Chapter 9 Flashcards
Deletions, insertions, inversions, translocations, and other changes that can affect one base pair to hundreds or thousands of base pairs
Gene mutations
Alterations of a single or a few base pairs
Point mutations
Types of gene mutation
Silent mutation
Conservatove mutation
Non-conservative mutation
Framshift mutation
Substitution of one nucleotide with a different nucleotide
Silent
Without changing the amino acid sequence
Silent
Substitutions may change the amino acid sequence
Conservative
But the replacement and the original amino acid have similar biochemical properties, e.g., leucine for valine, change will not affect protein function significantly
Conservative
Substitution of a biochemically different amino acid, e.g., proline for glutamine, which changes the biochemical nature of the protein
Non conservative
Terminates proteins prematurely when a nucleotide substitution produces a stop codon instead of an amino-acid
Nonsense
Insertion or deletion of more or efewer than three nucleotides throwing the triplet code out of frame
Frameshift
Detected from the most convenient and noninvasive specimen material, such as blood or buccal cells
Inherited mutation
Often more challenging to find because cells harboring mutations may be only a small fraction of the total specimen that consists of mostly normal cells
Somatic mutation
Under these circumstances, detection procedures must identify a single mutated gene from among thousands of normal genes
Somatic mutation
Three Broad Approaches:
- Hybridization - based methods
2 sequence ( polymerization) - based methods - Enzymatic or chemical cleavage methods
more frequently used mutation screening
SINGLE-STRAND CONFORMATION POLYMORPHISMS (SECP)
is determined by the migration of the single-stranded
conformers in polyacrylamide gels under precisely controlled denaturing and temperature conditions.
SINGLE-STRAND CONFORMATION POLYMORPHISMS (SECP)
Steps in SSCP
short,double-stranded PCR produts➡️Dilute
long ➡️Denatured followed by rapid cooling
SSCP DENATURATION OF DNA
10-10 mM NaH, 80% formamide for 5 mins at 95°c
SSCP DENATURATION IF EDTA
0.004 mM EDIA 10% formamide for 5 mins
at 55°- 60°C
the _____________________ depends on the complementary
nucleotides available for hydrogen bonding and folding
shape of the conformer
SSCP CAN BE RESOLVED in
polyacrylamide gel or by capillary electrophoresis
with temperature con tron
SSCP band or peak patterns are detected by
silver stain, radioactivity , or fluorescent
SSCP CAN BE Reported to detect ___________% of putative mutations.
Reported to detect 35-100% of putative mutations.
exploits differences in denaturation between a normal &
mutated DNA molecure caused by even one nucleotide
difference in a sequence
DENATURING GRADIENT GEL ELECTROPHORESIS (DEGE)
stacking can affect denaturation of asDNA.
DENATURING GRADIENT GEL ELECTROPHORESIS (DEGE)
DGGE ds DNA fragments________ bp
200-700
DGGE fragments are separated on polyacrylamide gels
containing a gradient of concentrations
urea and formamide
As the dsDNA fragments moves through the gel, the
denaturing conditions _____________, sequences reach their
denaturing point, and the complementary strands
begin to denature.
Increases
TWO gradient orientation used in D66P:
PERPENDICULAR DGGE
PARALLEL DGGE
PERPENDICULAR DGGE
Horizontal
Parallel DGGE
Parallel
TwO methods that are similar in design to DGGE:
CONSTANT GRADIENT GEL ELECTROPHORESIS (CDGE)
TEMPORAL TEMPERATURE GRADIENT GEL ELECTROPHORESIS
(IT6E)
for detecting known mutations than for screening
CONSTANT GRADIENT GEL ELECTROPHORESIS (CDGE)
Requires the initial determination of optimal denaturant concentration for a particular gene mutation
CONSTANT GRADIENT GEL ELECTROPHORESIS (CDGE)
ascertained by perpendicular DEGE or by using computer
programs designed to predict the melting characteristics of a nuleotide sequence for a range of temperature and
denaturing concentrations
CONSTANT GRADIENT GEL ELECTROPHORESIS (CDGE)
similar 10 CDGF (difference is temperature) In that
specific concentrations of formamide and urea are used to denature DNA dupiexes
TEMPORAL TEMPERATURE GRADIENT GEL ELECTROPHORESIS
(IT6E)
differences in denaturation unlike DG, are resolved by
slowly raising the temperature of the gel during migration
TEMPORAL TEMPERATURE GRADIENT GEL ELECTROPHORESIS
(TTGE)
utilizes the differences in melting temperatures of short
sequences of -20 bases with one or two mismatches and
those with no mismatches.
ALLELE - SPECIFIC OLIGOMER HIBRIDIZATON
a single- stranded probe will not bind to a near
complementacy target sequence with one or two
mismatched bases, whereas a probe perfectly complementary to the target sequence will bind
specific annealing temperatres and conditions (stringency),
a DOT BLOT method, similar to vouthern bior using
immobilized targer and jabered probe in solution-
ASO
AsO can also be carried out as a _____________
to capture probe methoas developed for infectious
disease testing.
Reverse dot blot
ASO method has been proposed for detection of fre
quently occuring mutations such as
factor V Leiden.
exploits tne sequence- and stacking - directed denaturation characteriStes of DNA duprexes.
MELT CURVE ANALYSIS
very useful as a post amplification step of RT-PCR.
Melt curve analysis
PR amplicons generated in the presence of a DNA-
specific fluorescent due such as
etmidium bromide
uses fluorescent resonance energy transfer (FRET)
propes that hybridize next to ong another across the
sequence position being anargned.
HIGH RESOLUTON MELI CURVE ANALYSIS (AR-MCA)
method for detection of SNPS in DNA
Inversion probe assay
the probe hybridizes to the targer sequence, the
two ends flanking the potential SP being tested
INVERSION PROBE ASSAY
formed when a single strands that
are not complementary hybridize to ano another
HETERODUPLEX ANALYSIS
Gel-based heteroduplex methods have been designed to
HN typing and hematological testing.
