Part 1: Lesson 10, 11, 12, 13 Flashcards
(35 cards)
Involves restriction enzymes(________________): _______ is commonly used, with ___ bp recognition/restriction sites, or binding/cutting sites on DNA
Used to identify the position of _____________ in a DNA fragment in relation to each other
Developed using _____________________________
RESTRICTION ENZYME MAPPING OF DNA
Involves restriction enzymes(endonucleases): type II is commonly used, with 4-6 bp recognition/restriction sites, or binding/cutting sites on DNA
Used to identify the position of restriction sites in a DNA fragment in relation to each other
Developed using small circular bacterial plasmids
____________________________
* Resulting differences in the size or number of restriction fragments
* Useful in epidemiological studies The basis of the 1st molecular-based human ID & mapping methods
* Clinical analysis of structural changes in ________________ associated with disease
Restriction Fragment Length Polymorphisms (RFLPs)
* Resulting differences in the size or number of restriction fragments
* Useful in epidemiological studies The basis of the 1st molecular-based human ID & mapping methods
* Clinical analysis of structural changes in
* chromosomes associated with disease
______________________________
Also uses endonuclease
Found in ________ & ___________
Guides a common enzyme to _____________ determined by RNA components Useful for manipulation of both ____ sequence & ____ expression
CRISPR ENZYME SYSTEMS
Also uses endonuclease
Found in archaea & bacteria
Guides a common enzyme to specific sites determined by RNA components Useful for manipulation of both DNA sequence & RNA expression
__________________________________________ (CRISPRS)
* Protective system using the invading DNA to target itself
* Encodes an endonuclease: ______________________ (Cas)
Clustered regularly interspaced short palindromic repeats (CRISPRS)
* Protective system using the invading DNA to target itself
* Encodes an endonuclease: CRISPR-associated protein (Cas)
Types:
* Type I & II - _______________
* Type III - _____________________
Types:
* Type I & II - target dsDNA
* Type III - targets ssDNA & RNA
___________________
Formation of H bonds between 2 complementary strands of nucleic acids
In hybridization technologies:
* 1 strand is ___________
* 1 strand is ________ (with isotopes/ fluorescence) = _______; used to detect the presence of the DNA fragment of interest
Northern blot and Western blot are modifications of _______________
HYBRIDIZATION TECHNOLOGIES
Formation of H bonds between 2 complementary strands of nucleic acids
In hybridization technologies:
* 1 strand is unlabeled
* 1 strand is labeled (with isotopes/ fluorescence) = probes; used to detect the presence of the DNA fragment of interest
Northern blot and Western blot are modifications of Southern blot
__________
ss fragment of nucleic acid(NA)/protein with a detectable signal that specifically binds to ___________________/target protein
Purpose: identify 1 or more sequences of interest within a large amount of NA
Other probes used: modified NA, such as ___________________ & ___________
PROBES
ss fragment of nucleic acid(NA)/protein with a detectable signal that specifically binds to complementary sequences/target protein
Purpose: identify 1 or more sequences of interest within a large amount of NA
Other probes used: modified NA, such as peptide nucleic acids (PNA) & locked NA
_______________
Sources:
1. Early methods Fragment of gene was cloned on a _______________ → isolated by restriction enzyme digestion & gel purification labeling & denaturation of fragment applied to _______________________
2. Isolation of sequence of interest from viral genomes
3. In vitro organic synthesis of a predetermined sequence → only for short, oligometric probes
4. Synthesized using ____
DNA PROBES
Sources:
1. Early methods Fragment of gene was cloned on a bacterial plasmid → isolated by restriction enzyme digestion & gel purification labeling & denaturation of fragment applied to Southern/Northern blot
2. Isolation of sequence of interest from viral genomes
3. In vitro organic synthesis of a predetermined sequence → only for short, oligometric probes
4. Synthesized using PCR
In order the probe to bind: ____________ has to contain the sequence of interest
Denaturation of dsDNA probes before use
* Heating the probe (_____, _______) in hybridization solution
* Treating with 50% formamide/2x SSC at a lower temperature for a shorter time (____, _________)
In order the probe to bind: target NA has to contain the sequence of interest
Denaturation of dsDNA probes before use
* Heating the probe (95°C, 10-15 min) in hybridization solution
* Treating with 50% formamide/2x SSC at a lower temperature for a shorter time (75°C, 5-6 min
___________________
Source: transcription from a ______________ template in vitro
Predesigned systems commercially available
Southern blot: _______________ transcript
Northern blot: ____________ transcript
Labeled with a radioactive/modified nucleotide for production of signal.
Should be performed in ____________________ environment
RNA PROBES
Source: transcription from a synthetic DNA template in vitro
Predesigned systems commercially available
Southern blot: coding strand transcript
Northern blot: antisense transcript
Labeled with a radioactive/modified nucleotide for production of signal.
Should be performed in Ribonuclease free environment
___________________
* Must generate a _______________ for visualization of the probe binding to the target fragments on a membrane
* Earlier labeling: ___________ 32P
* Present labeling: _____________ labels/tags (biotin & digoxigenin)
* 3 basic methods for DNA probe labeling:
a. ___________
b. _______________
c. ____________________
PROBE LABELING
* Must generate a detectable signal for visualization of the probe binding to the target fragments on a membrane
* Earlier labeling: radioactive 32P
* Present labeling: nonradioactive labels/tags (biotin & digoxigenin)
* 3 basic methods for DNA probe labeling:
a. End labeling
b. Nick translation
c. Random priming
- ____________ (_____-_________)
Greater specificity → less affected by point mutations/polymorphisms
Difficult & expensive to synthesize - _______________ (________)
Less specific
Higher chance of being repeated randomly in unrelated regions of the genome
Ideal for _________________
- Longer probes (500-5000bp)
Greater specificity → less affected by point mutations/polymorphisms
Difficult & expensive to synthesize - Shorter probes (<500 bp)
Less specific
Higher chance of being repeated randomly in unrelated regions of the genome
Ideal for mutational analysis
_____________________
_____________: 1st reported the procedure
Detects specific DNA sequences
SOUTHERN BLOTS
Edwin Southern: 1st reported the procedure
Detects specific DNA sequences
_______________: removes purines to loosen up larger fragments; before denaturation
____________________: mostly used membrane; where hybridization occurs
_______________: most common; rely with the capillary movement
Purination: removes purines to loosen up larger fragments; before denaturation
Nitrocellulose membrane: mostly used membrane; where hybridization occurs
Capillary transfer: most common; rely with the capillary movement
TYPE OF MEMBRANES:
* Nitrocellulose - bind __-___ μg of nucleic acids per cm2
mostly used
* Nylon
* Cellulose modified with a dimethyl aminoethyl Carboxymethyl (CM) chemical group
* Polyvinyl difluoride (PVDF) - __________
TYPE OF MEMBRANES:
* Nitrocellulose - bind 70-150 μg of nucleic acids per cm2
mostly used
* Nylon
* Cellulose modified with a dimethyl aminoethyl Carboxymethyl (CM) chemical group
* Polyvinyl difluoride (PVDF) - proteins
TRANSFER METHODS:
* ________________: rely on the capillary movement of the buffer
* _________________________: uses electric current to move the DNA from the gel to the membrane; expensive
* ________________: uses vacuum to move the DNA towards the nitrocellulose membrane. The fastest (2-3hrs). Expensive
TRANSFER METHODS:
* Capillary transfer: rely on the capillary movement of the buffer
* Electrophoretic transfer: uses electric current to move the DNA from the gel to the membrane; expensive
* Vacuum transfer: uses vacuum to move the DNA towards the nitrocellulose membrane. The fastest (2-3hrs). Expensive
_______________________
* Required following DNA immobilization to prevent the probe from binding to _________________sites on the membrane surface
* Allow us to visualize the ______of interest
PREHYBRIDIZATION
* Required following DNA immobilization to prevent the probe from binding to nonspecific sites on the membrane surface
* Allow us to visualize the gene of interest
______________________
Designed to investigate RNA structure & ___________.
1. Levels of gene expression (transcription from DNA) & ________
2. RNA structural abnormalities resulting from ____________ in synthesis/processing (alternative splicing)
Sample is cut out from the gel & soaked in __________________ to remove denaturant & will be stained
Stains: _____________________
NORTHERN BLOTS
Designed to investigate RNA structure & quantity.
1. Levels of gene expression (transcription from DNA) & stability
2. RNA structural abnormalities resulting from aberrations in synthesis/processing (alternative splicing)
Sample is cut out from the gel & soaked in ammonium acetate to remove denaturant & will be stained
Stains: acridine orange/ethidium
___________________
Immobilized target: ___________
SDS-polyacrylamide gel electrophoresis (SDS-PAGE): resolves proteins according to ______________ at ____-_____ concentrations
_________________________: resolves proteins according to charge
Other membranes used: PVDF & anion (DEAE) or cation (CM) exchange cellulose
Signals: _________________ or _______________
WESTERN BLOTS
Immobilized target: PROTEIN
SDS-polyacrylamide gel electrophoresis (SDS-PAGE): resolves proteins according to molecular weight at 5%-20% concentrations
Isoelectric focusing gels (IEF)/tube electrophoresis: resolves proteins according to charge
Other membranes used: PVDF & anion (DEAE) or cation (CM) exchange cellulose
Signals: chemiluminescent or color signals
HYBRIDIZATION CONDITIONS, STRINGENCY
Combination of conditions under which the target is exposed to the probe
________________________:
* High hybridization temperatures + low concentration of salt in the buffers
* Probe ________ bind to its target
___________________________:
* Low hybridization temperatures + a high concentration of salt in the buffer
* probe _________ to unrelated targets
HYBRIDIZATION CONDITIONS, STRINGENCY
Combination of conditions under which the target is exposed to the probe
High stringency conditions:
* High hybridization temperatures + low concentration of salt in the buffers
* Probe will not bind to its target
Low stringency conditions:
* Low hybridization temperatures + a high concentration of salt in the buffer
* probe will bind to unrelated targets
- ____________/with higher % of _________ bases will bind under more stringent conditions than ___________/with higher % of _________ bases.
- Long probe/with higher % of G & C bases will bind under more stringent conditions than short probe/with higher % of A & T bases.
- Southern Blot: comparing ___________________ lengths of selected regions in different samples
- Northern (or western) Blot: amount of _______________ is determined relative to the internal control
o usually lane 1
o help us know the specificity and target size
- Southern Blot: comparing restriction fragment lengths of selected regions in different samples
- Northern (or western) Blot: amount of gene expression is determined relative to the internal control
o usually lane 1
o help us know the specificity and target size
ARRAY-BASED HYBRIDIZATION
1. ___________________
* Applied to expression, mutation, & amplification/deletion analyses
* Target DNA/RNA is deposited directly on ______________ by means of various devices (vacuum systems/ pipet for few samples)
* Performed on cloned _________, PCR products, selected mRNA preparations
ARRAY-BASED HYBRIDIZATION
1. DOT/SLOT BLOTS
* Applied to expression, mutation, & amplification/deletion analyses
* Target DNA/RNA is deposited directly on membrane by means of various devices (vacuum systems/ pipet for few samples)
* Performed on cloned plasmids, PCR products, selected mRNA preparations
______________
* Target is deposited in a ___ or dot
* Useful for multiple qualitative analyses where many targets are being compared (____________________)
DOT BLOTS
* Target is deposited in a circle or dot
* Useful for multiple qualitative analyses where many targets are being compared (mutational screening)
