Genetics Flashcards
1
Q
gene
A
a DNA segment with a nucleotide sequence encoding an RNA product that is either directly functional or encodes a protein
2
Q
locus
A
location of a gene or a particular DNA sequence (e.g., promoter) on a chromosome
3
Q
allele
A
one of the variant forms a gene can have in a population (from a particular locus)
4
Q
wild-type allele
A
the allele that encodes for the most common phenotype in a population
5
Q
mutant allele
A
any allele that does not code for the most common phenotype in a population
6
Q
multiple alleles
A
the occurrence of more than two different alleles in a population (e.g., the ABO blood group system)
7
Q
allele frequency
A
the prevalence of a particular allele at a genetic locus within a population
E.g. If 10 individuals of in a population of 250 individuals (total of 500 gene copies) are homozygotes and 30 are heterozygotes for a certain mutant allele, then the total number of mutant copies is (10 x 2) + (30 x 1) = 50. Thus the allele frequency of the mutant allele will be 50/500 = 0.01 = 1%
8
Q
genetic polymorphism
A
the presence of two or more variant forms of an allele for a gene that can occur among different individuals or populations
the most common type involves variation at a single-nucleotide polymorphism (SNP), but can be much larger, involving longer stretches of DNA
9
Q
chromosome
A
a structure found in the nucleus of eukaryotic cells that contains part or all of the genetic information for a given organism
comprised of nucleic acids and associated proteins (e.g., nucleosomes)
each human cell contains 23 pairs of homologous chromosomes (corresponding in structure and genetic information, i.e., 23 chromosomes are inherited from each parent)
10
Q
allosome
A
AKA sex chromosome; a type of chromosome that carries the genes that determine chromosomal sex
human cells contain one pair of allosomes: XX in female individuals and XY in male individuals
11
Q
autosome
A
any chromosome of a cell genome that is not an allosome (sex chromosome)
human cells contain 22 pairs of homologous autosomes
12
Q
ploidy
A
the number of chromosome sets present in a cell
13
Q
haploid cell
A
contains one single unpaired set of chromosomes (n = 23)
14
Q
diploid cell
A
carries a complete set of paired chromosomes (2n = 46)
15
Q
chromatid
A
one of the two identical strands of a replicated chromosome
16
Q
sister chromatids
A
two identical chromatids joined at the centromere (i.e., the duplicated chromosome)
17
Q
centromere
A
a condensed region of chromosomes where sister chromatids join and mediates attachment of the chromosome to the meiotic or mitotic spindle
divides the chromatids into a short p arm and a long q arm
18
Q
metacentric
A
due to the position of the centromere, the p and q arms of chromosomes are of approximately identical length
19
Q
submetacentric
A
due to the position of the centromere, the p arm of the chromosome is short and the q arm of the chromosome is long
20
Q
acrocentric
A
due to the position of the centromere, the p arm of the chromosome is much shorter than the q arm the chromosome
21
Q
kinetochore
A
a protein complex found at the centromere that allows for the attachment of mitotic spindle microtubules during mitosis
22
Q
telomere
A
repetitive, noncoding DNA sequence at the ends of each chromosome, which prevents the loss of coding DNA sequences during DNA replication
23
Q
telomere shortening
A
occurs after each cell division; can reduce a cell’s life span
24
Q
mutations
A
alterations in a cell’s genome
may have endogenous causes (e.g., errors in DNA replication, cell division, and/or DNA repair mechanisms) or exogenous ones (e.g., a variety of physical, chemical, and biological agents)
25
germline mutation
AKA gametic mutation; a mutation of germline cells that can be passed on to offspring
26
somatic mutation
AKA acquired mutation; a mutation of somatic cells that typically affects only one allele of a gene
since it does not occur in a germline cell, cannot be passed down to offspring
common mechanism for carcinogenesis
27
mosaicism
the presence of two or more populations of cells within an organism, each with a different genetic composition
28
chromosomal mosaicism
the presence of cell populations with different karyotypes in one organism
E.g. sex chromosome mosaicism is frequently seen in Turner syndrome (i.e., one population of XO cells and one population of XX cells)
29
gonadal mosaicism
the selective presence of a mutation in individual germ cells; caused by a mutation in the DNA of a primordial germ cell during mitosis
Clinical application: Suspect this type of mosaicism if no blood relatives of the affected individual have the condition
30
somatic mosaicism
the selective presence of a mutation in individual somatic cells; caused by a mutation during mitosis after fertilization
usually, multiple tissues or organs are affected
E.g. McCune-Albright syndrome
31
chimerism
the presence of two genetically distinct cell lines that arise from two different zygotes that fused into one single embryo
[two cell lines sharing an embryo, like multiple animals sharing the body of a chimera]
32
chromosomal instability
a chromosomal state characterized by increased susceptibility to mutations; caused by mutations of DNA repair genes
results in multiple chromosomal translations, inversions, and deletions among daughter cells
E.g. Fanconi anemia, ataxia-telangiectasia
33
loss of heterozygosity (LoH)
loss of a normal allele of a gene with the exclusive expression of the abnormal allele
the occurrence of LoH in tumor suppressor genes leads to malignant transformation of the cell.
E.g. Lynch syndrome
34
two-hit hypothesis
states that two mutations (i.e., “hits”) must occur in the cellular DNA of tumor suppressor genes to induce oncogenesis (but does not apply to oncogenes)
first hit: One copy of a tumor suppressor gene is inactivated by mutation or epigenetic changes, and the cell becomes heterozygous but the second allele is functional and produces a normal tumor suppressor protein.
second hit: The second copy of a tumor suppressor gene is inactivated by the mutation, and LoH occurs (the cell becomes homozygous). No tumor suppressor protein is produced leading to increased risk of oncogenesis
E.g. retinoblastoma, Li-Fraumeni syndrome, Lynch syndrome, familial adenomatous polyposis
35
del
deletion
36
dup
duplication
37
46,XX, dup(q3)
duplication of the long arm of chromosome 3 in a female individual
38
46,XY, del(p5)
deletion of the short arm of chromosome 5 in a male individual
E.g. cri-du-chat syndrome
39
inv
inversion
40
46,XY, inv(3)(p23q27)
pericentric inversion of the chromosome 3 segment with breakpoints at position 23 on the short arm and 27 on the long arm in a male individual
41
t
translocation
42
46,XY, t(14;18)(q32;q21)
translocation between position 32 on chromosome 14 and position 21 on chromosome 18 in a male individual
e.g. follicular lymphoma
43
rob
Robertsonian translocation (a chromosomal translocation that involves the fusion of the long arms of two acrocentric chromosomes at the centromere with resulting loss of the short arms of the involved chromosomes)
44
46,XX, rob(14;21)
chromosomal translocation with the fusion of the long arms of the acrocentric chromosomes 14 and 21 in a female individual. The short arms of the two chromosomes involved are lost
45
chromosomal aberrations
mutations affecting large segments of DNA
can be detected by FISH (flourescence in situ) and not karyograms
46
numerical chromosomal aberrations
the presence of an abnormal number of copies of a single chromosome, which is usually caused by the failure of homologous chromosomes to separate during mitosis or meiosis (nondisjunction)
detected through a karyogram
47
aneuploidy
an abnormal number of chromosomes or parts of a chromosome within the cell
[an = not; eu = good; ploidy relates to chromosome number]
48
hypoploidy
the presence of fewer chromosomes within a cell compared to the expected number
49
monosomy
presence of a single copy of a chromosome
usually results in embryonic death due to the high probability of recessive trait expression of the respective chromosome (exception: Turner syndrome, which is characterized by a 45,XO genotype)
50
hyperploidy
the presence of extra chromosomes within a cell
51
trisomy
the presence of a triplicate instead of a duplicate set of chromosomes
E.g. trisomy 21 (Down syndrome), trisomy 13 (Patau syndrome), trisomy 18 (Edwards syndrome)
52
polysomy
the presence of ≥ 3 copies of a chromosome in a cell
E.g. trisomy, tetrasomy, pentasomy
53
structural chromosomal aberrations
an alteration of a chromosome structure with an identical number of chromosomes
(ALL translocations are classified as structural chromosomal aberrations)
54
deletion (chromosomal)
a loss of a chromosome segment
E.g.
cri-du-chat syndrome, which is characterized by 46,XX, del(5)
myelodysplastic syndrome, which is characterized by 5q deletion
55
duplication
duplication of a chromosome segment
56
inversion
chromosomal rearrangement involving end-to-end reversal of a chromosomal segment
E.g. 46,XY, inv(3)(p23q27)
57
chromosomal translocation
relocation of one chromosome segment onto another (non-homologous) chromosome
58
balanced translocation
a type of translocation in which no genetic material is lost or duplicated, thus expressing a normal phenotype
59
unbalanced translocation
a type of translocation in which genetic material is lost or duplicated, thus expressing an abnormal phenotype
can result in chromosomal imbalance (e.g. Patau syndrome), multiple malformations, stillbirth, and repeated miscarriages
60
Robertsonian translocation
a chromosomal translocation that involves the fusion of the long arms of two acrocentric chromosomes at the centromere with resulting loss of the short arms of the involved chromosomes
one of the most frequent translocations, and may be balanced or unbalanced
61
balanced Robertsonian translocation
the translocation between the long arms of the chromosomes occurs and the karyogram shows a total number of 45 chromosomes but results in a normal phenotype (pt is a carrier)
E.g. translocation of 21q to 14q with the elimination of the p arms
affected individuals inherit a balanced translocation from their phenotypically normal parent
pregnancies with balanced translocations have an increased risk of miscarriage, and children of carriers of balanced translocations are at higher risk of acquiring unbalanced translocation
62
leptotene
stage of prophase I (meiosis) during which chromosomes condense and become visible, and then the chromosomes attach to the nuclear envelope
63
unbalanced Robertsonian translocation
clinical features of trisomy 21 caused by inheritance of a translocation chromosome and a normal chromosome
although there are only 46 chromosomes present, three copies of genetic material from chromosome 21 exist
results in the karyotypes 46,XX,+21,t(14;21) and 46,XY,+21,t(14;21)
64
reciprocal translocation
a translocation between nonhomologous chromosomes
E.g. Philadelphia translocation
[you ask a favor (reciprocation) from strangers (nonhomologous)]
65
uniparental disomy
a chromosomal abnormality in which offspring receive two copies of one chromosome from one parent and no copies from the other parent; cannot be detected via karyotyping because the number of chromosomes is normal and there is no loss of genetic material
may be caused by errors in meiosis I (i.e., heterodisomy), meiosis II (i.e., isodisomy), or trisomic rescue
usually results in normal phenotype and euploidy in affected individuals and should be suspected if an individual presents with an autosomal recessive condition but only one parent is a carrier
E.g. Angelman syndrome (5% of cases); Prader-Willi syndrome (25% of cases)
66
isodisomy
when an individual has two identical homologous chromosomes inherited from one parent
either occurs in meiosis II d/t nondisjunction, or as a postzygotic chromosomal duplication that leads to duplication of one chromosome of one pair and subsequent loss of the other partent
["I"sod"I"somy: meiosis "II" error]
67
heterodisomy
when an individual has two different homologous chromosomes inherited from one parent
error in meiosis I d/t nondisjunction
Heterod"I"somy: meiosis "I" error
68
trisomy rescue
when a cell containing three copies of a chromosome from nondisjunction loses one of those chromosomes, resulting in two chromosomes
if both of the remaining chromosomes are from the same parent, this results in uniparental disomy
69
12–345 (mnemonic)
the chromosomes most frequently involved in Robertsonian translocations are chromosomes 2"1", 2"2", 1"3", 1"4", and 1"5" (21, 22, 13, 14, 15)
70
genetic transition
the replacement of one purine with another purine (e.g., G to A), or the replacement of a pyrimidine with another pyrimidine (e.g., T to C)
71
genetic transversion
the replacement of a purine with a pyrimidine (e.g., A to C, A to T, G to C, G to T) and vice versa
72
trinucleotide repeat expansion
increased repetition of base triplets that leads to faulty protein synthesis or folding; characterized by genetic anticipation
E.g. Friedreich ataxia, fragile X-syndrome, Huntington dz, myotonic dystophy
[Friedrich gave the fragile hunter my tonic]
73
Huntington disease mode of inheritance
autosomal dominant
74
fragile X syndrome mode of inheritance
X-linked dominant
75
Friedreich ataxia mode of inheritance
autosomal recessive
76
myotonic dystrophy mode of inheritance
autosomal dominant
77
Huntington disease affected gene
HTT
78
Huntington disease chromosome
4
79
Huntington disease trinucleotide repeat and typical features
CAG
features: chorea, akinesia, cognitive decline, behavioral change
[a CAG trinucleotide repeat leads to Chorea, Akinesia, and Grotesque behavior]
80
fragile X syndrome affected gene
FMR1
81
fragile X syndrome chromosome
X
82
fragile X syndrome trinucleotide repeat and typical features
CGG
features: large protruding chin, large genitalia (testes), hypermobile joints, mitral valve prolapse
[a CGG trinucleotide repeat leads to an X-tra large Chin and Giant Genitalia.]
83
Friedreich ataxia affected gene
FXN
[Friedreich ataXia Nucleotides]
84
Friedreich ataxia chromosome
9
85
Friedreich ataxia trinucleotide repeat and typical features
GAA
features: ataxic gait, dysarthria, kyphoscoliosis, hypertrophic cardiomyopathy
[a GAA trinucleotide expansion leads to an ataxic GAAit and slurred words like GAA]
86
myotonic dystrophy affected gene
DMPK
[Dystrophic Musculature Produces Kyphoscoliosis]
87
myotonic dystrophy chromosome
19
88
myotonic dystrophy trinucleotide repeat and typical features
CTG
features: cataracts, premature hair loss in men, myotonia, arrhythmia, gonadal atrophy (men), ovarian insufficiency (women)
[a CTG trinucleotide repeat leads to Cataracts, Thinning hair (premature hair loss), and Gonadal atrophy]
89
in-frame deletion or insertion
deletion or insertion of three, six, nine, or more base pairs (always in triplets) without a shift in the reading frame, but with deletion or insertion of one, two, three, or more amino acids in the protein during translation
90
gain-of-function mutation
a mutation that leads to either the expression of the larger amount of the gene product or increased function of the expressed gene product
[make more or work better]
91
pathogenicity severity ranking of mutations
silent < missense < nonsense < frame shift
92
silent mutation
a point mutation that forms a triplet that codes for the same amino acid; often d/t a base change in the third position of codon (TRNA wobble)
93
missense mutation
a point mutation resulting in the formation of a triplet that codes for another amino acid
called conservative if the new amino acid is similar to the chemical structure of the original amino acid
E.g. sickle cell dz (glutamate -> valine)
94
nonsense mutation
nucleotide substitution that produces an early stop codon (UAG, UAA, UGA); usually results in a non-functional protein
(Stop the nonsense!)
95
frameshift mutation
deletion or insertion of a number of nucleotides not divisible by 3, results in misreading of all nucleotides downstream
protein may be shorter or longer, and its function may be disrupted or altered.
E.g. Duchesne muscular dystrophy, Tay-Sachs disease
96
splice mutation
an alteration (especially point mutations) in the nucleotide sequence required for splicing (e.g., the exon-intron border or at the junction)
results in defective mRNA (e.g., due to a retained intron) → shortened proteins that are either defective or exert an altered function
E.g. Gaucher disease, Marfan syndrome, dementia, epilepsy, some types of β-thessalemia
97
loss-of-function mutations
a mutation resulting in the expression of the gene product with decreased or absent function
98
null mutation
When a lost-of-function mutation occurs, and the degree of function is entirely lost
99
dominant-negative mutation
a gene mutation that produces a nonfunctional protein that exerts a dominant effect and impairs the function of the normal protein encoded by the wild-type allele in heterozygous individuals
E.g. mutant, nonfunctional p53 binds DNA and prevents the attachment of the functional p53 protein
100
epigenetics
the study of how an individual's surrounding environment and behaviors influence gene expression and regulation, which is determined by chemical modifications of DNA bases (e.g., methylation) and histone proteins (e.g., various covalent modifications), which are carried out by specialized enzymes
epigenetic modifications influence the activation or deactivation of genes through reversible alterations to the chromatin structure but do not affect the DNA sequence or structure of DNA molecules
101
DNA methylation
the linkage of methyl groups to specific DNA cytosine bases with the subsequent formation of 5-methylcytosine
facilitated by DNA methyltransferases, and the resulting DNA segment has an unchanged genetic code but differs in epigenetic markers (e.g., methylation at transcriptional start sites) that alter DNA expression (inhibits transcriptional of the methylated gene)
102
DNA methyltransferases
a conserved enzyme family of cytosine methylases critical for epigenetic regulation that transfer a methyl group to DNA
103
process of DNA methylation
a newly synthesized DNA strand is methylated after DNA replication (using the matrix strand as a template)
in vertebrates, methylation most commonly occurs at CpG islands, a region of DNA enriched for repeating segments of a cytosine nucleotide that is followed by a guanine nucleotide, linked by a phosphodiester bond
methylation within the CpG islands will usually lead to repression of gene transcription
104
CpG islands
a region of DNA enriched for repeating segments of a cytosine nucleotide that is followed by a guanine nucleotide, linked by a phosphodiester bond
CpG islands are approx. 1000 base pairs in length, and many gene promoter regions are found within CpG islands
methylation within the CpG islands will usually lead to repression of gene transcription
105
inheritance of DNA methylation
specific methylation of CpG island transcription start sites can be inherited during somatic cell division and may be responsible for particular genomic processes (e.g., genomic imprinting)
106
Epigenetic regulation mechanisms mnemonic
```
Acetylation Activates (DNA)
Methylation Mutes (DNA)
```
AA, MM
107
histone modification
the covalent bonding and chemical modification of histone proteins with various molecular moieties that alter the dynamic structure of chromatin and influence genomic regulation
results in the modification of the gene expression
108
types of histone modifications
deamination
ubiquitination
sumoylation
ADP ribosylation
phosphorylation
acetylation -> increased DNA transcription
deacetylation -> decreased DNA transcription
[Acetylation accelerates, deacetylation decelerates]
methylation - increased or decreased gene transcription
other modifications include histone tail clipping, citrullination, and serotonylation
109
inheritance of histone modification
the histone modification pattern is passed on to daughter cells during cell division
110
regulatory RNA
a diverse class of RNA molecules that play a role in the regulation of chromatin structure and gene expression
these RNA molecules are typically noncoding and do not produce translated protein products
gene regulation occurs via RNA-interference (RNAi) pathways
111
RNA-interference (RNAi) pathways
molecular pathways that use PIWI and Argonaute proteins to influence histone and DNA modifications with subsequent transcriptional inhibition
[Pee Wee Herman and the Argonauts forged a PATHWAY through hist-ory and the DNA of Greece mythology]
112
types of regulatory RNAs
long noncoding RNAs (lncRNA)
small interfering RNAs (siRNA)
micro RNAs (miRNA)
PIWI-interacting RNA (piRNA)
113
unfortunate histone modifications examples
carcinogenesis: dysregulated histone deacetylases (HDACs) can decrease the transcription of cell cycle inhibitors (e.g., p21 in colorectal cancer) or cell differentiation factors (e.g., Runx1 in leukemia)
aging
114
examples of regulatory RNA effects
X chromosome inactivation
genomic imprinting
transposon repression and silencing
115
X inactivation definition
also called lyonization; inactivation of one of the X chromosomes in individuals with two or more X chromosomes (e.g., individuals with Klinefelter syndrome)
the selection of which X chromosome is inactivated occurs randomly, and prevents a toxic double-dose of X chromosome gene products
occurs early during embryogenesis with the affected X chromosome remaining inactive for the lifetime of the cell
116
X inactivation proposed mechanisms
DNA methylation
Histone posttranslational modifications
RNAi via lncRNAs such as X-inactive specific transcript RNA (Xist RNA [seXist RNA]), a functional RNA product that coats the affected X chromosome and serves as the initiating and, hence, most important event in the inactivation process
117
Barr body
contains the inactivated X chromosome packaged as heterochromatin which is transcriptionally inactive; located on the periphery of the nucleus
although most genes on the Barr body are inactive, some genes escape lyonization (X inactivation) and remain active
118
manifestation of X-linked disorders in female individuals
female individuals will only express alleles or genes located on the active X-chromosome, thus the extent to which heterozygous female carriers of X-linked recessive disorders express phenotypic characteristics of the disease depends on the genetic inactivation pattern of the mutant versus the normal X-chromosome
leads to phenotypic variation among heterozygous female carriers of X-linked disorders (e.g., one female carrier of an X-linked disorder may be completely asymptomatic, while another has severe manifestations of the involved disease).
in female individuals who are homozygous for an X-linked recessive disorder, it does not matter which chromosome is inactivated
119
genomic imprinting definition
a mechanism of gene regulation in which one allele of a gene is silenced and imprinted while the other allele is expressed depending on which parent it was inherited from (i.e., parent-of-origin effect)
must uphold the four principles of epigenetic imprinting
120
four principles of epigenetic imprinting
1. The product of gene transcription must be affected.
2. The imprinting must be heritable in a somatic lineage.
3. The process of genomic imprinting is initiated during gametogenesis.
4. The imprint must be eliminated in the germline for sex-specific reprogramming to occur in the next generation of gametes
[effective, heritable (in somatic), has genesis in gametogenesis, and is eventually eliminated (in germline)]
121
genomic imprinting mechanism
DNA methylation results, depending on the gene, in epigenetic silencing of either maternal or paternal chromosomes (e.g., if the paternal gene is silenced, only the maternal gene is expressed)
this then results in differentially methylated regions (DMRs)
122
Angelman syndrome symptoms
an example of a genomic imprinting disorder
causes delayed development, problems with speech and balance, intellectual disability, and, sometimes, seizures. People with Angelman syndrome often smile and laugh frequently, and have happy, excitable personalities
[ANGEL: Usually angels fly so they can't walk (Ataxia), but these angels lack wings, so they flap their hands to fly; Angels speak in tongues (Speech impairment), and angels don't cry (excitable/happy mood)
MANS: M: Microcephaly, A: Attention span short, N: Neurodevelopmental disorder, S: Seizures]
123
differentially methylated regions (DMRs)
the essential imprinting control regions, often exist within gene promoters, that are responsible for allelic expression
124
pedigree chart
depicts a group's genetic history in a family tree
Circles - Females
Squares - Males
125
zygoten
stage of prophase I (meiosis) during which synapsis or syndesis (the pairing of homologous chromosomes) occurs and the formation of the synaptonemal complex also occurs
[zyg = syn]
126
synapsis/syndesis
the pairing of homologous chromosomes during prophase I (zygotene specifically)]
[syn - same, desis - to bind, tie, fasten; bind together the same chromosomes]
127
pachytene
stage of prophase I (meiosis) during which crossing over occurs; the chromatids crossover (form chiasmata) and exchange genetic material, resulting in genetic recombination of homologous chromosomes
(the chromatids re-pach their genetic material with one another]
128
diplotene
stage of prophase I (meiosis) during which the synaptonemal complex disappears, then homologous chromosomes begin to separate (undoes synapsis/syndesis)) but the chromosomes remain held together by the chiasmata (crossover site)
129
diakinesis
stage of prophase I (meiosis) during which the nuclear membrane disintegrates and the spindle apparatus begins to form
[movement (kinesis) between (dia) the end of prophase and beginning of metaphase]
130
chiasmata
the crossover sites of chromatids during prophase I (meiosis)
131
Angelman syndrome main causes
normally, only the maternal copy of the UBE3A [you (U) be (B) an ethereal (E3) angel (A) ma!] gene is active in the brain, but most cases of Angelman syndrome occur when part of the maternal copy is missing or damaged
in a few cases, Angelman syndrome is caused when two paternal copies of the gene are inherited, instead of one from each parent
132
Prader-Willi syndrome symptoms
an example of a genomic imprinting disorder
characterized by behavior problems, intellectual disability, and short stature; hormonal symptoms include delayed puberty and wanting to eat constantly because they never feel full (hyperphagia) leading to obesity
[Weight Increased, Learning disabilities, Low muscle tone, Infertility (Due to hypogonadism), Short stature;WILLIS]
133
Prader-Willi syndrome main cause
usually caused by deletion of a part of chromosome 15 passed down by the father
[Pappas Region Absent or Deleted ERroneously; PRADER]
134
preanalytical phase
encompasses the selection of relevant diagnostic tests and the collection and transport of samples
specificity and sensitivity are important factors that should be considered when selecting a diagnostic test
135
commonly analyzed biofluids
blood
urine
cerebrospinal fluid
fluid collection from punctures (e.g., ascites, pleural effusion)
136
interference factors
affect sample quality
can be errors during sample collection (e.g., prolonged venous stasis during blood collection -> falsely elevated levels of K+ and Ca+)
can be errors during sample transport, such as:
exposure to strong light (e.g., leading to degradation of bilirubin -> in falsely low levels)
shaking of blood samples -> hemolysis
137
qualitative methods during the analytical phase
the result is either positive or negative
E.g. pregnancy test, rapid antigen detection test, urine dipstick
138
analytical phase
comprises sample processing and the generation of results
139
quantitative methods
the concentration of a substance is determined
E.g. immunochemistry (e.g., ELISA for the detection of PSA), photometry (e.g., indirect determination of glucose concentration), and microscopy (e.g., manual blood cell count with differential)
140
most important steps of the post-analytical phase
1. Ensuring the correct allocation of results to patient data
2. Plausibility check
3. Conveyance of results
4. Medical interpretation based on the combination of test results, patient history, and physical examination findings
141
blotting definition
a technique used to detect DNA, RNA, and proteins that involves transferring the DNA, RNA, or proteins onto a membrane, and then the sample of interest is visualized using marked detector molecules (e.g., radiolabeled DNA or chemiluminescent detector RNA)
142
most common blotting techniques employed in laboratory medicine
southern blot
western blot
northern blot
143
the similar process all blotting techniques follow
1. DNA, RNA, or protein molecules contained in the sample are separated by gel electrophoresis and/or electric charge, depending on their size (small molecules travel faster than bigger molecules).
2. The separated molecules are transferred from the gel onto a membrane.
3. The DNA, RNA, or protein molecule of interest is detected by using labeled, highly specific oligonucleotide probes, antibodies, or x-rays
144
sample types of each blot method
northern - RNA
southern - DNA
western - proteins
[SNoW flakes DRoP; Southern blot, Northern blot, and Western blot are used on DNA, RNA, and Proteins respectively]
145
northern blot principle of detection
annealing of radiolabeled or chemiluminescent detector RNA or DNA
146
assessment of gene expression by measuring mRNA levels (e.g., expression of the FMR1 gene in different tissues in fragile X syndrome)
northern blot use
147
southern blot detection
annealing of radiolabeled or chemiluminescent detector RNA or DNA
148
detection of specific genes and nucleotide sequences, such as restriction fragment length polymorphisms (RFLPs) or minisatellite DNA and microsatellite DNA (called “variable number of tandem repeats,” VNTRs)
southern blot use
149
western blot detection
direct or indirect detection using monoclonal antibodies (immunoglobulins that bind to a single, specific antigen)
150
western blot use
measuring the amount of antigens and antibodies, confirmation of the presence of a protein in a sample, and rough estimation of its amount
151
northern blot procedure
the RNA sample is cleaved by enzymes and separated by gel electrophoresis (commonly on agarose gels), then the separated and cleaved RNA is transferred (blotted) to a membrane
the membrane is incubated with labeled probes of RNA or DNA, and labeled probes recognize and anneal to the complementary strand if it is present on the membrane
152
northern blot result
double-stranded RNA
one unlabeled strand (cleaved RNA sample)
one labeled strand that can be visualized using special techniques
153
the two techniques for visualizing the one labeled strand produced with northern blot
when radiolabeled detector DNA/RNA is used, an x-ray film is placed against a western blot. This film develops when exposed to the label, creating dark regions that correspond to the target protein band.
in case of chemiluminescent DNA/RNA, a CCD camera that captures a digital image is used
154
southern blot procedure
the DNA sample is cleaved by restriction enzymes and separated by gel electrophoresis, then the separated and cleaved DNA is transferred (blotted) to a filter membrane
the membrane is exposed to (radio)labeled oligonucleotides: DNA probes recognize and anneal to the complementary strand if it is present on the membrane
155
southern blot results
double-stranded DNA
one unlabeled strand (cleaved DNA sample), and one labeled strand that can be visualized when the membrane is exposed to x-ray film
156
western blot procedure
the protein sample is separated by gel electrophoresis, and the separated proteins are then transferred (blotted) to a membrane
the protein of interest is detected by a specific antibody that can be labeled directly or detected by a secondary, radiolabeled antibody
157
southwestern blot sample
DNA-binding proteins
158
southwestern blot principle of detection
target proteins binding to radiolabeled double-stranded DNA probes
159
southwestern blot procedure
proteins are separated by gel electrophoresis and then blotted onto a nitrocellulose membrane (similar to western blot)
target proteins bind the radiolabeled double-stranded DNA probes (similar to southern blot)
160
study of DNA-binding proteins, such as transcription factors (e.g. c-Fos, c-Jun)
southwestern blot uses
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enzyme-linked immunosorbent assay (ELISA)
an enzyme immunoassay that employs enzyme-labeled immunoreactants and an immunoadsorbent to determine the presence and concentration of certain proteins (e.g., tumor markers, viral proteins, drugs, antibodies) in serum
based on the highly specific immunologic interaction between an antibody and its antigen (i.e., the protein of interest) (but the specificity of ELISA is lower compared to western blot)
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two main types and the indirect type of ELISA and their functions
direct ELISA: tests for the antigen directly
indirect ELISA: tests for the antibody, which indicates the presence of an antigen (indirectly)
sandwich ELISA: an indirect ELISA that employs two antibodies that bind to different epitopes on the antigen of interest
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ELISA basic steps
1. The antigen is fixed on a microtiter plate and bound by an enzyme-coupled antibody
2. The enzyme catalyzes a reaction when incubated with its substrate which is chromogenic, chemifluorescent, or chemiluminescent
3. The intensity of the signal is directly proportional to the amount of captured antigen
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direct ELISA steps
1. The patient's sample which supposedly contains the protein of interest (i.e., the antigen) is added to a well of microtiter plates with a buffered solution
2. The specific antibody-enzyme conjugate is added to the solution
3. A substrate for the enzyme is added
4. Spectrometry is used to detect the generated chromophore
Higher concentration of antibodies binding to the antigen → stronger signal
Lower concentration of antibodies binding to the antigen → weaker signal
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direct ELISA function
tests for the antigen directly
166
indirect ELISA function
tests for the antibody, which indicates the presence of an antigen (indirectly)
167
sandwich ELISA function
an indirect ELISA that employs two antibodies that bind to different epitopes on the antigen of interest
168
indirect ELISA steps
1. The patient's sample which supposedly contains the protein of interest (i.e., the antigen) is added to a well of microtiter plates with a buffered solution
2. The primary antibody (name is different from direct ELISA) conjugate is added to the solution
3. A substrate for the enzyme is added
4. The primary antibody is detected by a secondary, labeled antibody
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sandwich ELISA steps
1. A surface plate is coated with capture antibodies (not the patient's antibodies)
2. The sample is added to the coated plate where the captured antibodies bind the antigen of interest
3. Specific (labeled) antibodies for the antigen are added; if the antigen is present, the antibody binds to the antigen
4. A substrate for the enzyme is added (color, fluorescent, or electrochemical changes are due to the reaction between substrates and enzymes)
5. Spectrometry, fluorescence, or electrochemical studies are performed to assess for the amount of antigens present
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Screening for HIV antibodies (high sensitivity, low specificity)
ELISA use
Testing for West Nile virus antibodies
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Detection of the following organisms:
o Mycobacterium tuberculosis
o Rotavirus (in feces)
o Hepatitis B virus
o E. coli enterotoxin (in feces)
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Detection of PSA
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polymerase chain reaction (PCR) overview
a technique that allows amplification of specific chromosome segments by producing more than a billion copies; this makes testing of very small sequences of DNA that could not otherwise be studied possible
172
common sequences of DNA studied by PCR
mutations
microsatellite instability sequences
short tandem repeats (STRs)
173
PCR procedure overview
PCR usually consists of 25–50 cycles, which are divided into three phases:
denaturation
hybridization
elongation and amplification
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denaturation PCR step
the sample is heated at a target temperature of 90–98°C (194–208°F) for 20–30 seconds as high temperatures break the hydrogen bonds between complementary base pairs of the double-stranded DNA and produce two single DNA strands.
175
hybridization PCR step
the sample is cooled at a target temperature of 50–65°C (122–149°F) for 20–40 seconds, and then the following is added to the sample:
Complementary DNA primers that are necessary for annealing
Enzymes (for DNA synthesis): heat-stable DNA polymerase (Taq DNA polymerase)
Deoxyribonucleotides (dNTPs)
primers bind the 3′ ends of the DNA sequence that needs to be amplified
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elongation and amplification PCR step
the sample is heated at a target temperature of 70–80°C (158–176°F); 72°C (162°F) is most commonly used for DNA polymerase
DNA polymerase uses dNTPs to elongate the primers, thereby replicating the sequence of the sample DNA strands
The process is repeated until it yields an amplification to 106 –1010 copies of the original DNA fragment (approximately 25–50 cycles)
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detection of HIV (particularly when ELISA and western blot are inconclusive)
PCR use
178
reverse transcription polymerase chain reaction (RT-PCR) procedure
1. A sample mRNA is converted to complementary DNA (cDNA) by reverse transcriptase
2. cDNA is amplified by the standard PCR procedure (see above)
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reverse transcription polymerase chain reaction (RT-PCR) use
detection and quantification of mRNA levels in a sample
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real-time polymerase chain reaction procedure
also known as quantitative PCR or qPCR
a PCR technique that utilizes fluorescence (e.g., intercalating dyes or DNA probes) for monitoring the amplification of targeted DNA during the PCR via computer software (in a graph)
melting temperatures specific to the amplified fragment allow for high specificity.
there are many types, including quantitative reverse transcription PCR (RT-qPCR) and semiquantitative real-time PCR
181
real-time polymerase chain reaction uses
allows monitoring of a targeted product at any point throughout the amplification
rapidly detect nucleic acids for diagnosis (e.g., RT-qPCR for SARS-CoV2)
easily quantify gene expression (e.g., via comparing to a standard curve of serial dilutions)
quantify and genotype viruses
182
chromosome testing purposes
used in the (prenatal) diagnosis of inherited disorders, e.g. to detect gene mutations
used in the diagnosis of infectious diseases (e.g., diphtheria)
used in forensics
used in tumor diagnostics
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genetic markers
individual differences in the DNA sequence of a specific region of the genome
(repetitive sequences of various lengths which are present in several noncoding regions of the genome and which differ in sequence motif length
since the frequency of repetition differs in each individual, polymorphisms in DNA form the basis for the diagnosis of diseases and allow individuals to be identified)
184
three different markers for DNA analysis
microsatellites (short tandem repeats, STRs)
SNPs (single nucleotide polymorphism)
RFLP (restriction fragment length polymorphism)
185
microsatellites
AKA short tandem repeats (STRs); belong to the VNTRs (variable number tandem repeats)
repetitive sequences of several base pairs in DNA that are highly polymorphic
can be analyzed via PCR to identify individuals (very small amounts of DNA suffice for analysis)
186
VNTRs
variable number tandem repeats; short nucleotide sequences in the genome of an individual that are repeated a variable number of times
187
SNPs
single nucleotide polymorphism
DNA sequence variants in a population that differ by only a single base pair; usually caused by errors during DNA replication and are point mutations
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RFLP
restriction fragment length polymorphism
depending on the DNA sequence of an individual, cleaving of chromosomal DNA with restriction enzymes leads to DNA fragments of variable lengths
189
RFLP detection method
the DNA fragments of variable lengths s/p cleaving of chromosomal DNA with restriction enzymes are analyzed using Southern blot and detected by probes
190
genetic fingerprint
the DNA profile of an individual, especially as determined by microsatellite analysis
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DNA diagnostics uses
suitable for the direct or indirect detection of a gene mutation that results in disease
can also be used to exclude the presence of a gene mutation
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direct detection prerequisites
the causal gene for the (suspected) disease is known
193
restriction endonucleases
AKA restriction enzymes
enzymes that cleave double-stranded DNA at an enzyme-specific nucleotide sequence
194
direct detection process
amplification of a specific region of the affected gene using PCR
detection of a mutation, e.g., through sequencing or cleavage of DNA fragments using restriction enzymes
analysis via gel electrophoresis: detection is based on the altered mobility of the DNA fragments of mutated and normal alleles in gel electrophoresis
195
restriction site cleavage
most restriction sites are palindromes
cleaved by restriction enzymes (restriction endonucleases)
cleavage of DNA using restriction enzymes results in sticky ends or blunt ends.
196
origin of restriction enzymes
prokaryotic defense system against foreign DNA
197
genetic linkage analysis
AKA indirect detection
analysis of genetic markers associated with the mutated gene and comparison of the patient's genotype with that of unaffected family members
performed if direct detection is not possible
198
indirect detection prerequisite
the disease occurs in several family members and the suspected locus is known
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indirect detection result
there is no direct detection of a gene mutation, but a probability of the presence of a certain mutation that causes disease (genetic risk score) can be calculated
the validity of indirect DNA diagnostics depends on the pattern of inheritance and the number of family members being investigated
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karyotyping overview
can be used to visualize chromosomes for examining chromosome numbers and for an overview of potential structural changes within a chromosome
determines the number, size, morphology, banding pattern, and the arm-length ratio of chromosomes
helpful for the diagnosis of trisomies, monosomies, and sex chromosome disorders
201
banding pattern definition
transverse bands of various widths and distribution, which can be induced depending on the preparation and staining technique
202
banding pattern preparation
the cell sample is cultivated and cell division is stimulated.
to obtain chromosomes during metaphase, the cells are arrested using the spindle inhibitor colchicine
203
colchicine
interferes with the formation of the microtubules of the mitotic spindle, leaving the cell suspended in metaphase and arresting mitosis
204
banding techniques
staining with quinacrine (fluorescent bands; not used routinely in diagnostics)
Giemsa banding (standard banding technique, results in dark G bands with transcriptionally inactive chromatin and bright, transcriptionally active R bands)
205
banding pattern analysis
assessment of an average 10–15 metaphase chromosome pairs in 1250x magnification
206
staining with quinacrine
fluorescent bands; not used routinely in diagnostics
207
Giemsa banding
standard banding technique; results in dark G bands with transcriptionally inactive chromatin and bright, transcriptionally active R bands
208
karyotype overview
evaluation of a karyogram according to the number and the structure (morphology, length, arm-ratio, pattern of banding, size) of the chromosomes
the total number of chromosomes is stated first followed by the type of sex chromosome present (e.g., 46, XX = healthy, female karyotype)
anomalies, if present, are mentioned last (e.g., 47, XY+21: male karyotype with trisomy 21).
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FISH overview
fluorescence in situ hybridization
a method used for the staining of specific DNA sequences by a fluorescence-labeled DNA or RNA probe
E.g., to stain chromosomes in karyograms, in tumor diagnosis, or to map specific genes on chromosomes in metaphase
210
FISH procedure
denaturation of DNA in the prepared chromosomes
hybridization of the DNA with a single-stranded, fluorescence-labeled DNA probe that is complementary to a specific DNA sequence
analysis of the chromosome set in fluorescence microscopy (fluorescent signal indicates the site of the bound probe)
211
microdeletion in FISH
the deleted region does not exhibit fluorescence, compared to the region present in the same locus of the other copy of the chromosome
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translocation in FISH
fluorescence signal from one chromosome is present in a different chromosome
213
duplication in FISH
extra copies of chromosomes that exhibit fluorescence in addition to the normal ones
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phytohemagglutinin
a mitogen that increases the proliferation of peripheral blood lymphocytes
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FISH result
the specific staining increases the resolution compared to the classical staining methods of karyograms, thus even minor chromosomal aberrations (e.g., microdeletions) can be identified.
FISH is also possible on chromosomes in interphase
216
FISH uses
direct visualization of chromosomal anomalies at specific gene loci on a molecular level
detection of microdeletions (2–3 × 106 base pairs) such as in DiGeorge syndrome
search for evidence of a Philadelphia chromosome translocation t(9;22)
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array comparative genome hybridization (CGH)
another name for DNA microarray
218
DNA microarray
mainly used to simultaneously examine expression levels of multiple genes or to genotype many regions at the same time
219
DNA microarray prepartaion of the sample
the first step in microarray procedure
a sample (m)RNA and control (m)RNA are converted to complementary DNA (cDNA) by reverse transcriptase
cDNA is then labeled with a fluorescent dye, one color for the sample that is being tested, another color for the control (e.g., green for the control, red for the sample being tested)
mRNA is removed and samples are combined.
220
DNA microarray preparation of the chip
the second step in microarray procedure
thousands of genetic sequences of nucleic acid (DNA or RNA) probes are attached to a chip (e.g., glass, silicon).
oth patient and control DNA is applied to this chip and hybridizes to the probes on the chip, which is mounted to a scanner that can detect complementary binding of probes and sample sequences
each region of the chip stands for a known genetic sequence, and the higher the expression of the gene in one sample, the more intense the fluorescence.
221
DNA microarray uses
helpful for the detection of copy number variations (CNVs) and sequencing
222
DNA microarray sequencing
determination of the exact sequence of base pairs of a gene; e.g., to demonstrate an unknown mutation on a disease-causing gene
223
copy number variations (CNVs)
single nucleotide polymorphisms (SNPs) that are used for genotyping, forensic science, cancer mutations, genetic linkage analysis, and genetic testing
224
CRISPR gene editing
a technique in genetic engineering that employs the prokaryote CRISPR/Cas9 defense system directed against foreign gene sequences to modify the genomes of living organisms (e.g., adding or deleting genes in DNA sequences)
225
CRISPR
clustered regularly interspaced short palindromic repeats; a family of DNA sequences of prokaryotic origin
226
crRNA
CRISPR RNA
transcribed CRISPR sequence; binds to Cas9
227
Cas9
CRISPR-associated system 9
endonuclease that produces single or double-strand breaks at a specific nucleotide sequence guided by a site-specific RNA (targeted DNA double-strand break)
228
tracrRNA
transactivating crRNA
RNA sequence that is partially complementary to crRNA; also binds to Cas9 (needed for Cas9 maturation)
229
Cas9 in prokaryotes
the guide RNA consists of crRNA and tracrRNA
230
Cas9 for laboratory use
a single guide RNA is specifically designed and synthesized
231
cas9 gene sequence location
found adjacent to the CRISPR sequence
232
adaptive prokaryotic immune response
1. Foreign DNA is incorporated into own DNA at the CRISPR locus (acquisition)
2. The CRISPR locus is transcribed together with foreign DNA and forms the primary transcript.
3. The primary transcript binds tracrRNA and is processed to form a crRNA-tracrRNA hybrid, which contains a foreign genetic sequence
4. crRNA-tracrRNA hybrid forms a complex with Cas9.
5. Now foreign DNA that contains the sequence complementary to the one contained by the crRNA/tracrRNA/Cas9 complex can be recognized and cleaved
233
CRISPR/Cas9 in gene editing
for gene editing purposes, tracrRNA and crRNA are combined into one molecule, the single synthetic guide RNA (sgRNA) that is complementary to the DNA sequence of interest (target DNA)
234
three major variants of Cas9 used in CRISPR gene editing
wild-type Cas9
Cas9D10A
nuclease-deficient Cas9
235
wild-type Cas9
major variant of Cas9 used in CRISPR gene editing; cleaves dsDNA
can repair induced dsDNA breaks through nonhomologous end joining (NHEJ) pathway or homology-directed repair (HDR) pathway
236
nonhomologous end joining (NHEJ) pathway
pathway for wild-type Cas9 to repair dsDNA
deletion of the targeted gene (gene knock-out) → accidental frameshift mutations
237
homology-directed repair (HDR) pathway
pathway for wild-type Cas9 to repair dsDNA
insertion of donor DNA (gene knock-in) → mutation in the gene of interest
238
Cas9D10A
major variant of Cas9 used in CRISPR gene editing; cleaves only one DNA strand (nickase activity)
more specific than wild-type Cas9 because it does not activate NHEJ (nonhomologous end joining) but only the high-fidelity HDR (homology-directed repair) pathway
239
nuclease-deficient Cas9
major variant of Cas9 used in CRISPR gene editing; mutations in nuclease domains prevent cleavage but not binding
can be used to activate or silence genes by creating fusion proteins of Cas9 with effector proteins
240
potential applications of CRISPR/Cas9
immuno-oncology (i.e. using manipulated immune cells to fight cancer)
curing genetic diseases by replacing the alleles of genes associated with disease phenotypes with unaffected variants
eliminating virulence factors of pathogens
241
hemoglobin electrophoresis definition
a screening test that detects and quantifies the types of hemoglobins present in a sample by separating them based on their electrical charge
242
hemoglobin electrophoresis applications
diagnostic work-up of hemolytic anemias
screening for or evaluation of hemoglobinopathies in high-risk individuals (e.g., positive family history)
evaluation of abnormal erythrocytes on peripheral blood smear
243
hemoglobin electrophoresis procedure
a sample of the patient's hemoglobin is obtained by hemolyzing a sample of blood using a hemolysate reagent and the sample is added to the gel electrophoresis buffer
an electric field is applied to the buffer that causes the different hemoglobin types to separate according to their electrical charge; hemoglobin is negatively charged at an alkaline pH and migrates on the gel towards the anode, forming a band with the degree of negative charge of the hemoglobin molecule determining the migration speed and distance from the cathode to the anode
a stain is applied to the gel to make the charged molecules visible
244
hemoglobin electrophoresis speeds
HbA migrates the fastest and therefore the greatest distance, followed by Hb F, Hb S, and Hb A2, and Hb C (A > F > S > A2 and C)
[A FaSt TouCan can go far (A > F > S > A2 > C)]
245
hemoglobin C migration during electrophoresis
HbC migrates the least because a missense mutation replaces the negatively charged glutamic acid with the positively charged lysine
246
hemoglobin S migration during electrophoresis
migrates less than HbA because of the replacement of the negatively charged glutamic acid with the neutral valine
247
expected hemoglobin electrophoresis finding for normal adult
wide HbA band (AA)
248
expected hemoglobin electrophoresis finding for normal fetus
HbA and widened HbF band (AF)
249
expected hemoglobin electrophoresis finding for beta thalassemia minor (trait)
narrowed HbA band and widened HbA2 band
250
expected hemoglobin electrophoresis finding for beta thalassemia major
no HbA band; widened HbA2 and HbF bands
251
expected hemoglobin electrophoresis finding for sickle cell trait
HbA and HbS bands (AS)
252
expected hemoglobin electrophoresis finding for sickle cell anemia
no HbA band; wide HbS band (SS)
253
expected hemoglobin electrophoresis finding for HbC trait
HbA and HbC bands (AC)
254
expected hemoglobin electrophoresis finding for HbC disease
no HbA band; wide HbC band (CC)
255
expected hemoglobin electrophoresis finding for HbSC disease
no HbA band; HbS and HbC bands (SC)
256
RNA interference definition
inhibition of gene expression via small, non-coding RNA molecules that bind target RNA (mostly mRNA)
257
RNA interference functions
inhibition of gene expression
defense mechanism against foreign RNA molecules
258
RNA interference mechanisms
inhibition of translation
mRNA destabilization via removal of poly(A) tail at the 3′OH-end of target mRNA (deadenylation) and decapping
mRNA degradation
259
Cre-Lox system
used to induce recombination between specific DNA sites (e.g., to study the effect of gene insertions or deletions)
consists of Cre recombinase and LoxP sequences
260
Cre recombinase
an enzyme that catalyzes recombination between LoxP sequences
261
LoxP sequences
short DNA sequences within the cell genome that have sites for Cre recombinase binding
262
molecular cloning definition
experimental production of recombinant DNA molecules within host organisms (e.g., bacterial hosts)
263
molecular cloning process
1. Isolation of eukaryotic target mRNA
2. Production of complementary DNA (cDNA) using reverse transcriptase
3. Insertion of cDNA fragments into a cloning vector (e.g., bacterial plasmids carrying the antibiotic resistance genes)
4. Transformation of the produced recombinant plasmid into bacteria
5. Selection of bacteria that contains the plasmid (e.g., via antibiotic exposure when cloning antibiotic resistance genes)
264
molecular cloning result
synthesis of multiple copies of target cDNA (cloned DNA)
265
assessment of RNA splicing (including splicing errors)
northern blot use
266
southern blot use examples
restriction fragment length polymorphisms (RFLPs)
minisatellite DNA and microsatellite DNA (called “variable number of tandem repeats,” VNTRs)
267
testing for West Nile virus antibodies
ELISA use
268
detection of Mycobacterium tuberculosis
ELISA use
269
detection of Rotavirus (in feces)
ELISA use
270
detection of Hepatitis B virus
ELISA use
271
detection of E. coli enterotoxin (in feces)
ELISA use
272
detection of PSA
ELISA use
273
method useful for the detection of neonatal exposure to HIV
PCR use
274
PCR use benefits
positive early after infection and is not affected by the immune status of the patient (e.g., immunocompromised patients may not produce an antibody response)
275
diagnosis of bacterial and viral infections (e.g., Lyme disease, HSV encephalitis)
PCR use
276
diagnosis of immunodeficiency (e.g., severe combined immunodeficiency)
PCR use
277
forensic analysis (e.g., comparison of DNA samples from suspects, paternity testing)
PCR use
278
sequencing of mutations
PCR use
