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ABB HCLD Mol Diag (passed) > ABB HCLD Molecular Diagnostics > Flashcards

ABB HCLD Molecular Diagnostics Flashcards

1
Q

Molecular Diagnostics is a universal Technology affecting what areas?

A

Genetics, Infectious Disease, Heme Oncology, Identity Testing

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2
Q

What is an infectious disease impacted by molecular diagnostics?

A

TB and other hard to culture organisms. These could take weeks to culture.

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3
Q

What is a diploid genome?

A

Two sets of chromosomes

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4
Q

How many pairs of chromosomes?

A

23 pairs, 22 pairs autosomes, 2 sex chromosomes

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5
Q

How many bases make up the human Genome?

A

3 billion

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6
Q

How many genes in the human genome?

A

22,000

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7
Q

How much of the human genome codes for proteins?

A

10%

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8
Q

Heterochromain

A

Densely packed region of chromosomes, like centromeres, not transcribed

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9
Q

Euchromain

A

Less densely packed region of chromosomes, transcribed

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10
Q

Chromosomes are characterized based on the position of the centromere.

A

Telocentric = centromere is located at the proximal end or tip of the chromosome
Acrocentric = centromere is located at one end of the chromosome, resulting in a very short arm and a very log arm (named after grasshoppers).
Sub-metacentric = centromere is located near but not exactly at the center of the chromosome (majority of human chromosomes).
Metacentric = centromere is located exactly at the center of the chromosome, leading to 2 equal size arms (seen in primitive organisms, amphibians) (human chromosomes 1 and 3).

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11
Q

Describe the centromeres of chromosomes.

A

Centromeres are variable in size, humans and plants are megabases and budding yeast are only 125bp. Centromeres are rich in repetitive AT sequences and are essential for mitosis and meiosis. The chromatin structure is unique because histone H3 is replaced by centromere specific histone H3, CENP-A which may affect DNA binding ability.

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12
Q

Antiparallel structure

A

5’ - 3’ and 3’ - 5’

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13
Q

A NUCEOTIDE is made up of :

A

A 5-carbon sugar, a phosphate and a nitrogenous base

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14
Q

Pyrimidine

A

A 6-member ring, Cytosine, Thymine, Uracil

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15
Q

Purine

A

Fused 5 and 6-member rings, Adenine, and Guanine

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16
Q

Difference between deoxyribose and ribose

A

Deoxyribose is missing an hydroxyl group in lower right corner

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17
Q

Mitochondrial DNA, how many bases and how many genes?

A

16.5K bases, 37 genes

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18
Q

What are some mitochondrial genes?

A

rRNA, tRNA, cytochrome C and ATP synthase

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19
Q

What do the large and small subunits of ribosomes do during translation?

A

The small subunit (40S) decodes the genetic message. The large subunit (60S) catalyzes the peptide bond formation. Both subunits are composed of rRNA and other proteins.

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20
Q

What is a locus?

A

The location of the gene on the chromosome

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21
Q

What is an allele?

A

Version of the gene present at any locus

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22
Q

Polymorphism

A

Benign change in the genome, almost everyone has them

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23
Q

Mutation

A

Present in a low percentage of people, role in disease

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24
Q

How often do SNP’s occur in the genome?

A

Every 300bp or 10 million per genome

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25
Q

What is a synonymous polymorphism?

A

A silent variant, the substitution does not change the amino acid.

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26
Q

What is a non-synonymous polymorphism?

A

The substitution does change the amino acid.

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27
Q

What is a somatic mutation?

A

Mutation that is acquired in non-germline cells, not inheritable,

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28
Q

What is a germline mutation?

A

Mutations occur in germline cells, they are inheritable,

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29
Q

What are structural mutations?

A

Translocations, Rearrangements, gene amplifications

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30
Q

What are molecular mutations?

A

Insertions, deletions, point mutations, nucleotide repeats

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31
Q

What does an exonuclease do?

A

Exonucleases are enzymes that cleave DNA sequences in a polynucleotide chain from either the 5’ or 3’ end, one at a time. DNA polymerase I exonuclease activity plays a role in correcting polymerase errors. Also, in replication they work with RNA pol II to degrade RNA primes which are replaced with DNA nucleotides.

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32
Q

The venom of snakes and lizards contains which group of enzymes?

A

Exonucleases are present in their toxins leading to cleavage of DNA molecules which code for essential proteins.

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33
Q

What is aneuploidy?

A

Whole chromosome loss or gain

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34
Q

What is a duplication?

A

An area of gene sequence is repeated, can visualize with FISH

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35
Q

What does a chromosome microarray show you?

A

Virtual or digital karyotype, millions of probes on a chip highlight areas of the chromosome where there could be abnormalities. Allows you to look for gains or losses in the genome.

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36
Q

What is a Duplication?

A

An area of gene sequence is repeated, visualize with FISH.

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37
Q

What is a Translocation?

A

An area of one chromosome is transferred to another chromosome, like BCR-ABL. Visualize with FISH or real-time PCR or NGS.

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38
Q

What is a point mutation?

A

A single nucleotide substitution, insertion or deletion.

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39
Q

What is a transition mutation?

A

A purine is substituted for a purine. A pyrimidine is substituted for a pyrimidine.

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40
Q

What is a transversion mutation?

A

A purine is substituted for a pyrimidine. A pyrimidine is substituted for a purine.

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41
Q

What is a missense mutation?

A

A base substitution that alters the codon resulting in a different amino acid.

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42
Q

What is a nonsense mutation?

A

A base substitution that alters the codon resulting in a stop codon.

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43
Q

What is a silent mutation?

A

Alters the codon, but not the amino acid

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44
Q

What is a neutral mutation?

A

Alters the amino acid to a similar amino acid which does not disrupt the protein.

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45
Q

What is a frameshift mutation?

A

Insertion or deletion of one or more bases which alters the codon reading frame changes protein structure

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46
Q

Sickle cell anemia is caused by:

A

A single point mutation in the hemoglobin gene

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47
Q

What is a splice site mutation:

A

Mutation alters the exon intron splice site, leads to exon skipping or intron inclusion

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48
Q

What is the universal genetic code?

A

The different codons that form the amino acids.

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49
Q

What a loss of function mutation?

A

Leads to complete or partial loss of protein function. Frequently recessive.

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50
Q

What a gain of function mutation?

A

Causes an entirely new trait which can appear in inappropriate tissue or at inappropriate time in development. Frequently dominant.

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51
Q

Quantity of DNA from UV Spectrophotometry?

A

(A260-A320) * dilution factor * 50 ug/mL

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52
Q

Quantity of RNA from UV Spectrophotometry?

A

(A260-A320) * dilution factor * 40 ug/mL

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53
Q

Quality of DNA by UV Spectrophotometry?

A

(A260/A280) = purity
(A260-A320) / (A280-A320)
1.7-2.0 = good quality
<1.7 = too much protein

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54
Q

How to look at quality of DNA/RNA with respect to size?

A

Gel electrophoresis

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55
Q

Maxam - Gilbert sequencing

A

Used a toxic chemical and never really caught on

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56
Q

Sanger sequencing technique

A

Chain termination sequencing using dideoxy method

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57
Q

Difference between deoxy and dideoxy

A

Dideoxyribonucleic acid is missing a hydroxl group and more dNTP’s cant add on so chain terminates

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58
Q

Capillary Sequencing Technique

A

Separate bands in the gel capillary, excite fluorescent tag with Argon laser, Fluorescent light is shown through a prism to separated the colors.

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59
Q

How does Ion Torrent Sequencing work?

A

Tagged DNA fragments are mixed with oil droplets, inside each oil droplet there is a sphere with DNA sequences that match the adaptors. Fragments bind to sphere and are PCR amplified inside the oil droplet. Each sphere is spun down into the chip. Sequencer floods the chip with a certain dNTP and measures the pH.

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60
Q

How does Illumina work?

A

Seguencing by synthesis. Adapters cause DNA fragments to bind to the surface of the flow cell. Adapter at other end of the fragment bends over and binds forming a bridge. Synthesis occurs and amplifies the fragments, makes many copies and you visualize fluorescence.

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61
Q

What is the clinical exome?

A

About 4000 genes associated with human disease

62
Q

What is the whole exome?

A

Where you sequence all 20,000 genes

63
Q

Whole genomes

A

All introns and exons and everything

64
Q

GC rich regions are difficult to sequence, how can you improve sequencing of these regions?

A

Adjust magnesium concentration or use additives such as DMSO, glycerol, or BSA. Use slow-down PCR which requires the addition of 7-deaza-2’deoxyguanosine, a GTP analog.

65
Q

What does a endonuclease do?

A

Endonucleases belong to a group of enzymes which cleave the phosphodiester bond present within a polynucleotide chain. These enzymes can be sequence specific (restriction endonucleases) or not. They are critical for DNA repair and the cleaved sites form single-stranded ends called “sticky ends” which are hybridized together by DNA ligase.

66
Q

What are the steps in a Southern Blot?

A
  1. Extract DNA
  2. Restriction Enzyme digest
  3. Gel electrophoresis
  4. Denature DNA and transfer the gel to a membrane
  5. Denature and hybridize a tagged probe to the membrane
  6. Detect tagged probe
67
Q

Limitations of Southern Blot?

A
  1. Need high molecular weight DNA
  2. Takes a long time
  3. Labor intensive
68
Q

How do you label your Southern probe

A

Nick translation (radioactive nucleotides) (Chemiluminescent), random primers

69
Q

What types of genetic abnormalities will a Southern detect?

A

Large insertions and deletions.

70
Q

What is probe Stringency?

A

Conditions of hybridization that control the specificity of binding between the probe and the target. Increase temperature or ionic strength to increase stringency.

71
Q

What is LCR?

A

Ligase chain reaction

72
Q

What is NASBA?

A

Nucleic Acid Sequence based amplification

73
Q

What is TMA?

A

Transcription mediated amplification

74
Q

What is SDA?

A

Strand displacement Amplification

75
Q

What is bDNA?

A

Branched DNA, signal amplification

76
Q

What is HCA?

A

Hybrid capture, signal amplification

77
Q

What does Magnesium do in a PCR reaction?

A

Stabilizes primer template interactions.
Stabilizes replication complex polymerase with the primer template.
Can increase non-specific annealing.

78
Q

Does Taq polymerase have proofreading?

A

No, it does not have proofreading 3’-5’ exonuclease activity, but it does have very low mis-incorporation rate.

79
Q

What do you have to consider when selecting primers?

A

Keep them around 50% GC, Avoid G and C at 3’ end (decreases primer dimers and non-specific annealing), Check primer end for complementarity.

80
Q

What is the formula for calculating how many copies of amplicon are present in a PCR reaction?

A

Amplicons = Starting copy number * 2^(# cycles-2)

81
Q

Advantages of real-time PCR over PCR and gel electrophoresis.

A
  1. Rapid target ID
  2. Eliminates post-PCR processing
  3. Highly specific with probes
  4. Allows for multiplexing
  5. Allows for quantification, amount of signal is proportional to the amount of DNA
82
Q

Delta Ct formula for fold differences

A

Delta Ct = (Ct of A) - (Ct of B)
Fold difference = 2^ delta Ct

83
Q

Digital Droplet PCR

A

Single molecule amplification in droplets of oil, count positive droplets to get absolute quantification of target sequence

84
Q

MPN stands for

A

Myeloproliferative Neoplasm

85
Q

BCR-ABL positive indicates

A

CML

86
Q

BCR-ABL negative indicates

A

PV, ET, MF

87
Q

Gleevec

A

Small molecule drug that binds to the BCR-ABL fusion gene and shuts off the overactive kinase activity.

88
Q

Why is BCR-ABL RT-PCR better at monitoring these patients than FISH or Karyotype

A

Its more sensitive and quantitative.

89
Q

Polycythemia Vera (PV) is

A

A myeloproliferative disease, MPD characterized by overproduction of RBC’s

90
Q

Essential Thrombocythemia (ET) and Chronic Idiopathic Myelofibrosis (CIMF)

A

Are related MPD’s

91
Q

The JAK2 mutation is present in:

A

The majority of PV and about half of ET and CIMF.

92
Q

The role of JAK2 is

A

a tyrosine kinase involved in signal transduction for multiple hematopoietic growth factors

93
Q

Myeloid Diseases

A

Are clonal diseases of hematopoietic stem cells or progenitor cells, derived from a single progenitor cell with a genetic abnormality

94
Q

Myeloid diseases are caused by:

A

Genetic or epigenetic alterations that perturb key processes such as self-renewal, proliferation, and differentiation

95
Q

What genes are associated with AML?

A

FLT3, NPM1, CEBPA
Prognostic: IDH1, IDH2, TET2, RUNX1

96
Q

What genes are associated with APL (acute promyelocytic leukemia)?

A

APL is characterized by a unique fusion mRNA produced in leukemic cells from a t(15; 17) translocation involving the RARA gene (retinoic acid receptor a) and the PML gene (promyelocytic leukemia gene).

97
Q

What genes are associated with MDS?

A

KRAS, NRAS, IDH’s, TET2, RUNX1

98
Q

PGXm, pharmacokinetic

A

M = metabolism
What the body does to the drug. Absorption, distribution, excretion.

99
Q

Mutations affecting PGXm

A

Polymorphisms, not typically disease causing mutations

100
Q

PGXt, targeted therapeutic

A

T = whether you have the target
Presence or absence of the target, resistance, lack of response

101
Q

Mutations affecting PGXt

A

Mutations in disease causing genes, driver and passenger genes. Germline or somatic

102
Q

Using polymorphisms affecting drug metabolism:

A

You can predict the spectrum of drug metabolism.
1. Ultra rapid metabolizers will have to take 100x more to get any affect.
2. Poor metabolizers will probably overdose

103
Q

Irinotecan for colon cancer (PGXm)

A

Toxicity to UGT1A1 deficiency. Normally the drug is inactivated by UGT1A1.

104
Q

Promoter of UGT1A1

A

Has TA repeats in the promoter in varying numbers TA7 causes reduced activity of UGT1A1. Detect by capillary electrophoresis.

105
Q

Tamoxifen for breast cancer (PGXm)

A

Toxicity to CYP2D6 deficiency

106
Q

Examples of PGXt drugs:

A

Gleevec for BCR-ABL
Herceptin for HER2 receptor in amplified breast cancer
Cetuximab for KRAS mutations
Tarceva for EGFR mutations

107
Q

Mutations can be detected in any sample type

A

Germline mutations

108
Q

Autosomal dominant inheritance

A
  1. Affected individuals have at least one affected parent.
  2. Each child of an affected parent has a 50% chance of inheriting the mutation
  3. Unaffected people have unaffected children
  4. males and females are affected equally
  5. Inheritance pattern is vertical
109
Q

Retinoblastoma gene mutations:

A

Can be hereditary or non-hereditary caused by mutations in the RB1 tumor suppressor gene. Autosomal dominant inheritance or one mutation and another mutation is acquired in the retinal cells in early childhood. Greatly increases the risk of developing cancer of the eye.

110
Q

Mutations in these genes are associated with hereditary colon cancer

A

Lynch Syndrome is caused by mutations in MLH1, MSH2, MSH6, PMS2, and EPCAM.

111
Q

Mutations in RAS genes

A

RAS genes are involved in cytoplasmic cell signaling. Mutations in these genes make them constitutively active leading to uncontrolled cell growth. About one third of cancers have mutations in RAS genes, including colon, lung and pancreatic cancers.

112
Q

MLH1, MSH2, MSH6

A

These are mismatch repair genes and mutations in these genes lead to high MSI and HNPCC. Methylation of the promoters of these genes leads to transcriptional silencing.

113
Q

Mutations associated with lung cancer

A

EGFR and KRAS mutations render them constitutively active, signaling cell proliferation

114
Q

EGFR in NSCLC’s

A

Mutations render this tyrosine kinase constitutively active leading to rapid cell growth. Exons 18-21 may be involved. Most therapies for these cancers are tyrosine kinase inhibitors, but there is one monoclonal antibody.

115
Q

Autosomal recessive inheritance

A
  1. Affected members of the family are usually siblings of the proband.
  2. Carrier parents have a 25% chance of having an affected child
  3. Affected people may have unaffected parents (skips generations)
  4. Inheritance pattern is horizontal
116
Q

What gene is mutated in Cystic Fibrosis?

A

The CFTR gene. The resulting protein normally transports chloride (a component of salt) to the cell surface. Mutations in the CFTR gene render the protein dysfunctional and without enough salt on the cell surface, sufficient water is not attracted and mucus becomes thick and sticky (thus trapping germs).

117
Q

X-Linked Dominant

A
  1. Female offspring of affected males will be affected
  2. No male offspring of affected males will be affected
  3. An affected female has a 50% chance of having an affected child.
  4. All affected individuals have an affected parent
118
Q

X-Linked Recessive

A
  1. Affected individuals are primarily males
  2. All female offspring of affected males will be carriers
  3. Male offspring of affected males will be unaffected
  4. Inheritance pattern is not vertical of horizontal
119
Q

Muscular Dystrophy

A

Duchenne MD is caused by mutations in the dystrophin gene and are inherited in an X-linked recessive pattern. Myotonic MD is caused by expansion of repetitive sequences in the DMPK or CNBP genes (normal = 40, MD = 4000 repeats).

120
Q

Heterogeneity

A

When a disease is caused by multiple mutations in the same gene or different genes

121
Q

Anticipation

A

A progressive increase in severity in future generations

122
Q

Imprinting

A

Different expression of a mutant gene depending on if it was inherited by the mother or the father

123
Q

Angelman Syndrome vs Prader-Willi Syndrome

A
124
Q

Retinoblastoma gene mutations:

A

Can be hereditary or non-hereditary caused by mutations in the RB1 tumor suppressor gene. Autosomal dominant inheritance or one mutation and another mutation is acquired in the retinal cells in early childhood. Greatly increases the risk of developing cancer of the eye.

125
Q

Alpha-1-trypsin deficiency (ATT)

A

Causes genetic COPD and early onset emphysema
One of the most common genetic disorders affecting Caucasians and associated with childhood liver disorder. Only 6% of severe ATT is diagnosed

126
Q

Normal A1AT

A

Keeps neutrophil elastase in check

127
Q

A1AT Deficiency

A

Neutrophil elastase becomes a burden and attacks lung tissue

128
Q

Detect A1AT mutations by

A

allelic Discrimination

129
Q

Mutations in these genes are associated with hereditary colon cancer

A

Lynch Syndrome is caused by mutations in MLH1, MSH2, MSH6, PMS2, and EPCAM.

130
Q

Fragile X Syndrome

A

Most common form of inherited mental retardation and autism.
First example of trinucleotide repeat (CGG) expansion mutation
Maternally inherited with incomplete penetrance

131
Q

Symptoms of Fragile X Syndrome

A

Mental retardation, delayed speech, hyperactivity, anxiety, attention deficit, long narrow face, big ears, large testes

132
Q

Incidence of Fragile X

A

1:4000 males, 1:6000 Females

133
Q

FMR-1 gene encodes for

A

Fragile X mental retardation protein (FXMRP), role in protein synthesis in neurons, and dendritic development

134
Q

CGG repeat involved in upstream methylation

A

Normal people have 5-45 copies
Fragile X individuals have >200 copies
Premutation or carrier state have 55-200

135
Q

How to detect fragile X?

A

Capillary electrophoresis, and PCR

136
Q

Another Trinucleotide repeat disorder

A

Huntington Disease

137
Q

Affymetrix chromosomal microarray

A

2.6 million probes, 750,000 SNPs

138
Q

Digitized karyotype

A

The software looks at probe binding and flags areas of the chromosome where there might be issues.

139
Q

LCSH

A

Long Continuous Stretches of Homozygosity
Indicates parents were related.

140
Q

WES

A

Whole Exome Sequencing

141
Q

What is Parvovirus B19?

A
  1. Small single stranded DNA virus.
  2. Non-enveloped icosahedral particles
  3. Replicates in actively dividing committed erythroid precursor cells in bone marrow
  4. Destroys RBC’s
  5. Transmitted via respiratory route
142
Q

Parvovirus B19 symptoms

A
  1. Fever chills headache myalgia, muscle soreness (First 7 days)
  2. Rash, arthralgia, joint stiffness (day 21-28)
  3. Anemia
143
Q

Utility of PCR testing for parvovirus?

A
  1. Diagnose parvovirus infection as the causative agent in fetal hydrops (fluid retention)
  2. Diagnose parvo in immunosuppressed patients
  3. Diagnose parvo prior to seroconversion
  4. Determine ideology of acute and chronic anemias
144
Q

q PCR

A

Quantitative Real-Time PCR

145
Q

Fold difference in amount of target

A

Delta Ct method, 2^ delta Ct - fold difference

146
Q

Delta delta Ct

A

delta Ct = Ct(target A-treated) - Ct(Ref B-treated)
delta Ct = Ct(target A-control - Ct(Ref B-control)
delta delta Ct= delta Ct (treated) - delta Ct (control)
= Normalized target gene expression =

147
Q

Absolute Quantification

A

Using a standard curve to calculate how much of a target is present

148
Q

Characteristics of HIV

A

Icosahedral (20 sided), enveloped virus in the retrovirus family (RNA transcribed to DNA).
Two viral strands of RNA in core surrounded by outer protein coat.
Outer envelope has a lipid matrix embedded with glycoproteins which bind to target cell.

149
Q

Three main structural genes in HIV

A
  1. Gag gene - Group Specific Antigen
  2. Env - Envelope
  3. Pol - Polymerase
150
Q

HIV life cycle

A

Virus enters host cell, RNA is reverse transcribed into DNA which incorporates into host DNA. As host cell replicates, it starts churning out viral particles.

151
Q

HIV drugs that target life cycle steps

A

Nucleoside Analogues and Non-nucleosides target the reverse transcriptase.
Protease Inhibitors stop virus particle building.

152
Q

Units of HIV Viral Load

A

log (copies/mL)

ABB HCLD Mol Diag (passed) (2 decks)

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