MUTATION DETECTION Flashcards
CTGGAG
CTGGGG
A arrow to 4th G
what mutation is this?
Point mutation (base substitution)
DETECTION OF THE KNOWN
give me the 4
- Allele-Specific Oligonucleotide Hybridization
- DNA Microarray
- DNA Sequencing
- Multiplex Ligation-Dependent Probe Amplification (MLPA)
ALLELE-SPECIFIC OLIGONUCLEOTIDE
HYBRIDIZATION also called
“DOT-BLOT” Method
Alternative form of a gene due to
mutations in the same place on a chromosome or
DNA sequence
ALlele
Relies on specific binding of known chromogenic
probes on DNA and detection of probes thereafter
ALLELE-SPECIFIC OLIGONUCLEOTIDE
HYBRIDIZATION
The healthy cell is probed with green fluorescence and the pathologic one is probed with red. And then, you mixed them together on a tube and placed it on each of those dots. And in each of those dots, they have specific genes that they detect or probe for.
DNA MICROARRAY
We need to know the sequence that we want to look for so that we can make a primer for it.
DNA SEQUENCING
5’ - TCGTGTCGATAGCGCT - 3’
The type of sequencing above is Maxam-Gilbert
tama ang question? :(
automated sequencing
electropherogram
Multiplex PCR makes use of _____
multiple primer
MLPA will only detect ______________________
insertions and deletions (base indels)
MLPA cannot detect
single base changes
Under MLPA
When amplified, a ratio of 1:1 will be the result if ____________
there are no mutations detected = NORMAL
DETECTION OF THE UNKNOWN
give me the 6
- Single-Strand Conformation Polymorphism
- Denaturing Gradient Gel Electrophoresis
- Temperature Gradient Gel Electrophoresis
- Restriction Fragment Length Polymorphism
- Heteroduplex Analysis
- Single Strand Specific Nucleases
takes advantage of the secondary structures that form for single-stranded DNA in non-denaturing
conditions
SINGLE-STRAND CONFORMATION
POLYMORPHISM
SINGLE-STRAND CONFORMATION
POLYMORPHISM
Different folding pattern = Different _______
migration pattern
SINGLE-STRAND CONFORMATION
POLYMORPHISM
Fragment size limit: ___
150-200 bp (highly limited)
SINGLE-STRAND CONFORMATION
POLYMORPHISM
Detects ___%
80-90% of unknown mutations
Modification of gel electrophoresis that uses different intensities of a denaturing environment in the same set-up
DENATURING GRADIENT GEL
ELECTROPHORESIS
WHAT ARE EXAMPLES OF DENATURING CHEMICALS?
○ Formamide
○ Urea
DENATURING GRADIENT GEL
ELECTROPHORESIS
Fragments melt in a ___ manner based on its melting profile
stepwise
DNA MELTING PROFILE
__ = weaker bonds, hence, they melt first
AT bond
DNA MELTING PROFILE
__ = last to melt due to their strong bond
GC bond
DNA MELTING PROFILE
Lowest denaturing concentration =
______
UPPERMOST PART OF THE GEL
○ They have lesser GC and more AT
DNA MELTING PROFILE
Highest denaturing concentration =
_____
LOWERMOST PART OF THE GEL
○ They have more GC, thus, they denature earlier.
Modification of DGGE that uses temperature instead of a denaturing chemical.
TEMPERATURE GRADIENT GEL
ELECTROPHORESIS
T or F
chemical gradient is more reproducible and reliable than temperature gradient.
F
Uses the different restriction cut sites to detect mutations.
RESTRICTION FRAGMENT LENGTH
POLYMORPHISM
HETERODUPLEX ANALYSIS
○ dsDNA with one or more mismatched
pairs.■ Wt – Mu
Heteroduplex
HETERODUPLEX ANALYSIS
○ Perfectly paired dsDNA.
■ Wt (Wild Type) – Wt
■ Mu (Mutant) – Mu
○ Faster mobility in gel.
Homoduplex
These ____ will cut kung merong naging bulge. Kung merong naging
mutation na hindi nag-perfectly pair. It will cut on those sides. Then, it will be release. After that, ang naiwan na lang ang need pang i-detect. So, the
reading will be much easier.
this is all i can find sowwy.
single-strand specific nucleases
