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Pathophysiology > Molecular Diagnostics (exam 2) > Flashcards

Molecular Diagnostics (exam 2) Flashcards

1
Q

3 methods of amplification

A

target amplification systems
probe amplification systems
signal amplification

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2
Q

target amplification

A

copy directly from the target (RNA/DNA)

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3
Q

probe amplification systems

A

not copying off the organism, but something else initially bound to the target is being copied

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4
Q

signal amplification

A

can see if something is amplified or not; amount of signaling

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5
Q

Target amplification methods

A

PCR
TMA - transcription mediated
SDA - strand displacement

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6
Q

Types of PCR

A

PCR with specific probes
Nested PCR
Multiplex PCR

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7
Q

Nested PCR

A

use two sets of amplification primers, one internal to the other
start off wide, narrow in on a region

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8
Q

Multiplex PCR

A

two or more sets of primers specific for different targets
multiple PCR reactions at once

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9
Q

signal amplification example

A

bDNA - branched DNA probes

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10
Q

probe amplification example

A

LCR - ligase chain reaction

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11
Q

how many targeted sequences are produced?

A

10^8 - 10^9 copies

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12
Q

How did Kary Mullis invent PCR?

A

knew that template DNA could be exposed
knew that primers initiate DNA synthesis
Knew that cheap, commercial enzyme was available

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13
Q

Mullis wanted a way for PCR to

A

generate large amounts of DNA from a single copy

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14
Q

Steps of PCR

A
  1. denaturation
  2. annealing of primers
  3. extension of new strand
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15
Q

what is needed for a PCR reaction?

A

primers
thermostable polymerase
target nucleic acid (RNA/DNA)

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16
Q

primers may be

A

specific or random

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17
Q

what can dictate primer length?

A

annealing temperature and specificity

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18
Q

PCR primers

A

ss 18-30 b DNA fragments complementary to sequences flanking the region to be amplified

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19
Q

the larger the distance between the primers,

A

the larger the size of the PCR product

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20
Q

Tm

A

for short (14-20 bp) oligomers
= 4 (GC) + 2 (AT)

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21
Q

Most common thermostable polymerase used and the source it came from

A

Taq polymerase

from T. aquaticus

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22
Q

a reaction mix contains

A

all the components necessary for DNA synthesis

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23
Q

what is in a standard PCR reaction mix?

A

primer
nucleotides (dNTPs)
KCl
Tris buffer
MgCl2
polymerase
copies of template

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24
Q

Temperatures at each step of PCR

A

denaturation - 90-96, 20 seconds
annealing - 40 -68, 20 seconds
extension - 70-75, 30 seconds

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25
Q

for Primer design,

A

avoid inter-strand homologies
avoid intra-strand homologies
Tm forward primer = Tm reverse primer
avoid longer than GGGG
product size - 100bp-700bp
target specificity

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26
Q

for long primers in PCR,

A

94-60-72

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27
Q

for adequate primers in PCR,

A

94-50,72

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28
Q

for uncontrolled results of PCR,

A

94-37-65

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29
Q

the amplification program consists of a series of

A

20-50 PCR cycles

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30
Q

1st major advancement of PCR

A

thermostable polymerase

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31
Q

2nd major advancement of PCR

A

automation

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32
Q

why are thermostable polymerases used in PCR?

A

because they can withstand repeated high denaturation temperatures

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33
Q

3rd advancement in PCR

A

detection
gel electrophoresis –> micrometers –> microassays

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34
Q

microarrays can

A

detect thousands of samples

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35
Q

the most dangerous contamination is

A

PCR product from a previous reaction

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36
Q

what is a potential source of contamination

A

any molecule of DNA containing the intended target sequence

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37
Q

most common cause of contamination

A

carelessness and bad technique

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38
Q

ways to reduce contamination

A

separate pre and post PCR labs
dUTP and uracil-N-glycosylase
Psoralen and UV
10% bleach

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39
Q

dUTP and uracil-N-glycosylase

A

chews up DNA with uracil in it
degrades contaminants from previous PCR reactions

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40
Q

controls for PCR and what they control

A

blank - contamination
negative - specificity
positive - sensitivity

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41
Q

blank reaction

A

all reagents except DNA template

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42
Q

negative control reaction

A

all reagents and a DNA template lacking the target sequence

43
Q

positive control reaction

A

all reagents and a known target containing DNA template

44
Q

COBAS Amplicor Analyzer

A

samples are amplified and products detected automatically after the PCR reaction

45
Q

Real time/quantitative PCR

A

products are detected by fluorescence during the PCR reaction
allows for quantification of starting material

46
Q

4th advancement of PCR and example

A

combining amplification and detection
ex: COBAS amplicor

47
Q

5th advancement of PCR and example

A

combining everything!
ex: COBAS 6800/8800

48
Q

the length of the lag phase in qPCR is

A

inversely proportional to the amount of starting material

49
Q

difference between end point PCR and real time PCR

A

end point - simple +/- results
real time - complex results

50
Q

hybridization analysis

A

probe hybridization
only bind and fluoresce the intended PCR product

51
Q

HIV case study led to

A

a perfect match between PCR and immunodiagnostic product
PCR is much faster, more efficient

52
Q

Lyme disease case study led to

A

PCR being able to detect differences between types of infections (new vs recurrent)

53
Q

Sars CoV2 case study led to

A

PCR having great results in sensitivity and it is very specific so no false positives

54
Q

advantages of PCR

A

specific
simple and fast
can amplify low quantities
works on damaged DNA
sensitive
flexible

55
Q

limitations of PCR

A

contamination risk
primer complexities
detection methods
primer-binding site complexities

56
Q

transcription amplification method

A

isothermal RNA amplification modeled after retroviral replication

57
Q

transcription amplification method process

A

RNA is reverse transcribed into cDNA followed by RNA synthesis via RNA polymerase
primer contains the T7 RNA pol promoter sequence

58
Q

3 enzymes in TMA

A

reverse transcriptase
T7 RNA pol
RNAse H

59
Q

Hologic Analyzer

A

shuffles through RNA and DNA
TMA approach

60
Q

strand displacement amplification

A

isothermal
enzyme comes in, cuts and then pushes the strand out of the way

61
Q

ligase chain reaction

A

probe amplification
probes bind immediately adjacent to one another on a template
bound probes are ligates and become templates for the binding of more probes

62
Q

branched DNA detection

A

captures probe hybrid
2nd probe binds to first probe
hybridize bDNA amplifier
addition of alkaline phosphate molecules

63
Q

RFLP analysis

A

Restriction fragment length polymorphism

use of restriction enzymes to recognize specific DNA sequences and cut them

64
Q

RFLP means that there are

A

polymorphisms between people in the number of restriction sites

65
Q

issues with RFLP analysis

A

can not detect all mutations - it has to coincide with a RE cut site

66
Q

genetic testing examples

A

predictive testing
newborn screening
carrier testing

67
Q

predictive testing

A

carries a mutation that will cause or put them at a higher risk for disease later in life

68
Q

newborn screening

A

detects common disorders in newborns

69
Q

carrier testing

A

carries a mutation that could be passed on to offspring

70
Q

Thalassemia

A

blood related genetic disorder
involves errors in hemoglobin genes

71
Q

most common inherited single gene disorders

A

alpha and beta thalassemias

72
Q

CRISPR

A

DNA editing technique

73
Q

CRISPR gene therapy for sickle cell anemia

A
  1. harvest bone marrow stem cells
  2. Cas9 guides RNA
  3. CRISPR/Cas9 genome editing
  4. implant edited stem cells in patient
74
Q

hemophilia

A

partial/total lack of blood clotting factor
can be diagnosed in infants after 9 months

75
Q

hemophilia A vs hemophilia B

A

A - deficiency in factor 8
B - deficiency in factor 9

76
Q

Cystic fibrosis

A

affects respiratory, digestive and reproductive systems
thick mucous linings produced in the lungs

77
Q

CFTR gene contains

A

27 exons, 250 kb of DNA

78
Q

how many mutations in the CFTR gene result in CF disease?

A

over 200

79
Q

the most common mutation in CFTR gene causing CF

A

3 base pair deletion at codon 508
loss of phenylalanine residue blocks transport of CFTR protein

80
Q

what is used to diagnose cystic fibrosis?

A

dot blot

81
Q

dot blot

A

DNA from a patient is spotted out twice
one used with a probe complementary to the normal sequence
other with a probe complementary to a mutated sequence

82
Q

what if someone is a carrier for CF? how will their dot blot look?

A

faint dots in both normal and mutant probe

83
Q

Pulmozyme

A

therapeutic used for CF patients

84
Q

Tay-Sachs

A

autosomal recessive disorder fatal genetic disorder
mutation in the HEXA gene on chromosome 15

85
Q

is there a cure for Tay-Sachs? what has been done to decrease the number of children born with it?

A

no cure

carrier screening and genetic counseling

86
Q

characteristics of Tay Sachs at birth?

A

appear normal

87
Q

characteristics of Tay Sachs at 6 months?

A

development slows

88
Q

characteristics of Tay Sachs at 2 years?

A

seizures and fading mental functions

89
Q

characteristics of Tay Sachs at 3 years?

A

blindness, mental retardation, paralysis, and non responsiveness

90
Q

detection methods for tay-sachs

A

amniocentesis
chorionic villus sampling
blood samples to detect carriers

91
Q

Tay-Sachs disease is the inability to

A

synthesize an enzyme that prevents lipid build up in brain cells

92
Q

Tay-Sachs causes

A

the breakdown of the nervous system

93
Q

Parent help for Tay-Sachs

A

genetic counseling and support groups

94
Q

Fragile X is the

A

most common cause of inherited mental retardation

95
Q

Fragile X

A

loss of function mutations in the fragile X mental retardation 1, FMR1 gene
neurodegenerative, trinucleotide repeat disorder
repeats cause high methylation, blocks transcription

96
Q

Huntington Disease

A

autosomal dominant genetic disorder
neurodegenerative - loss of ability to walk, talk, etc.
over 36 CAG repeat in long polyglutamine tract

97
Q

is there a cure for Huntington disease?

A

no treatment or cure

98
Q

examples of polygenic genetic disorders

A

cancer
diabetes
heart disease
hypertension
obesity
infertility
asthma

99
Q

Mitochondrial disorders

A

heart failure, autism, Alzheimers, etc.
caused by mitochondrial genome and not nuclear genome

100
Q

Next-Gen sequencing

A

computer aligns sequence at the end, can get the whole genome
high powered sequencing

101
Q

Why are pharmacists interested in DNA sequencing?

A

personalized medicine

102
Q

personalized medicine

A

tailoring treatments to specific characteristics of each patient

103
Q

Herceptin (Trastuzumab)

A

humanized monoclonal antibody designed to target the HER2 receptor domain

104
Q

will Trastuzumab work for breast cancers that aren’t HER-2 positive tumors?

A

NO!

only for the specific mutation

Pathophysiology (15 decks)

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