NUCLEIC ACID HYBRIDIZATION Flashcards
Takes advantage of the bonding of singlestranded nucleic acids with their complementary sequences
to identify specific DNA sequences.
nucleic acid hybridization
uses of nucleic acid hybridization
✓ Detection of specific sequences
in a mixture of DNA fragments or
gDNA
✓ Determination of base sequence
similarities and changes
Single-stranded
fragment of nucleic
acid attached to a
signal-producing
moiety to identify
sequence/s of interest
Probe
Denatured DNA or RNA
that has good
complementarity
to the probe for
identification
TARGET
PROBE TYPE: DNA
Early method of production:
- Cloning into a plasmid
- Isolation by RE digestion
- Gel purification
- Labeling
- Denaturation (heating alone or
with formamide)
T OR F???
* Shorter probes are more specific
- Longer probes are better for
mutational analyses
F
* Longer probes are more specific
* Shorter probes are better for
mutational analyses
- Made by in vitro transcription from DNA (from plasmid template or PCR amplicons)
PROBE TYPE: RNA
LABELING THE PROBE
END Labeling
Labeled molecules added to the end using ___
terminal transferase
or T4 polynucleotide
kinase
Introduction of nucleotides with
radioactive phosphorus (32P), sulfur (35S), or nitrogen to the probe
Radioisotopes
Non Radioactive labels
- Most common: ___ and
___ attached to a UTP or CTP - Can also be ___
biotin and digoxygenin (1)
fluorescent dyes (FISH) (2)
Non Radioactive labels
Detection:
1. Washing off of unbound probe
2. ____ or ____ are conjugated to ____ or ____
3. Washing off of unbound conjugate
4. Bathing in substrate (dioxetane
or tetrazolium dye)
Antidioxygenin (dioxygenin) or streptavidin (biotin) (1)
alkaline phosphatase (AP) or horseradish peroxidase (HRP) (2)
Southern Blot
* Target: DNA
* Probe: ____
* First done by ____
Nucleic acid (1)
Edwin Southern (2)
Southern Blot
unsa tung 1-6????
- RE digestion/cutting
- Resolution
- Denaturation of fragments
- Blotting (Transfer)
- Probing
- Detection
Restriction enzyme digestion
____ have sequence-specific activity with 4-6 bp palindromic recognition sites
Restriction endonucleases
Resolution of the cut fragments are done by ___
gel electrophoresis
RESOLUTION
* COMMON PROBLEMS:
* Large aggregates near the top –
incomplete digestion
RESOLUTION
* Smear in the lower region –
degraded DNA
- Preparation before blotting
- Double-stranded DNA are separated into their single-stranded form for later
probing
Denaturation
Can be done directly using ___ or using ___ for depurination prior to denaturation (for >500 bp fragments)
NaOH or using HCl
“Transfer” of denatured DNA to a solid substrate (usually nitrocellulose or nylon)
Blotting
T OR F?
Blotting
CAPILLARY TRANSFER
* Gel is placed on the bottom of the high salt buffer (10X saline sodium citrate)
F : Gel is placed on top of high salt buffer
T OR F?
Blotting is faster than capillary transfer
T
Immobilization of the DNA is done by baking in a ___ or by ___ to the membrane
vacuum oven or by UV-crosslinking
- Target: RNA
- Probe: Nucleic acid
Northern Blot
