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MOL. BIO LEC > PRE FI GENE MUTATION > Flashcards

PRE FI GENE MUTATION Flashcards

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Biology 101 flashcards
1
Q
  • 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)
A

GENE MUTATION

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2
Q

TYPE OF MUTATION:
- substitution of 1 nucleotide, will NOT CHANGE the amino acid sequence

A

SILENT

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3
Q

TYPE OF MUTATION:
- CHANGE the amino acid sequence, but the replacement & the original amino acid HAVE SIMILAR BIOCHEMICAL PROPERTIES

A

CONSERVATIVE

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4
Q

TYPE OF MUTATION:
- replacement of amino acid w/ a BIOCHEMICALLY DIFFERENT AMINO ACID

A

NONCONSERVATIVE

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5
Q

TYPE OF MUTATION:
- terminates proteins prematurely when a nucleotide substitution produces a STOP CODON

A

NONSENSE

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6
Q

TYPE OF MUTATION:
- addition/deletion of nucleotides in a DNA sequence disrupts the READING FRAME

A

FRAMESHIFT

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7
Q

purine replaces a purine or pyrimidine with a
pyrimidine.

A

TRANSITION

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8
Q

purine replaces a pyrimidine or
pyrimidine replaces a purine.

A

TRANSVERSION

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9
Q

 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.

A

BIOCHEMICAL METHODS

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10
Q

BIOCHEMICAL METHODS INLUDE

A
  1. Enzyme Immunoassays
  2. Immunohistochemistry
  3. High-performance liquid chromatography
    (HPLC)
  4. Gas chromatography
  5. Mass spectrometry
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11
Q

 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)

A

Enzyme Immunoassays

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12
Q

Widely used EIA (Enzyme Immunoassays)

A

enzyme-linked
immunosorbent assay (ELISA)

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13
Q

 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

A

Immunohistochemistry

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14
Q

To avoid the effects of formalin fixation, ____________________
may be used

A

snap frozen tissue

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15
Q

Substances such as ______, _________, or__________ in the tissue may interfere with IHC results

A
  • endogenous peroxidase
  • fluorescence
  • nonspecific antibodies
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16
Q

blocking solution with serum protein (albumin),
detergent (Tween 20), and unlabeled antibodies

PURPOSE?

A

TO MINIMIZE NONSPECIFIC BINDING

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17
Q

included with samples to ensure the ADEQUACY and SPECIFICITY of staining.

A

POSITIVE AND NEGATIVE CONTROLS

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18
Q

Imaging/ microscopic observation of antibody
requires a signal from the antibody:
- fluorescent molecules
(fluorescein, Cy5, phycoerythrin)

A

FLUORESCENT SIGNAL

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19
Q

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

A

COLORIMETRIC SIGNAL

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20
Q

 SEPARATION OF MOLECULES (nucleic acids and proteins) in solution through interaction with a solid support in the column.

A

High-performance liquid chromatography
(HPLC)

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21
Q

2 PHASES OF High-performance liquid chromatography
(HPLC)

A
  • MOBILE PHASE (SOLVENT)
  • STATIONARY PHASE (SOLID SUPPORT)
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22
Q

INCREASE RESOLUTION and LOWER SEPARATION TIME while using less solvent; faster flow rates (5mL/ min).

A

Ultra-HPLC (UHPLC)

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23
Q

HPLC Types of detectors:

A
  • LIGHT SCATTERING
  • FLUORESCENCE
  • REFRACTIVE INDEX
  • UV LIGHT ABSORPTION
  • MASS SPECTROMETRY
24
Q
  • 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
A

Gas chromatography

25
Q

Gas chromatography DETECTOR?

A

FLAME IONIZATION DETECTOR

26
Q
  • 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
A

Mass spectrometry

27
Q

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

A

Electrospray ionization (ESI)

28
Q

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)

A

Matrix-assisted laser desorption/ ionization (MALDI)

29
Q

MS:
- high MW molecules
- ionized molecules are accelerated at a fixed point & allowed to drift through the flight tube to the detector

A

MALDI - TOF (time-of-flight) spectrometer

30
Q

MS:
- combined w/ TOF
- offers flexibility in the ID & quantification of properties

A

Surface - enhanced laser desorption/ionization (SELDI)

31
Q

 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.

A

NUCLEIC ACID ANALYSES

32
Q

simplified mutation detection (limiting specimens).

A

PCR amplification

33
Q

most definitive method for detecting mutations.

A

DNA sequencing

34
Q

Hybridization-based Methods

A

a. Single-Strand Conformation Polymorphism (SSCP)
b. Allele-Specific Oligomer Hybridization (ASO)
c. Melt-Curve Analysis (MCA)
d. Heteroduplex Analysis
e. Array Technology

35
Q

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)

A

c. Single-Strand Conformation

36
Q

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)

A

Allele-Specific Oligomer Hybridization (ASO)

37
Q

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.

A

horseradish peroxidase- anti biotin Fab fragment

38
Q

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)

A

Melt-Curve Analysis (MCA)

39
Q

Melt-Curve Analysis (MCA)
- overlaying peaks at expected Tm

A

Specimen with identical sequences

40
Q

Melt-Curve Analysis (MCA)
– 2 or more peaks at different temperatures

A

Specimen with different sequences

41
Q

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)

A

Heteroduplex Analysis

42
Q

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)

A

Array Technology

43
Q

Array Technology Hybridization formats:
– base substitution is always in the 12th position from the 3’ end of the probe.

A

Standard tiling

44
Q

Array Technology Hybridization formats:
– same mutation ID placed at different positions in the probe.

A

Redundant tiling

45
Q

Array Technology Hybridization formats:
– uses sets of color-
coded polystyrene beads in suspension as the solid matrix.

A

Bead array technology

46
Q

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

A

Sequence-Specific (Primer) PCR (SSP-
PCR)

47
Q

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

A

Allelic Discrimination with Fluorogenic
Probe

48
Q

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)

A

Restriction Fragment Length Polymorphisms (RFLPs)

49
Q

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)

A

Nonisotopic RNase Cleavage Assay (NIRCA)

50
Q

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)

A

Cleavage Assay

51
Q

provide specific multiplex detection and sensitivity required for clinical applications.

A

Array-based methods and massive parallel
sequencing methods

52
Q

Method selected will depend on the _________________, ______________, and the
_____________

A
  • available instrumentation
  • genetic target
  • nature of mutation
53
Q

Descriptive and consistent system of expressing
mutations/ polymorphisms.

A

GENE VARIANT NOMENCLATURE

54
Q

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

A

INTRONS OF GENOMIC DNA

55
Q
  • Set by the Human Genome Organization
    (HUGO) gene nomenclature committee
  • should be capitalized and set in italics with no hyphens
A

GENE NAMES

MOL. BIO LEC (15 decks)

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