PRE FI GENE MUTATION Flashcards
- PERMANENT ALTERATION in the DNA sequence that makes up a gene
- Causes: ERRORS IN DNA REPLICATION, EXPOSURE to various ENVIRONMENTAL FACTORS (radiation, chemicals, or certain viruses)
- Wide range of effects: harmless, genetic disorders, development of diseases (cancer)
GENE MUTATION
TYPE OF MUTATION:
- substitution of 1 nucleotide, will NOT CHANGE the amino acid sequence
SILENT
TYPE OF MUTATION:
- CHANGE the amino acid sequence, but the replacement & the original amino acid HAVE SIMILAR BIOCHEMICAL PROPERTIES
CONSERVATIVE
TYPE OF MUTATION:
- replacement of amino acid w/ a BIOCHEMICALLY DIFFERENT AMINO ACID
NONCONSERVATIVE
TYPE OF MUTATION:
- terminates proteins prematurely when a nucleotide substitution produces a STOP CODON
NONSENSE
TYPE OF MUTATION:
- addition/deletion of nucleotides in a DNA sequence disrupts the READING FRAME
FRAMESHIFT
purine replaces a purine or pyrimidine with a
pyrimidine.
TRANSITION
purine replaces a pyrimidine or
pyrimidine replaces a purine.
TRANSVERSION
Used to DIRECTLY ANALYZE the CHANGE in protein structure of function.
Other uses:
o Metabolic defects where several genes are
involved in the disease phenotype.
o Detection of the actual protein/ amino acid alterations.
BIOCHEMICAL METHODS
BIOCHEMICAL METHODS INLUDE
- Enzyme Immunoassays
- Immunohistochemistry
- High-performance liquid chromatography
(HPLC) - Gas chromatography
- Mass spectrometry
Detects the PRESENCE OF METABOLITES in the blood, urine, or other biological fluids.
Involve the USE OF SPECIFIC ANTIBODIES or other ligands to detect the presence of the target molecules.
- useful in the detection of antibodies against infectious agents
- Antibody specific for the target analyte is immobilized in plate wells. If present, antigen binds to the antibody and is detected with a secondary antibody conjugated to enzyme (AP, alkaline phosphatase)
Enzyme Immunoassays
Widely used EIA (Enzyme Immunoassays)
enzyme-linked
immunosorbent assay (ELISA)
Longstanding method that allows DETECTION OF PROTEIN ABNORMALITIES on situ.
Formalin-fixed paraffin-embedded tissue: <5 microns slices (microtome)
Fixation can affect tissue antigens –> altering/covering some EPITOPES–> can be solved by antigen retrieval:
a. ENZYME DIGESTION (proteinase K, chymotrypsin, pepsin, pronase)
b. HEATING TISSUE SECTIONS IN WATER/BUFFER
Snap frozen tissue (in isopentane, at -160C): 5 to 15-micron slices (cryostat inside of a chamber held at 20C)
Fixation: acetone
Sections are dried and stored frozen
Rehydration of the dried sections in PHOSPHATE-BUFFERED SALINE
Immunohistochemistry
To avoid the effects of formalin fixation, ____________________
may be used
snap frozen tissue
Substances such as ______, _________, or__________ in the tissue may interfere with IHC results
- endogenous peroxidase
- fluorescence
- nonspecific antibodies
blocking solution with serum protein (albumin),
detergent (Tween 20), and unlabeled antibodies
PURPOSE?
TO MINIMIZE NONSPECIFIC BINDING
included with samples to ensure the ADEQUACY and SPECIFICITY of staining.
POSITIVE AND NEGATIVE CONTROLS
Imaging/ microscopic observation of antibody
requires a signal from the antibody:
- fluorescent molecules
(fluorescein, Cy5, phycoerythrin)
FLUORESCENT SIGNAL
Imaging/ microscopic observation of antibody
requires a signal from the antibody:
- substrate solution is
added, oxidized by the enzymes (horseradish peroxidase/
o Most frequently used: red/ brown IHC staining
COLORIMETRIC SIGNAL
SEPARATION OF MOLECULES (nucleic acids and proteins) in solution through interaction with a solid support in the column.
High-performance liquid chromatography
(HPLC)
2 PHASES OF High-performance liquid chromatography
(HPLC)
- MOBILE PHASE (SOLVENT)
- STATIONARY PHASE (SOLID SUPPORT)
INCREASE RESOLUTION and LOWER SEPARATION TIME while using less solvent; faster flow rates (5mL/ min).
Ultra-HPLC (UHPLC)
HPLC Types of detectors:
- LIGHT SCATTERING
- FLUORESCENCE
- REFRACTIVE INDEX
- UV LIGHT ABSORPTION
- MASS SPECTROMETRY
- SEPARATION OF VAPORIZED SAMPLE through a column of inert carrier gas (mobile phase) & liquid (stationary phase)
- used for DETECTION OF DRUGS & POISONS & their metabolites in biological samples
- may be COUPLE W/ MS to detect BIOMARKERS OF DISEASE
Gas chromatography
Gas chromatography DETECTOR?
FLAME IONIZATION DETECTOR
- CONVERTS MOLECULES TO ION that can be moved in
a magnetic field based on their CHARGE and MASS. - Readout: spectrum with mass/ charge value on the x-axis and abundance of the ion on the y-axis
ID of molecule: by their characteristic spectrum/ set of peaks
Mass spectrometry
Mass spectrometry
2 ionization methods for large biomolecules
(proteins):
- test sample is converted into a FINE SPRAY OF CHARGED DROPLETS that are electrostatically directed to the mass spectrometer inlet
- MULTIPLE IONIZED SPECIES FROM ONE PROTEIN are separated by mass and charge.
- results are plotted as mass/charge ratio (m/z) and relative abundance
Electrospray ionization (ESI)
Mass spectrometry
2 ionization methods for large biomolecules
(proteins):
- produces ions by firing a laser pulse into the sample coated with a matrix (organic compound)
- sample is adhered to the matrix on a sample plate, ionized (protonated)
Matrix-assisted laser desorption/ ionization (MALDI)
MS:
- high MW molecules
- ionized molecules are accelerated at a fixed point & allowed to drift through the flight tube to the detector
MALDI - TOF (time-of-flight) spectrometer
MS:
- combined w/ TOF
- offers flexibility in the ID & quantification of properties
Surface - enhanced laser desorption/ionization (SELDI)
Performed on a variety of specimen types: blood or buccal cells.
DNA mutations from single-base pair changes to large chromosomal rearrangements can be detected.
NUCLEIC ACID ANALYSES
simplified mutation detection (limiting specimens).
PCR amplification
most definitive method for detecting mutations.
DNA sequencing
Hybridization-based Methods
a. Single-Strand Conformation Polymorphism (SSCP)
b. Allele-Specific Oligomer Hybridization (ASO)
c. Melt-Curve Analysis (MCA)
d. Heteroduplex Analysis
e. Array Technology
Hybridization-based Methods:
Based on the PREFERENCE OF DNA to EXIST in a double-stranded state.
ABSENCE OF THE COMPLEMENTARY STRAND: nucleic acids form intrastrand duplexes, 3D structure (conformer).
o Shape: kinks, loops, bubbles, tail
Bands/peaks pattern
- different from the normal sequence (control) conformers –> presence of gene mutation
- detected by SILVER STAIN, RADIOACTIVITY, or FLUORESCENCE
a. Melt-Curve Analysis (MCA)
b. Heteroduplex Analysis
c. Single-Strand Conformation Polymorphism (SSCP)
d. Array Technology
e. Allele-Specific Oligomer Hybridization (ASO)
c. Single-Strand Conformation
Hybridization-based Methods:
Utilizes DIFFERENCES in the Tm OF SHORT SEQUENCES
(20 bases) with 1 or 2 mismatches and those
with no mismatches.
SYNTHETIC SS-PROBES (labeled) with normal/ mutant target DNA sequence (immobilized) in a solution.
At specific annealing temperatures and conditions (STRINGENCY)
a. Probe will not bind to a near complementary
target sequences with 1 or 2 mismatched bases
b. Probe with perfect complementary sequence will bind
a. Melt-Curve Analysis (MCA)
b. Heteroduplex Analysis
c. Single-Strand Conformation Polymorphism (SSCP)
d. Array Technology
e. Allele-Specific Oligomer Hybridization (ASO)
Allele-Specific Oligomer Hybridization (ASO)
Allele-Specific Oligomer Hybridization (ASO)
Bound probes will be detected with a conjugated _______________ and will be exposed to a CHROMOGENIC/CHEMILUMINESCENT SUBSTRATE generating a COLOR/ LIGHT SIGNAL
indicating the binding of the test DNA to the probe.
horseradish peroxidase- anti biotin Fab fragment
Hybridization-based Methods:
Method of analyzing the DISSOCIATION OF ds-DNA during the HEATING CYCLES
PCR amplicons in the presence of a DNA specific fluorescent dye (EtBr, SYBR green, LC green) are heated (0.3C/ sec).
Rise in the temperature, DNA duplexes begin to
separate into single strands, losing the dye.
Black line –> targets with different mismatches to the hybridization probe.
Sequence differences result in different melting characteristics and Tm for each sequences.
Interpretation of results by the temperature peak placement with respect to the temperature on the x-axis
a. Melt-Curve Analysis (MCA)
b. Heteroduplex Analysis
c. Single-Strand Conformation Polymorphism (SSCP)
d. Array Technology
e. Allele-Specific Oligomer Hybridization (ASO)
Melt-Curve Analysis (MCA)
Melt-Curve Analysis (MCA)
- overlaying peaks at expected Tm
Specimen with identical sequences
Melt-Curve Analysis (MCA)
– 2 or more peaks at different temperatures
Specimen with different sequences
Hybridization-based Methods:
Formed when single strands that are NOT COMPLETELY COMPLEMENTARY HYBRIDIZE TO ONE ANOTHER
Can be resolved through POLYACRYLAMIDE/
AGAROSE GEL ELECTROPHORESIS: presence of bands different from a homozygous reference
control is indicative of mutation.
Can also be resolved in denaturing high-
performance liquid chromatography (DHPLC).
a. Melt-Curve Analysis (MCA)
b. Heteroduplex Analysis
c. Single-Strand Conformation Polymorphism (SSCP)
d. Array Technology
e. Allele-Specific Oligomer Hybridization (ASO)
Heteroduplex Analysis
Hybridization-based Methods:
- HIGH DENSITY OLIGONUCLEOTIDE ARRAYS: test DNA is
fragmented by treatment with DNase before binding to the complementary probes on the array.
a. Melt-Curve Analysis (MCA)
b. Heteroduplex Analysis
c. Single-Strand Conformation Polymorphism (SSCP)
d. Array Technology
e. Allele-Specific Oligomer Hybridization (ASO)
Array Technology
Array Technology Hybridization formats:
– base substitution is always in the 12th position from the 3’ end of the probe.
Standard tiling
Array Technology Hybridization formats:
– same mutation ID placed at different positions in the probe.
Redundant tiling
Array Technology Hybridization formats:
– uses sets of color-
coded polystyrene beads in suspension as the solid matrix.
Bead array technology
Sequence (polymerization) - Based Methods
- DETECT POINT MUTATIONS & other SNPS
- Primer 3’ end falls on the nucleotide to be analyzed
o Must match the template perfectly to be
extended by Taq polymerase.
o Presence/ absence of the product = presence/
absence of the mutation.
a. Sequence-Specific (Primer) PCR (SSP-
PCR)
b. Allelic Discrimination with Fluorogenic
Probes
Sequence-Specific (Primer) PCR (SSP-
PCR)
Sequence (polymerization) - Based Methods
Thermal cyclers with fluorescent detection.
RT-PCT, using 2 PROBES labeled 3’ quencher molecules and different fluors on the 5’ ends (complementary to either normal/ mutant sequence).
Prescence of corresponding fluorescent signal
indicates whether the test sequences is normal/ mutant.
a. Sequence-Specific (Primer) PCR (SSP-
PCR)
b. Allelic Discrimination with Fluorogenic
Probes
Allelic Discrimination with Fluorogenic
Probe
Enzymatic/ chemical Cleavage Methods:
Can detect sequence alterations
Mutand changes the structure of a restriction
enzyme target site/ changes the size of a
fragment.
PCR-RFLR is used
A. Nonisotopic RNase Cleavage Assay (NIRCA)
B. Cleavage Assay
C. Restriction Fragment Length Polymorphisms (RFLPs)
Restriction Fragment Length Polymorphisms (RFLPs)
Enzymatic/ chemical Cleavage Methods:
Heteroduplex analysis using duplex RNA
T7 or SP6 phage RNA polymerase
Detection mutation: heteroduplexes form
between normal and mutant transcripts
targets for cleavage by RNase enzymes (E.
coli RNaw and Aspergillus RNase T1)
Remaining dsRNA fragments can then be
separated by agarose gel electrophoresis.
A. Nonisotopic RNase Cleavage Assay (NIRCA)
B. Cleavage Assay
C. Restriction Fragment Length Polymorphisms (RFLPs)
Nonisotopic RNase Cleavage Assay (NIRCA)
Enzymatic/ chemical Cleavage Methods:
Bases on the characteristic enzymatic activity of CLEAVASE .
Premixed reagents (including cleavase) +
standard 96 well-plate + test specimens +
controls
Clevaase: recognizes the structure formed by
hybridization of the normal/ mutant probes to the test sequences
If the probe and test reactions occur –>
fluorescent signal (standard fluorometer)
A. Nonisotopic RNase Cleavage Assay (NIRCA)
B. Cleavage Assay
C. Restriction Fragment Length Polymorphisms (RFLPs)
Cleavage Assay
provide specific multiplex detection and sensitivity required for clinical applications.
Array-based methods and massive parallel
sequencing methods
Method selected will depend on the _________________, ______________, and the
_____________
- available instrumentation
- genetic target
- nature of mutation
Descriptive and consistent system of expressing
mutations/ polymorphisms.
GENE VARIANT NOMENCLATURE
Indicated by:
o Position of the mutation in the genomic sequence of the DNA/ position from the end of the coding sequence (exons) + position in the intron
INTRONS OF GENOMIC DNA
- Set by the Human Genome Organization
(HUGO) gene nomenclature committee - should be capitalized and set in italics with no hyphens
GENE NAMES
