3.1 - MOLECULAR PATHOLOGY Flashcards
1
Q
PCR is abbreviation for
A
polymerase chain reaction
2
Q
BDNA is abbreviation for
A
branched-DNA
3
Q
FISH is abbreviation for
A
fluorescent in situ hybridization
4
Q
highly effective for the identification of proteins that are implicated in causing disease
A
mass spectroscopy
5
Q
enables detection of gene rearrangements and gene deletions in a number of diseases, especially in cancers
A
FISH
6
Q
genes active in carcinogenesis
A
oncogenes
7
Q
proteins active in carcinogenesis.
A
oncoproteins
8
Q
When one or more of the proteins on mitogenic signal transduction pathways become mutated, the result is…
A
the cells in which they are expressed become transformed into cancer cells
9
Q
proteomics
A
the large-scale study of proteomes. A proteome is a set of proteins produced in an organism, system, or biological context
10
Q
(PSA) detects
A
Prostate-specific antigen - prostate cancer
11
Q
(CEA) detects
A
Carcinoembryonic antigen (CEA) - colon cancer and other gastrointestinal tumors
12
Q
(AFP) detects
A
Alpha-feto protein (AFP) - hepatocellular
carcinoma.
13
Q
Two essential components of quality assurance programs:
A
- Standardized methods (published by the Clinical and Laboratory Standards Institute)
- Interlaboratory comparison of test (provided by the College of American Pathologists)
14
Q
Carries information from DNA to the cytoplasm of a cell.
A
Ribonucleic acid (RNA)
15
Q
DNA - long, double-stranded polymeric molecule (dsDNA) that exists predominantly in the form of a (left-handed /right- handed) double helix
A
right-handed helix
16
Q
Backbone of the ssDNA polymer
A
- sugar deoxyribose connected by phosphate groups
17
Q
Phosphodiester bonds between 3’ to 5’ gives
a. directionality
b. invariant structure
A
b. 3’ to 5’ gives invariant structure
18
Q
Phosphodiester bonds between 5’ to 3’ gives
a. directionality
b. invariant structure
A
5’ to 3’ gives directionality
19
Q
Purine bases
adenine (A)
cytosine (C)
guanine (G)
thymine (T)
A
PURGA
Adenine (A) and guanine (G) (purines)
20
Q
Pyrimidine bases
adenine (A)
cytosine (C)
guanine (G)
thymine (T)
A
PRCaT
21
Q
Building blocks of the single-stranded polymer:
(deoxyribonucleotide triphosphates) - 4
A
o dTTP
o dCTP
o dATP
o dGTP
22
Q
Building blocks of the single-stranded polymer consist of
A
sugar molecule + triphosphate
group + 1base
23
Q
Steps of DNA synthesis in order
a. chain
b. linked together by phosphodiester
c. nucleotides
d. stripped to two phosphate groups
A
Nucleotides → stripped to two phosphate groups → linked together by phosphodiester bonds → chain
24
Q
Only ways DNA loses its normal conformational structure
A
extremes of heat
pH
destabilizing agents
25
most energetically favorable state for DNA
a. dsDNA
b. ssDNA
Double-stranded helix - most energetically favorable state for DNA
26
sugar and phosphate groups are
a. hydrophilic
b. hydrophobic
Both sugar and phosphate groups are hydrophilic, forming stable hydrogen bonds
27
What forms hydrogen bonds in DNA?
Both sugar and phosphate groups are hydrophilic, forming stable hydrogen bonds
28
bases are
a. hydrophilic
b. hydrophobic
Bases - hydrophobic and are insoluble in water at neutral pH
29
Repeating nucleotide units are linked by phosphodiester bonds are formed between...
the 5′ carbon of one sugar to the 3′ carbon of the next
30
What allows the ladder to twist
a. carbon-oxygen linkages in the phosphodiester bond
b. hydrogen-hydrogen linkages in the hydrogen bond
a. carbon-oxygen linkages in the phosphodiester bond
31
What mechanism protect DNA bases from water?
1. ladder twisting and flexibility leaves no room for water molecules in between
2. helical conformation - protects the base pairs from water exposing only the hydrophilic backbones
32
Helical dsDNA is stable at the pH of
a. 2-7
b. 4-9
b. 6-11
Helical dsDNA - stable at a pH of 4–9
| o Solutions with pH outside the limit may cause DNA denaturation and unwinding
33
Formamide cause DNA denaturation (melting) and unwinding by
a. disrupting hydrogen bones
b. disrupting phosphodiester bonds
c. changing pH
a. hydrogen bond disrupters (e.g. formamide)
34
length of a fully extended eukaryotic DNA molecule
3m per genome
35
Chromosomes made of
DNA strand wound around
| DNA associated proteins (chromatin)
36
Human cells have 2 sets of how many genes?
a. 22
b. 23
c. 24
Human cell nucleus - contains two sets of 23 chromosomes (human genome)
37
What is the sugar of DNA and RNA?
DNA: Deoxyribose
RNA: Ribose
38
What is the base pairs of DNA and RNA?
DNA: Thymine–adenine Cytosine–guanine
RNA: Uracil–adenine Cytosine–guanine
39
What is the 3D structure of DNA and RNA?
DNA: Double-stranded Alpha helix
RNA: Single-stranded
Random
40
What is the stability of DNA and RNA?
DNA: Stable; Degraded by DNase
RNA: Subject to base hydrolysis; Degraded by RNase
41
What is the function of DNA and RNA?
DNA: Maintains genetic information in nucleus
RNA: Carries genetic information to cytoplasm
42
Polymerases
a. catalyzes the formation of
phosphodiester bonds during synthesis
b. hydrolyzes phosphodiester bonds
c. found only in bacteria that functions to destroy foreign DNA
a. Polymerases- catalyzes the formation of phosphodiester bonds during synthesis
43
Nucleases
a. catalyzes the formation of
phosphodiester bonds during synthesis
b. hydrolyzes phosphodiester bonds
c. found only in bacteria that functions to destroy foreign DNA
b. hydrolyzes phosphodiester bonds
44
Restriction endonuclease
a. catalyzes the formation of
phosphodiester bonds during synthesis
b. hydrolyzes phosphodiester bonds
c. found only in bacteria that functions to destroy foreign DNA
c. found only in bacteria that functions to destroy foreign DNA
45
In vivo function of
| Polymerases
DNA polymerases, RNA polymerases
Polymerases join DNA or RNA nucleotides together to form a single-stranded daughter molecule using a stretch of single-stranded parent molecule as a template. These enzymes perform syntheses according to base pair rules and proceed in the 5′ to 3′ direction.
Some polymerases also have nuclease activity.
46
In vivo function of
Reverse transcriptase
Mostly of viral origin, reverse transcriptase catalyzes the synthesis of DNA from either an RNA or DNA template.
47
In vivo function of
DNA ligases
Joins DNA fragments formed by discontinuous synthesis in DNA replication or by DNA repair pathways.
48
In vivo function of
| Nucleases
DNases, RNases
Nucleases “digest” nucleic acid molecules by breaking phosphodiester bonds.
Nucleases may have single-stranded, double-stranded, DNA, or RNA specificity. Some polymerases also have nuclease activity.
49
In vivo function of
Endonucleases
Endonucleases digest nucleic acids from the middle of the molecule.
Nucleases may have single-stranded, double-stranded, DNA, or RNA speci city. Some polymerases also have nuclease activity.
50
In vivo function of
Exonucleases
Exonucleases digest nucleic acids by begining at a free end and may require a 3′ or 5′ end.
Nucleases may have single-stranded, double-stranded, DNA, or RNA speci city. Some polymerases also have nuclease activity.
51
In vivo function of
Restriction endonuclease
Bacterial endonucleases that recognize specific short DNA base pair sequences and cleave the DNA molecule only at the recognition site
52
Steps in synthesis
a. DNA polymerase III proceeds with DNA synthesis
b. Production of a small single-stranded region
c. RNA primer is excised and replaced with DNA by
DNA polymerase I
d. Short RNA primer synthesis complementary to the single stranded sequence
Synthesis:
1. Production of a small single-stranded region
2. Short RNA primer synthesis complementary to the single stranded sequence
3. DNA polymerase III proceeds with DNA synthesis
4. RNA primer is excised and replaced with DNA by
DNA polymerase I
53
DNA polymerase III
a. birectional enzyme
b. unidirectional enzyme
DNA polymerase III - unidirectional enzyme
can synthesize DNA only in the 5’ to 3’ direction because
it requires a free 3’ OH end
54
Why can DNA only be synthesized in the 5' to 3' directions?
can synthesize DNA only in the 5’ to 3’ direction because it requires a free 3’ OH end
55
What has proofreading and exonuclease activity?
a. DNA polymerase I
b. DNA polymerase II
c. DNA polymerase III
c. DNA polymerase III
if an incorrect nucleotide is added to the growing chain, it is detected and excised by the nuclease portion of the
enzyme, and the correct nucleotide is then added
56
What joins Okazaki fragments together?
a. DNA ligase
b. DNA polymerase II
c. DNA polymerase III
d. DNases
a. DNA ligase
57
Contains the amino acid sequence code for one
protein as well as DNA sequences necessary for
the regulation of the production of that protein
Gene
58
Coding sequences makes up this much of nucleotides:
a. <5%
b. <7%
c. <9%
Coding sequences - make up <5% of nucleotides
59
Noncoding DNA regions are also known as
junk DNA
60
Protein synthesis begins with
Protein synthesis begins with the activation of the appropriate gene. A copy of the gene is made from DNA in the form of RNA.
61
mRNA function
carries the code from the DNA in the cell nucleus to the cytoplasm where amino acid synthesis takes place
62
What is transcription?
mRNA synthesis from one strand of DNA gene
63
mRNA contains
both amino acid coding sequences (exons) and noncoding sequences (introns)
64
These are excised from the mRNA molecule before protein synthesis.
a. exons
b. introns
c. promoters
Introns- excised from the mRNA molecule before protein synthesis
65
Function of spliceosome
o composed of both low molecular weight RNA and protein
o recognizes mRNA sequences that
identify the boundaries of an intron
o joins the flanking exons and releases the intron
66
Cap step of posttranscriptional modification
- addition of 7-methyl guanosine residues to the 5’ end in a unique 5’-5’ phosphodiester bond
- aids in the binding of the ribosome to the mRNA molecule for initiation of protein synthesis
67
Purpose of poly-A tail
may be necessary for stability and transport to the cytoplasm
68
Where is the poly-A trail added?
added to the 3’ end
69
Reading frame of a protein coding sequence occurs in which direction?
running in the 5’ to 3’ direction
70
Stop codons
UAG, UGA, or UAA
71
when an amino acid can be encoded by more than one codon
Degenerate code
72
Mediates translation from the mRNA nucleotide code to protein
ribosomes in the cytoplasm of the cell
73
adaptor RNA molecules that link amino acids in the correct sequence:
a. mRNA
b. rRNA
c. tRNA
c. tRNA
74
a region of tRNA molecule that is complementary to a particular mRNA codon
a. anticodon
b. initiation codon
c. termination codons
a. anticodon
75
What is always the first codon in the mRNA sequence?
A tRNA with the correct complementary anticodon binds to the first codon in the mRNA sequence, which is always AUG.
76
What type of bonds are formed between amino acids linked to the tRNA during translation?
peptide bond
77
What is the purpose of transcriptional control?
For cellular differentiation and response to environmental stimuli
78
What is upstream?
a. toward the 5' end
b. toward the 3' end
a. toward the 5' end
79
TATA boxes
Most common promoters are rich in adenine and thymine
80
Why are promoters rich in adenine and thymine bonds?
Because A-T base pair bonds are weaker than G-C base pair bonds, DNA unwinds more easily at repeat A-T sequences
81
Enhancers are
DNA sequences that can augment mRNA transcription and may be found in different locations relative to the gene that they affect
82
Why is the half-life of mRNA is very short?
Because mRNA is far less stable than DNA
83
How does the cell respond to changes to its environment?
Cell responds to changes in transcriptional signals → genes that are transcribed into mRNA can be quickly changed → immediate synthesis of new proteins → ability of the cell to rapidly adjust its protein output in response to its environment.
84
Small RNAs are
short, noncoding ribonucleotides that function as posttranscriptional regulators of gene expression
85
Subgroups of small RNA
small interfering RNA (siRNA)
micro RNA (miRNA)
86
The DNA sequences for siRNAs and miRNAs
| are often located in (coding/noncoding) regions between genes
The DNA sequences for siRNAs and miRNAs
| are often located in noncoding regions between genes
87
Transcription of siRNAs and miRNAs is mediated by
a. DNA ligase
b. DNA polymerase II
c. DNA polymerase III
d. DNases
Generally have their own promoters, and transcription is mediated by polymerase II
88
RNA-induced silencing complex (RISC)
mediates binding to complementary sequences of
| mRNAs
89
What attaches to RISC?
Small RNA strand attaches to an RNA-induced silencing complex (RISC), which mediates binding to complementary sequences of
mRNAs
90
RNA interference (RNAi) function
siRNAs pair perfectly with
| the target mRNA and trigger a series of molecular mechanisms that lead to its degradation
91
DNA methylation
a. enzymes responsible for DNA methylation
b. result in condensed chromatin and reduce transcription
b. result in condensed chromatin and reduce transcription
A methyl group added to the fifth carbon of cytosine results in 5methyl cytosine
92
DNA methyltransferases
a. enzymes responsible for DNA methylation
b. result in condensed chromatin and reduce transcription
a. enzymes responsible for DNA methylation
93
Direct repair
a. functions immediately after DNA replication to replace mismatched bases with the correct ones
b. repair lesions in a single-step reaction
c. uses an enzyme system composed of many proteins to excise a single-stranded oligonucleotide containing the lesion
b. repair lesions in a single-step reaction
Ex: 6-methylguanine DNA methyltransferase repairs alkylation lesions by transferring the alkyl group from the lesion to the active site of the enzyme
94
Mismatch repair (MMR)
a. functions immediately after DNA replication to replace mismatched bases with the correct ones
b. repair lesions in a single-step reaction
c. uses an enzyme system composed of many proteins to excise a single-stranded oligonucleotide containing the lesion
a. functions immediately after DNA replication to replace mismatched bases with the correct ones
95
Nucleotide excision repair (NER)
a. functions immediately after DNA replication to replace mismatched bases with the correct ones
b. repair lesions in a single-step reaction
c. uses an enzyme system composed of many proteins to excise a single-stranded oligonucleotide containing the lesion
c. uses an enzyme system composed of many proteins to excise a single-stranded oligonucleotide containing the lesion
96
Xeroderma pigmentosum (XP)
which is caused by mutations in NER
results in extreme sensitivity to sunlight, with skin cancers occurring at an early age
97
In electrophoretic separations, the samples move towards the (negative/positive) electrode in a linear fashion.
the samples move toward the positive electrode in a linear fashion
98
DNA or RNA ladders
mixtures of nucleic acids of
| known fragment length that are analyzed in one or more lanes of the gel
99
Hybridization is interaction between two two single-stranded nucleic acid molecules to form a duplex (double-stranded) molecule. The strands are:
a. one strand is labeled
b. both strands are labeled
c. neither strand is labeled
a. one strand is labeled
If one strand is labeled, the labeled strand is referred to as a probe and the process is called hybridization because a hybrid molecule is formed between a labeled and unlabeled strand
---
Annealing - process of reforming the stable double- stranded structure when neither DNA strand is labeled
100
Positive sample control is:
a. known to contain sequences complementary to the probe
b. one known not to contain sequences complementary to the probe
a. known to contain sequences complementary to the probe; used to establish that sample preparation is adequate to release target for the hybridization assay, and to ensure that probe will hybridize with the specific target under the assay conditions
- ---
b. Negative sample control - one known not to contain sequences complementary to the probe; used to monitor specificity of the probe target interactions
101
Occurs in a biphasic environment, a solid phase (usually sample) and a liquid phase (usually probe)
a. Dot/Blot Hybridization
b. Liquid or Solution Phase Hybridization
c. Solid-Support Hybridization
c. Solid-Support Hybridization
---
b. Liquid or Solution Phase Hybridization
- Both the sample and probe interact in solution which maximizes the kinetics of the reaction
a. Dot/Blot Hybridization
- multiple samples are immobilized in a geometric array on a nitrocellulose or nylon membrane
102
involves taking morphologically intact tissue, cells, or chromosomes affixed to a glass microscope slide through the hybridization process
a. Dot/Blot Hybridization
b. In Situ Hybridization
c. Microarray Hybridization
b. In Situ Hybridization
---
a. Dot/Blot Hybridization
- multiple samples are immobilized in a geometric array on a nitrocellulose or nylon membrane
c. Microarray Hybridization (“DNA Chip Technology”)
- A variation of the dot-blot format in which the dotted material is arranged in a regular grid-like pattern with each feature reduced to a very small size so that hundreds to thousands of features can be placed on one solid surface, currently most often a glass microscopic slide
103
Southern and northern hybridizations steps
1. electrophoretic separation of test nucleic acid
| 2. transfer to a solid support and subsequent hybridization
104
Southern blotting sample is:
a. DNA
b. DNA binding proteins
c. Proteins
d. RNA
a. Southern blotting - sample is DNA
105
Northern blotting sample is:
a. DNA
b. DNA binding proteins
c. Proteins
d. RNA
d. Northern blotting – sample is RNA
106
Western blot sample is:
a. DNA
b. DNA binding proteins
c. Proteins
d. RNA
c. Western blot - similar procedure in which
proteins are subjected to electrophoresis and
transfer
107
Southwestern blot sample is:
a. DNA
b. DNA binding proteins
c. Proteins
d. RNA
b. Southwestern blot - technique separating and
| blotting DNA followed by incubation with protein solutions to permit evaluation of specific DNA binding proteins
108
Genotyping
comprehensive mapping of an individual’s SNP pattern
109
Polymerase Chain Reaction steps
Denaturation
Annealing
Extension
110
Developed to amplify ribonucleic acid (RNA) targets
a. Multiple Polymerase Chain Reactions
b. Nested Polymerase Chain Reaction
c. Reverse-Transcriptase Polymerase Chain Reaction
d. Polymerase Chain Reaction
c. Reverse-Transcriptase Polymerase Chain Reaction
Complementary DNA (cDNA) is first produced from RNA targets by reverse transcription and then amplified by PCR
employed two enzymes:
1. Heat-labile RTs
2. Thermostable DNA polymerase
111
Developed to increase both the sensitivity and specificity of PCR
a. Multiple Polymerase Chain Reactions
b. Nested Polymerase Chain Reaction
c. Reverse-Transcriptase Polymerase Chain Reaction
d. Polymerase Chain Reaction
b. Nested Polymerase Chain Reaction
Employs two pairs of amplification primers and two rounds of PCR
Increase in sensitivity – arises from the high total cycle number.
Increase in specificity – arises from the annealing of the second primer set to sequences found only in the first- round products.
112
Two or more primer sets designed for amplification of different targets are included in the same reaction mixture.
a. Digital Polymerase Chain Reaction
b. End-Point Quantitative Polymerase Chain Reaction
c. Multiple Polymerase Chain Reactions
d. Nested Polymerase Chain Reaction
e. Rapid-Cycle Polymerase Chain Reaction
f. Real-Time (Homogenous, Kinetic) Polymerase Chain Reaction
g. Reverse-Transcriptase Polymerase Chain Reaction
c. Multiple Polymerase Chain Reactions
113
co-amplification in the same reaction tube of two different templates of equal or similar lengths and with the same primer binding sequences.
a. Digital Polymerase Chain Reaction
b. End-Point Quantitative Polymerase Chain Reaction
c. Multiple Polymerase Chain Reactions
d. Nested Polymerase Chain Reaction
e. Rapid-Cycle Polymerase Chain Reaction
f. Real-Time (Homogenous, Kinetic) Polymerase Chain Reaction
g. Reverse-Transcriptase Polymerase Chain Reaction
b. End-Point Quantitative Polymerase Chain Reaction
114
Target amplification and detection steps occur simultaneously in the same tube
Advantage: decrease the time required to perform nucleic acid assays because there are no post- PCR steps
a. Digital Polymerase Chain Reaction
b. End-Point Quantitative Polymerase Chain Reaction
c. Multiple Polymerase Chain Reactions
d. Nested Polymerase Chain Reaction
e. Rapid-Cycle Polymerase Chain Reaction
f. Real-Time (Homogenous, Kinetic) Polymerase Chain Reaction
g. Reverse-Transcriptase Polymerase Chain Reaction
f. Real-Time (Homogenous, Kinetic) Polymerase Chain Reaction
115
Advantage: reducing the thermal profile of the solution using thin- walled tubes or capillaries that force the reaction solution into thin columns or sheets of fluid, it is possible to improve the thermal transfer rates as well as the equilibration rates such that thermocycling time can be significantly reduced
a. Digital Polymerase Chain Reaction
b. End-Point Quantitative Polymerase Chain Reaction
c. Multiple Polymerase Chain Reactions
d. Nested Polymerase Chain Reaction
e. Rapid-Cycle Polymerase Chain Reaction
f. Real-Time (Homogenous, Kinetic) Polymerase Chain Reaction
g. Reverse-Transcriptase Polymerase Chain Reaction
e. Rapid-Cycle Polymerase Chain Reaction
- Not chemically different from any of the standard PCR formats.
- Most of the time consumed in performing PCR is temperature equilibrium for the solution so that efficient annealing and extension occurs, as well as significant amount of time changing the temperature.
116
Accomplished by capturing or isolating each individual nucleic acid molecule present in a sample within many chambers, zones, or regions that are able to localize and concentrate the amplification product to detectable levels.
Used for:
o detection and quantification of low levels of pathogen sequences
o rare genetic sequences,
o gene expression in single cells
a. Digital Polymerase Chain Reaction
b. End-Point Quantitative Polymerase Chain Reaction
c. Multiple Polymerase Chain Reactions
d. Nested Polymerase Chain Reaction
e. Rapid-Cycle Polymerase Chain Reaction
f. Real-Time (Homogenous, Kinetic) Polymerase Chain Reaction
g. Reverse-Transcriptase Polymerase Chain Reaction
a. Digital Polymerase Chain Reaction
After PCR amplification, a count of areas containing PCR products is a direct measure of the absolute quantity of nucleic acid in the sample.
Capture or isolation of individual nucleic acid molecules may be done in capillaries, microemulsions, arrays of miniaturized chambers, or on surfaces that bind nucleic acids.
117
These techniques essentially recapitulate retroviral replication in vitro, converting RNA into DNA and then using the DNA as a template for transcription of multiple copies of RNA.
transcription-mediated amplification (TMA)
nucleic acid sequence-based amplification (NASBA)
118
In the case of TMA
c. a separate enzyme (RNAse H) degrages the initial RNA template
b. the reverse-transcriptase enzyme itself degrades the initial RNA template as it synthesizes its complementary DNA
b. In the case of TMA, the reverse-transcriptase enzyme itself degrades the initial RNA template as it synthesizes its complementary DNA.
119
TMA and NASBA have several advantages over other RNA amplification techniques.
o No initial denaturation is required for the amplification to occur.
o Uses isothermal processes that obviate the need for sophisticated thermocyclers because the whole process occurs at a single temperature.
o Both have been used successfully in a wide variety of applications.
o Both are isothermal RNA amplification techniques with a widespread applicability and several advantages over other amplification methods, especially for the elimination of DNA contamination issues, particularly those related to retroviral and intronless genes.
120
Strand - Displacement Amplification
An isothermal template amplification technique that can be used to detect trace amounts of DNA or RNA of a particular sequence
• Occurs in two distinct phases:
o Target generation
o Exponentialtargetamplification
121
dsDNA target is denatured and hybridized to two
different primer pairs, designated as bumper and
amplification primers.
Advantage: isothermal process that it can be performed at a single temperature after initial target denaturation
a. Helicase - Dependent Amplification
b. Loop - Mediated Amplification (LAMP)
c. nucleic acid sequence-based amplification (NASBA)
d. Strand - Displacement Amplification
e. transcription-mediated amplification (TMA)
Strand - Displacement Amplification
122
In the presence of BsoB1, and a dNTP mixture consisting
of dUPT, dATP, dGTP and thiolated dCTP (Cs), simultaneous extension products of both the bumper and amplification primers are generated
a. Helicase - Dependent Amplification
b. Loop - Mediated Amplification
c. nucleic acid sequence-based amplification (NASBA)
d. Strand - Displacement Amplification
e. transcription-mediated amplification (TMA)
d. Strand - Displacement Amplification
123
Isothermal amplification technology
transcription-mediated amplification (TMA)
nucleic acid sequence-based amplification (NASBA)
Strand - Displacement Amplification
Loop - Mediated Amplification (LAMP)
Helicase - Dependent Amplification
124
An isothermal method that relies on auto-cycling strand displacement DNA synthesis by Bst DNA polymerase and a set of four to six primers.
Advantage: Requires no expensive equipment
a. Helicase - Dependent Amplification
b. Loop - Mediated Amplification
c. nucleic acid sequence-based amplification (NASBA)
d. Strand - Displacement Amplification
e. transcription-mediated amplification (TMA)
b. Loop - Mediated Amplification
125
Signal amplification assays have several advantages over target amplification assays.
o The number of target molecules is not altered and, as a result, the signal is directly proportional to the amount of target sequence present in the clinical specimen.
o This reduces concerns about false-positives due to cross contamination and simplifies the development of quantitative assays.
• Because signal amplification systems are not dependent on enzymatic processes to amplify target sequences, they are not affected by the presence of enzyme inhibitors in clinical setting.
126
A probe amplification method that relies on the specific region and cleavage of particular DNA structures by members of the flap endonuclease-1 family of DNA polymerases.
Advantage: This technology can easily be adapted to detect point mutations of interest by designing the overlap region to encompass the mutation to be detected.
a. Branched DNA
b. Cleavase/Invader technology
c. Hybrid Capture Assays
b. Cleavase/Invader technology
127
If in signal amplification, the concentration of the probe or target does not increase how is there sensitivity?
The increase analytical sensitivity comes from the increasing concentration of label molecules attached to the target nucleic acid.
128
The key to this technology is the amplifier molecule, a DNA molecule with 15 identical branches, each of which can bind three labeled probes.
a. Branched DNA
b. Hybrid Capture Assays
a. Branched DNA
129
A solution hybridization antibody capture assay that uses chemiluminiscent detection.
a. Branched DNA
b. Hybrid Capture Assays
b. Hybrid Capture Assays
DNA-RNA hybrids are captured by antibodies specific for DNA-RNA hybrids that are coated on the surface of the tube.
• Alkalinephosphatase–conjugated antihybrid antibodies bind to the immobilized hybrids. The bound enzyme– antibody conjugate is detected with a chemiluminescent substrate and the light emitted is measured in a luminometer.
130
Advantages of microarrays
Saves time and cuts costs ·
Reduce reagent consumption ·
increases sample concentration
·Improves reaction kinetics
131
General schema of microarrays
o Generation of nucleic acid complementary to genes of interest
o Laid out in microscopic quantities on solid surfaces at defined positions nucleic acid
o cDNA is added to the surface and binds in the presence of DNA o detected by fluorescence – laser excitation
132
Limitations of microarray
· Ensuring reproducibility of high-density data presented in microarrays is unprecedented in the clinical laboratory
·Varying methods used for nucleic acid isolation or detection, tissue type, type of specimen, and time of processing can affect results of gene expression profiling.
·Need to further validate and confirm array-based results
·Microarrays have limited sensitivity for minority of alleles ·Caution must be exercised in interpreting differentially regulated genes during tumor progression (staging microarray).
·Biopsy tissues as source of nucleic acid may only represent homogenous tissue components only after tumor mass have reached enough size.
133
Gene
a sequence of nucleotides that represents a functional unit of inheritance; a region of DNA that codes for a product, either RNA or protein
134
§Chromosome
o highly ordered structure composed of DNA and proteins that carries the genetic information
o Humans: 46 chromosomes ordered in pairs
135
§Autosome
o all chromosomes other than X and Y chromosomes which are designated the sex chromosomes.
136
§Homologous chromosomes or homologs
o sister chromosomes, the members of a pair of chromosomes in which one is inherited from the mother and the other from the father
137
§Locus
o position of a gene on a chromosome
138
§Mutation
o a permanent heritable change in the sequence of genomic DNA
o may manifest at both molecular and cytogenic levels
o not all are negative events
o many are benign and some have positive effects
139
§Allele
o alternative form of a gene occupying the same locus. An allele may be the result of mutation.
o Maximum of 2 alleles per diploid chromosome complement (1 allele/chromosome)
o Multiple alleles may exist within population
140
§Constitutional mutation
o May pass mutation on their progeny by germline transmission §Acquired mutation
o Arise in a single somatic cell, which then divides mitotically, giving rise to a new clone of cells (ex. Cancer)
141
§Gonadal mosaicism
o Mutation will be limited to the clone
o Will not be transmitted to progeny of the individual
o May arise in the gonads, resulting in a mixed population of normal and mutant gametes
o Progeny receiving the new mutation may display a phenotype present in either parent
142
§Karyotype
o chromosome constitution of an individual
143
§Karyogram
o a figure showing the paired chromosomes from a cell arrayed in a standard sequence
144
§Diploid (2N)
o the presence of two copies of each unique chromosome per cell
o humans: chromosome occurs in pairs and the diploid number: 46
145
§Haploid (N)
o Humans: gametes are haploid: N=23
146
§Hemizygous
o presence of only one chromosome or chromosome segment rather than the usual two; applies to males with a single X chromosome
147
§Homozygous
o both alleles at locus are the same
148
§Heterozygous
o two alleles at locus are different
149
§Genotype
o genetic constitution of and individual or organism: what alleles are present
150
§Recessive allele
o In a diploid organism, an allele that is only expressed when homozygous
151
§Phenotype
o the appearance of an individual that results from the interaction of environment and genotype
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§Dominant allele
o an allele that is expressed when present in only a single dose
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§Codominant allele
o In a diploid organism, alleles that show no dominance or recessivity to each other but, when present together, are both fully expressed
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§Crossing Over
o physical exchange of genetic material between homologous chromosomes
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§Independent assortment
o Random assortment of chromosomes in the gametes; 50:50 chance of inheriting a given chromosome from one parent
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§Linkage
o The presence of two or more genes on the same chromosome that tend to be linked together
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§Recombination
o generation of new allelic combinations on chromosomes, usually by crossing over
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§Non disjunction
o Failure of chromosomes or chromatids to separate to opposite poles in cell division. Usually results in one too many or one too few chromosomes in a cell
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§Meiosis
o Cell division in the gonads that produces the gametes. A single DNA replication is followed by two cell divisions which reduces the total DNA content of a cell from 2N to N
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§Mitosis
o Somatic cell division in which the DNA replicates and I evenly distributed to two equal daughter cells
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The only clinical laboratory test to be able to survey the cellular genetic constitution of an individual with a single assay
CYTOGENETICS
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basis for most cytogenetic studies
most condensed form: METAPHASE
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o Also called primary constriction
o Hold two chromatids together
o Unreplicated region of DNA
o Acts as a landmark and divides the chromosome into two distinct regions known as ARMS
Centromere
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Chromatid
o Each double helix
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P arm
a. longer arm
b. shorter arm
b. shorter arm
----
Q arm
·Longer arm
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BANDING PATTERNS
§G banding:
§Q banding:
§C banding:
§R banding:
G banding for Geimsa stain
§ Q banding: quinacrine florescence staining for rapid identification of the Y chromosome
§C banding: constitutive or centromere banding used to evaluate constitutive heterochromatin / whether a chromosome has 2 centromeres
§R banding: reverse banding light and dark bands in G banding are reversed
