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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
What are two methods of creating a targeted sequencing library for NGS?
Biotinylated oligonucleotide probes PCR
26
Tagmentation
TN5 transposase w/ adapters cleaves DNA and anneals adapaters to each end
27
Contig
The final sequence built from a bunch of smaller sequences
28
Read depth
Average coverage on each nucleotide during sequencing
29
Sonication
Ultrasonic disruption
30
Quinolone
inhibit DNA gyrase --> too much supercoiling --> Bacteriocidal
31
Trimethoprim
Blocks reduction of dihydrofolate to tetrahydrofolate --> prevents thymine synthesis
32
Sulfonamide
Inhibits conversion of dUMP to thymine through thymidylate synthetase competition with its cofactor tetrahydrofolate
33
DNA Pol III
Normal DNA polymerase - Proofreading ability - Mismatch repair mechanisms - 3' exonuclease
34
DNA Pol I
Fills in Okazaki fragment gaps - 3' --> 5' exonuclease (proofreading) - 5' --> 3' exonuclease activity
35
Ribonucleotide reductase
Converts dinucleotides to their deoxy version
36
Thymidylate synthetase
Converts dUMP to thymine using tetrahydrofolate as a cofactor
37
Where does the sigma sununit bind?
-10 and -35 region on a gene promoter
38
What are the major sections of RNA polymerase
Core enzyme (polymerase activity) Sigma subunit: Promoter binding
39
Rifampicin
Inhibits RNA polymerase through binding to the DNA channel, which blocks elongation of the growing RNA
40
RNA Pol I
Transcribes large ribosomal RNAs
41
RNA Pol II
General Housekeeping
42
RNA Pol III
Transcribes genes for 5S rRNA and tRNA
43
Which protein adds 5' cap to mRNA?
TFIIH
44
What makes up the 5' cap?
Guanine nucleotide in teh reverse confirmation (phosphate group binds to the 5' end of the transcript)
45
What composes the tail signal on mRNA?
AAUAAA + CA dinucleotide + GU Rich tract
46
How is the Poly A tail added to mRNA
Tail signal recruits poly adenylation complex, adds tail after transcript is cut from DNA
47
What recognizes a splice site to recruit the spliceosome?
snRNPs
48
What are the 3 mechanisms of alternative splicing?
1. Alternative promoter selection 2. Alternative tail site selection 3. Exon cassette
49
Alternative promoter selection
Mechanism of alternative splicing Different promoter is chosen = more/less gene being transcribed = different protein
50
Alternative tail site selection
Mechanism of alternative splicing Different tail signals cause different exons to be included in the mRNA
51
Exon Cassette
Splicing out introns / exons results in different proteins
52
Shine-Dalgarno sequence
Site at which mRNA binds the ribosome (prokayotes)
53
Wobble rule
Variation in the third nucleotide of a codon results in different amino acids
54
What are the major loops of tRNA
DHU loop TψC loop Variable loop Anti-codon region
55
What is the modified base found in the DHU loop of tRNA
dihydrouridine (D)
56
What is the modified base found in the TψC loop of tRNA?
pseduouridine (ψ)
57
What is the importance of the modified bases in tRNA?
Important for the 3D structure / folding of the protein
58
What makes a bacterial ribosome?
30S and 50S subunit Total: 70S
59
What makes up a eukaryotic ribosome?
40S and 60S subunits Total: 80S
60
Major features of prokaryotic protein synthesis?
Polycistronic mRNA Coupled transcription / translation Termination through RF1/RF2
61
Major features of eukaryotic protein synthesis
Monocistronic mRNA Uncoupled transcription/translation Termination through IF1/2/3
62
What is a signal sequence?
Marks a protein for cellular export
63
Major function of chaperone proteins?
Aid in protein folding
64
Types of post-translational modifications
Methylation Acetylation Phosphorylation Ubiquitination
65
2D-PAGE
1. Gel separated based on charge 2. Treat with SDS 3. Run horizontally to separate based on size
66
His tagged proteins
Used to bind nickel resin in affinity chromatography
67
Sigma factor
localizes RNA polymerase to a gene promoter
68
anti-sigma factor
binds sigma factor / prevents DNA binding
69
What is the role of RpoH in heat shock?
RpoH is a TF that results in expression of heat shock response proteins
70
Describe the cellular state of bacteria at 37°C
Low levels of misfolded proteins --> DNAK/J bind RpoH and degrade it = no heat shock response
71
Describe the cellular state at >42°C
High levels of misfolded proteins --> DNAJ/K bind protein --> RpoH is free to bind DNA and transcribe heat shock response proteins
72
What triggers sporulation?
Stressful conditions
73
How does sporulation affect cell division?
Mother cell dies, daughter cell enters a protective state until conditions improve
74
What governs the sporulation process?
Sigma / anti-sigma factors traveling across the sporulation wall
75
What kind of transcriptional regulation does araBAD represent?
Represses in the absense of arabinose Acitvates when arabinose is present
76
What kind of transcriptional regulation is the maltose operon?
Positive regulation (malT + maltose promote transcription)
77
What kind of transcriptional regulation does the lactose operon represent?
Negative regulation (lacI)
78
Describe two component regulatory systems
1. Sensory kinase detects environmental changes and phoshphorylate another protein 2. Signal cascade leads to secondary protein binding DNA and promoting transcription
79
Quorum signaling at low levels
Autoinducer is expressed at low levels, cells act as individuals
80
Quorum signaling at high density
Autoinducer expressed at high levels, enters the cell and binds DNA Cells act as a community
81
Nucleosome
Subunit of DNA wrapped around histone proteins
82
Imprinting
Methylation that carries over to the next generation
83
Heterochromatin
Tightly wound DNA --> Less transcription
84
Euchromatin
Loosely wound DNA --> more trnascription
85
Acetylation of histone
Promotes chromatin aggregation --> more transcription
86
List the affects of the four major epigenetic modifications
Acetylation: Increases transcription Methylation: Decreases transcription Phosphorylation: Does both Ubiquitination: Does both
87
What does a transposon contain?
transposase gene surrounded by inverted repeats, gene of interest
88
Describe conservative transposition
Transposon is cut out of the host by transposase and inserted into target DNA
89
Describe Replicative transposition
Host DNA retains transposon, target DNA gets a copy through DNA crossover events
90
What are the Ac/Ds elements in maize?
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
What is the function of CsrA
Prevents glycogen synthesis by promoting an unstable confirmation that leads to degradation of the mRNA
92
how is adhE regulated at the protein level?
mRNA forms a confirmation that blocks the RBS, cleavage in this area reveals the RBS, allowing for translation
93
How is ferritin controlled at the protein level?
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
How does cPABP control translation of mRNA?
Activated by light, holds mRNA in a confirmation that allows for translation during the daytime (plants)
95
How does antisense RNA regulate protein synthesis?
Through binding ssRNA, prevents translation
96
Describe the bfr/anti-bfr system
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
How does methylation affect protein synthesis?
Increases the rate of export of mRNA, increases binding to the ribosome
98
How does lux/luc act as a reporter?
Luciferase modifies luciferin structure, which releases light
99
How does PhoA act as a reporter?
Cleaves phosphate groups, releasing a fluorophore (protein is alkaline phosphotase)
100
Describe Gel retardation
Gene promoter is digested into fragments of known sizes, when a protein is bound, that segment moves slower through the gel
101
Describe footprint analysis
When protein binds DNA, it blocks enzyme digestion, resulting in a missing section of the gel
102
Dropseq
PolyT beads are used to isolate all cellular RNA, converted to cDNA and synthesized using NGS

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