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CMB2000 > STRAND A > Flashcards

STRAND A Flashcards

1
Q

no. H bonds between CG

A

3

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2
Q

no. H bonds between AT

A

2

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3
Q

primase function

A

synthesizes RNA primers

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4
Q

PCR benefits

A

sensitive
robust
cheap
rapid
specific

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5
Q

PCR tube contents

A

template (ds DNA)
2 primers
polymerase
dNTP’s
Magnesium
buffer (8-9.5)

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6
Q

3 regions of Taq polymerase

A

synthesis
proof-reading
primer removal

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7
Q

Taq polymerase characteristics

A

heat-stable
3 regions
accurate DNA copying

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8
Q

implications of too long of a primer in PCR

A

slow hybridization

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9
Q

implications of too short of a primer in PCR

A

not specific

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10
Q

primer size range in PCR

A

18-24 bp

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11
Q

primer characteristics in PCR

A

oligonucleotide/ ssDNA
start/ finish with G/C pairs
Tm = 50-60 degrees C (5 degrees between pairs)
3’ comp to template

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12
Q

Magnesium role in PCR

A

non-protein co-factor allowing catalysis for enzymatic activity of DNA polymerase

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13
Q

potassium ions role in PCR

A

promote annealing

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14
Q

PCR process 3 stages

A

denaturation
annealing
elongation

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15
Q

PCR 1st cycle

A

1 strand synthesis
(boil, anneal and extend w polymerase/ dNTPS)

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16
Q

PCR 2nd cycle

A

synthesis of 2 strands
(boiling and annealing different primer comp to new/original DNA strand, polymerase extends)

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17
Q

final PCR cycles

A

simultaneous synthesis of both strands
30 repeats

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18
Q

PCR product detection

A

molecular weight markers
PCR products
primers
template
(agarose gel w intercalating dye)

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19
Q

uses of PCR

A

DNA manipulation/ quantification/ amplification
genetic disease diagnosis
pathogen detection
ancient DNA
gene function study
knock-out genes
biotechnology

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20
Q

reverse transcriptase PCR

A
  1. RNA converted to cDNA via reverse transcriptase
  2. amplification via PCR
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21
Q

RNA sources in reverse transcriptase PCR

A

gene expression
RNA virus

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22
Q

reverse transcriptase PCR ingredients

A

reverse transcriptase
dNTPs
buffer
primer
RNA template

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23
Q

end-point vs qPCR
age?
price?
precision

A

qPCR newer (1996 vs 1983), more expensive, more precise

24
Q

end-point vs qPCR quantification

A

end-point semi-quantitative, measuring densitometry
qPCR amount proportionate to template amount
end-point measurement at end (plateau) and qPCR continuous (exponential phase) measurement

25
Q

end-point PCR uses

A

cloning
genotyping
sequencing

26
Q

qPCR uses

A

gene expression
quantification
microarray verification
quality control assay validation
SNP genotyping
copy number variation
viral quantification
siRNA/ RNA experiments

27
Q

THERMOS LIGHTCYCLER

A

thermal cycler incorporating fluorometer for detection and quantification of PCR products

28
Q

master mix

A

pre-measured solution at optimal concentration for each reagent

29
Q

2 fluorescence reagents

A

SYBR green
TAQman

30
Q

SYBR green

A

binds to groove of dsDNA

31
Q

TAQman

A

probes with fluorescence reporter and quencher
probe hybridizes as FUP/RUP anneal and extend
DNA pol cleaves probe and fluorescence increases

32
Q

3 phases of PCR logarithmic standard curve

A

exponential
linear
plateau

33
Q

Ct

A

cycle threshold
no. cycles required for the PCR to reach the threshold/ exceed background level.

34
Q

Ct level indication

A

lower Ct value, higher amount of cDNA (starting material)

35
Q

reference gene

A

control to normalize gene expression levels, constant gene expression, unaffected by experimental factors

36
Q

house-keeping gene

A

normalizes mRNA levels between samples to for sensitive comparison
^reliability and reproducibility of experimental results

37
Q

PCR standard curve

A

semi-log regression line plot of Ct value vs log of nucleic acid input

38
Q

reference gene examples for PCR

A

Beta actin
GAPDH
Albumin
18S rRNA
TATA sequence binding protein

39
Q

3 examples of clinical applications of PCR

A

Genotyping patient
genotyping pathogen
phenotyping disease

40
Q

PCR patient genotyping components

A

genetic trait diagnosis
carrier detection
tissue matching (HLA typing)
predicting response to drugs

41
Q

DNA sources for patient phenotyping

A

blood, hair, buccal smear, amniotic fluid cells

42
Q

2 PCR based techniques for genotyping

A

PCR-RFLP (Restriction Fragment polymorphism)
ARMS-PCR (Amplification Refractory Mutation System)

43
Q

PCR-RFLP process

A
  1. amplify substrate to 2 strands of dsDNA
  2. add the restriction enzyme
  3. analysis with electrophoresis
44
Q

clinical example of PCR-RFLP

A

Diagnosis of Sorsby’s Fundus dystrophy
degenerative eye disease leading to blindness
autosomal dominant
TIMP3 mutation (tissue inhibitor of metalloproteinase 3) introduces premature stop codon

45
Q

PCR RFLP positives

A

cheap
easy design
microindel/SNP application
simple resources
commonly used techniques

46
Q

PCR RFLP negatives

A

only possible with known restriction site
some RE expensive
requires single nucleotide polymorphism
time-consuming
not suitable for high-throughput

47
Q

clinical example of ARMS-PCR

A

diagnosis of cystic fibrosis
mutation in CFTR gene leading to Cl- transport imbalance across PM
F508 common mutation

47
Q

ARMS-PCR

A

use of allele specific primers to detect alelic variants

48
Q

RFLP vs ARMS
primers?
dependents?
alternatives?

A

RFLP has locus-specific primers vs ARMS allele specific primers
RFLP relies on presence/ absence of restriction site vs ARMS relies on PCR stringency
ARMS has tetra primer alternative w non-allele specific primers in addition

49
Q

DNA sources for pathogen phenotyping

A

blood
sputum
urine
faeces
skin swab
tissue biopsy

50
Q

pathogen phenotyping influence

A

patient management and infection control measures

51
Q

microscopy disadvantages to PCR

A

less sensitive (high levels required)
difficult to distinguish strain/ species

52
Q

culture disadvantages to PCR`

A

not all organisms can be cultured (PCR doesn’t require culture)
takes weeks (rather than hours)
less specific

53
Q

patient antibody response disadvantages to PCR

A

May not illicit strong response whereas PCR not dependent on immune response

54
Q

pathogen phenotyping clinical example

A

TB smear test w acid fast stain > dependent on bacterial load/ quality/ expertise
culture for mycobacteria then molecular testing

55
Q

disease phenotyping technique

A

RT-PCR

56
Q

clinical example of disease phenotyping

A

HIV viral load measurement with RT-PCR

CMB2000 (3 decks)

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