeled dNTPs elongates the DNA
  5. Align the genome by the colors of the dNTPs
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22
Q

What are the basic steps of Ion Torrent Sequencing?

A

DNA isolation and fragmentation, Partition each fragment to a microbead, emulsion PCR, adding dNTPs one at a time

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23
Q

What are the basic steps for Nanopore sequencing?

A

Small pore allows one DNA strand through, each base creates a unique signal

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24
Q

What are the basic steps of SMRT Sequencing?

A

Fluorescent label is attached to a pyrophosphate

Light flash as pyrophosphate is discarded = analysis

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25
Q

What are two methods of creating a targeted sequencing library for NGS?

A

Biotinylated oligonucleotide probes

PCR

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26
Q

Tagmentation

A

TN5 transposase w/ adapters cleaves DNA and anneals adapaters to each end

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27
Q

Contig

A

The final sequence built from a bunch of smaller sequences

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28
Q

Read depth

A

Average coverage on each nucleotide during sequencing

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29
Q

Sonication

A

Ultrasonic disruption

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30
Q

Quinolone

A

inhibit DNA gyrase –> too much supercoiling –> Bacteriocidal

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31
Q

Trimethoprim

A

Blocks reduction of dihydrofolate to tetrahydrofolate –> prevents thymine synthesis

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32
Q

Sulfonamide

A

Inhibits conversion of dUMP to thymine through thymidylate synthetase competition with its cofactor tetrahydrofolate

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33
Q

DNA Pol III

A

Normal DNA polymerase
- Proofreading ability
- Mismatch repair mechanisms
- 3’ exonuclease

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34
Q

DNA Pol I

A

Fills in Okazaki fragment gaps
- 3’ –> 5’ exonuclease (proofreading)
- 5’ –> 3’ exonuclease activity

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35
Q

Ribonucleotide reductase

A

Converts dinucleotides to their deoxy version

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36
Q

Thymidylate synthetase

A

Converts dUMP to thymine using tetrahydrofolate as a cofactor

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37
Q

Where does the sigma sununit bind?

A

-10 and -35 region on a gene promoter

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38
Q

What are the major sections of RNA polymerase

A

Core enzyme (polymerase activity)

Sigma subunit: Promoter binding

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39
Q

Rifampicin

A

Inhibits RNA polymerase through binding to the DNA channel, which blocks elongation of the growing RNA

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40
Q

RNA Pol I

A

Transcribes large ribosomal RNAs

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41
Q

RNA Pol II

A

General Housekeeping

42
Q

RNA Pol III

A

Transcribes genes for 5S rRNA and tRNA

43
Q

Which protein adds 5’ cap to mRNA?

A

TFIIH

44
Q

What makes up the 5’ cap?

A

Guanine nucleotide in teh reverse confirmation (phosphate group binds to the 5’ end of the transcript)

45
Q

What composes the tail signal on mRNA?

A

AAUAAA + CA dinucleotide + GU Rich tract

46
Q

How is the Poly A tail added to mRNA

A

Tail signal recruits poly adenylation complex, adds tail after transcript is cut from DNA

47
Q

What recognizes a splice site to recruit the spliceosome?

A

snRNPs

48
Q

What are the 3 mechanisms of alternative splicing?

A
  1. Alternative promoter selection
  2. Alternative tail site selection
  3. Exon cassette
49
Q

Alternative promoter selection

A

Mechanism of alternative splicing

Different promoter is chosen = more/less gene being transcribed = different protein

50
Q

Alternative tail site selection

A

Mechanism of alternative splicing

Different tail signals cause different exons to be included in the mRNA

51
Q

Exon Cassette

A

Splicing out introns / exons results in different proteins

52
Q

Shine-Dalgarno sequence

A

Site at which mRNA binds the ribosome (prokayotes)

53
Q

Wobble rule

A

Variation in the third nucleotide of a codon results in different amino acids

54
Q

What are the major loops of tRNA

A

DHU loop
TψC loop
Variable loop
Anti-codon region

55
Q

What is the modified base found in the DHU loop of tRNA

A

dihydrouridine (D)

56
Q

What is the modified base found in the TψC loop of tRNA?

A

pseduouridine (ψ)

57
Q

What is the importance of the modified bases in tRNA?

A

Important for the 3D structure / folding of the protein

58
Q

What makes a bacterial ribosome?

A

30S and 50S subunit
Total: 70S

59
Q

What makes up a eukaryotic ribosome?

A

40S and 60S subunits
Total: 80S

60
Q

Major features of prokaryotic protein synthesis?

A

Polycistronic mRNA
Coupled transcription / translation
Termination through RF1/RF2

61
Q

Major features of eukaryotic protein synthesis

A

Monocistronic mRNA
Uncoupled transcription/translation
Termination through IF1/2/3

62
Q

What is a signal sequence?

A

Marks a protein for cellular export

63
Q

Major function of chaperone proteins?

A

Aid in protein folding

64
Q

Types of post-translational modifications

A

Methylation
Acetylation
Phosphorylation
Ubiquitination

65
Q

2D-PAGE

A
  1. Gel separated based on charge
  2. Treat with SDS
  3. Run horizontally to separate based on size
66
Q

His tagged proteins

A

Used to bind nickel resin in affinity chromatography

67
Q

Sigma factor

A

localizes RNA polymerase to a gene promoter

68
Q

anti-sigma factor

A

binds sigma factor / prevents DNA binding

69
Q

What is the role of RpoH in heat shock?

A

RpoH is a TF that results in expression of heat shock response proteins

70
Q

Describe the cellular state of bacteria at 37°C

A

Low levels of misfolded proteins –> DNAK/J bind RpoH and degrade it = no heat shock response

71
Q

Describe the cellular state at >42°C

A

High levels of misfolded proteins –> DNAJ/K bind protein –> RpoH is free to bind DNA and transcribe heat shock response proteins

72
Q

What triggers sporulation?

A

Stressful conditions

73
Q

How does sporulation affect cell division?

A

Mother cell dies, daughter cell enters a protective state until conditions improve

74
Q

What governs the sporulation process?

A

Sigma / anti-sigma factors traveling across the sporulation wall

75
Q

What kind of transcriptional regulation does araBAD represent?

A

Represses in the absense of arabinose

Acitvates when arabinose is present

76
Q

What kind of transcriptional regulation is the maltose operon?

A

Positive regulation (malT + maltose promote transcription)

77
Q

What kind of transcriptional regulation does the lactose operon represent?

A

Negative regulation (lacI)

78
Q

Describe two component regulatory systems

A
  1. Sensory kinase detects environmental changes and phoshphorylate another protein
  2. Signal cascade leads to secondary protein binding DNA and promoting transcription
79
Q

Quorum signaling at low levels

A

Autoinducer is expressed at low levels, cells act as individuals

80
Q

Quorum signaling at high density

A

Autoinducer expressed at high levels, enters the cell and binds DNA

Cells act as a community

81
Q

Nucleosome

A

Subunit of DNA wrapped around histone proteins

82
Q

Imprinting

A

Methylation that carries over to the next generation

83
Q

Heterochromatin

A

Tightly wound DNA –> Less transcription

84
Q

Euchromatin

A

Loosely wound DNA –> more trnascription

85
Q

Acetylation of histone

A

Promotes chromatin aggregation –> more transcription

86
Q

List the affects of the four major epigenetic modifications

A

Acetylation: Increases transcription

Methylation: Decreases transcription

Phosphorylation: Does both

Ubiquitination: Does both

87
Q

What does a transposon contain?

A

transposase gene surrounded by inverted repeats, gene of interest

88
Q

Describe conservative transposition

A

Transposon is cut out of the host by transposase and inserted into target DNA

89
Q

Describe Replicative transposition

A

Host DNA retains transposon, target DNA gets a copy through DNA crossover events

90
Q

What are the Ac/Ds elements in maize?

A

Ac: Autonomous transposable element that requires no input to move around

Ds: Requires an activator to transpose stuff, results in color change in corn kernels

91
Q

What is the function of CsrA

A

Prevents glycogen synthesis by promoting an unstable confirmation that leads to degradation of the mRNA

92
Q

how is adhE regulated at the protein level?

A

mRNA forms a confirmation that blocks the RBS, cleavage in this area reveals the RBS, allowing for translation

93
Q

How is ferritin controlled at the protein level?

A

IRE is a loop that binds IRP, which inhibits translation when iron is low. At high iron, the loop dissapears and allows for translation

94
Q

How does cPABP control translation of mRNA?

A

Activated by light, holds mRNA in a confirmation that allows for translation during the daytime (plants)

95
Q

How does antisense RNA regulate protein synthesis?

A

Through binding ssRNA, prevents translation

96
Q

Describe the bfr/anti-bfr system

A

In low iron: anti-bfr is transcribed –> prevents ferric uptake proteins

In high iron: no anti-bfr, bfr gene sigma factor is present and results in bacterioferritin protein being made

97
Q

How does methylation affect protein synthesis?

A

Increases the rate of export of mRNA, increases binding to the ribosome

98
Q

How does lux/luc act as a reporter?

A

Luciferase modifies luciferin structure, which releases light

99
Q

How does PhoA act as a reporter?

A

Cleaves phosphate groups, releasing a fluorophore (protein is alkaline phosphotase)

100
Q

Describe Gel retardation

A

Gene promoter is digested into fragments of known sizes, when a protein is bound, that segment moves slower through the gel

101
Q

Describe footprint analysis

A

When protein binds DNA, it blocks enzyme digestion, resulting in a missing section of the gel

102
Q

Dropseq

A

PolyT beads are used to isolate all cellular RNA, converted to cDNA and synthesized using NGS

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