MIDTERM LEC 3 Flashcards
→ Set of techniques & methods used to study and understand the structure, interaction, & characteristics of nucleic acids & proteins in biological systems
NUCLEIC ACID/PROTEIN ANALYSIS
NUCLEIC ACID/PROTEIN ANALYSIS 3 MAIN METHODS
- Restriction enzyme mapping of DNA
- CRISPR enzyme systems
- Hybridization technology
- Involves restriction enzyme such as endonucleases
- Used to identify the position of restriction sites in DNA fragments in relation to each other.
o Used for DNA fingerprinting, paternity testing - Developed using small circular bacterial plasmids.
Restriction enzyme mapping of DNA
commonly used endonuclease in restriction enzyme mapping of DNA
TYPE II ENDONUCLEASE
*Resulting DIFFERENCES IN SIZE OR NUMBER OF RESTRICTION FRAGMENTS
*Useful in epidemiologic studies.
*The basis of the first molecular-based human ID and mapping methods.
*Clinical analysis of structural changes in chromosomes associated with disease
Restriction Fragment Length Polymorphisms (RFLPs)
RESTRICTION MAPPING USES/APPLICATIONS
1.Gives an idea of genome sequence
2. Used to map genomes when sequence information is unknown.
3. Can be used to introduce primers for sequencing.
4. Used for ID and characterization of naturally-occurring plasmids and to engineer the construction of recombinant plasmids.
- Found in archaea and bacteria
- guides a common enzyme to specific sites determined by RNA components
- Useful for manipulation of both DNA sequence and RNA expression.
CRISPR enzyme systems
CRISPR stands for
CLUSTERED REGULARLY INTERSPACED REPEATS
CRISPR enzyme systems TYPES:
Type I & II - target ______
dsDNA
CRISPR enzyme systems TYPES:
Type III - target ______
ssDNA & RNA
- used to analyze the nucleic acid content of an unknown sample
Hybridization TECHNOLOGY
- Formation of H bonds between 2 complementary strands of nucleic acid.
HYBRIDIZATION
IN HYBRIDIZATION TECHNIQUE:
- First strand is _________
UNLABELED
TARGET: DNA
PROBE: NUCLEIC ACID (NA)
PURPOSE: Gene structure
Southern blot
TARGET: RNA
PROBE: NA
PURPOSE: Transcript structure, processing, gene expression
Northern blot
TARGET: PROTEIN
PROBE: PROTEIN
PURPOSE: Protein processing, gene expression
WESTERN BLOT
TARGET: PROTEIN
PROBE: DNA
PURPOSE: DNA – binding proteins, gene regulation
Southwestern blot
TARGET: PROTEIN
PROBE: PROTEIN
PURPOSE: Modification of western blot using enzymatic detection (PathHunter); also, detection of specific agriculturally important proteins
Eastern blot
TARGET: LIPIDS
PROBE: NONE
PURPOSE: Transfer high performance liquid chromatography (HPLC)- separate lipids to play difluoride (PVDF) membrane for analysis by mass spectrometry
Far-eastern blot
Southern blot – procedure was first reported by
Edwin Southern
- ss fragment of nucleic acid/protein with a DETECTABLE SIGNAL that specifically binds to complementary sequences/target protein.
- PURPOSE: identify one or more sequences of interest with a large amount of nucleic acids.
- OTHER USED: modified nucleic acid, such as peptide nucleic acids and locked nucleic acids.
PROBE
TYPES OF PROBES?
- SOUTHERN BLOTS
- Complementary to the target gene
Nucleic Acid (NA) Probes (DNA probes)
TYPES OF PROBES?
- NORTHERN BLOTS
- Complementary to the target sequence
Nucleic Acid (NA) Probes (RNA probes)
TYPES OF PROBES?
- WESTERN BLOTS
- Antibodies bind to the target protein
Protein Probes
In order for the probe to bind: target NA has to contain the _________
SEQUENCE OF INTEREST
Denaturation of dsDNA probes before use:
- Heating the probe (TEMP & TIME?) in hybridization solution
95°C, 10 – 15min
Denaturation of dsDNA probes before use:
o Treating with 50% formamide/2x SSC at a lower temp. for a shorter time (TEMP & TIME?)
75°C; 5-6 min
EARLY METHODS: fragment of gene was cloned on bacterial plasmid — isolated by restriction enzyme digestion and gel purification — labeling and denaturation of fragment — applied to southern/northern blot.
Isolation of sequence of interest from viral genomes.
In vitro organic synthesis of predetermined sequence — only for short, oligometric probes.
Synthesized using PCR
DNA PROBES
must generate a detectable signal for visualization of the probe binding to the target (it could be in the form of isotope/fluorescent dye)
PROBE LABELLING
3 BASIC METHODS FOR DNA PROBE LABELING:
END LABELING
NICK TRANSLATION
RANDOM PRIMING
o 3 BASIC METHODS FOR DNA PROBE LABELING
- labeled nucleotides are added to the END OF THE PROBE using terminal transferase or T4 polynucleotide kinase
END LABELING
o 3 BASIC METHODS FOR DNA PROBE LABELING
- labeled nucleotides are incorporated into single-stranded breaks, or nicks, that are substrates for nucleotide addition by DNA polymerase
NICK TRANSLATION
o 3 BASIC METHODS FOR DNA PROBE LABELING
- generates new single-stranded versions of the probe with the incorporation of the labeled nucleotides
RANDOM PRIMING
SOURCE:
- Transcription from a synthetic DNA temperature in vitro.
- Predesigned systems commercially available
RNA PROBES
modified nucleic acids that has advantage of being resistant to nucleases that degrade DNA and RNA by breaking the phosphodiester backbone
(Synthetic and resistant to nucleases)
such as:
- PEPTIDE NUCLEIC ACIDS
- LOCKED NUCLEIC ACIDS
- UNLOCKED NUCLEIC ACIDS
OTHER NUCLEIC ACID PROBE TYPES
Polyvinyl difluoride (PVDF) - proteins T
- products of a generalized response to a specific antigen.
POLYCLONAL ANTIBODIES
Polyvinyl difluoride (PVDF) - proteins T
- products of a SINGLE clone of plasma B cells
MONOCLONAL ANTIBODIES
Determine the specificity of the results
DESIGN & OPTIMAL HYBRIDIZATION OF PROBE
- 500-5000bp
- GREATER SPECIFICITY — less affected by points.
- mutations/polymorphisms
- Difficult and expensive to synthesize.
LONGER PROBES
- less than 500bp
- LESS SPECIFIC
- Higher chance of being repeated randomly in unrelated religions of the genome.
- Ideal for mutational analysis
SHORTER PROBES
- The procedure was first reported by Edwin Southern.
- Detects specific DNA sequences.
SOUTHERN BLOTS
SOUTHERN BLOTS TRANSFER METHODS:
- simple and relatively inexpensive; no requirements are required
- Driven by capillary movement of buffer from the soaked paper to the dry paper
- procedure is slow
CAPILLARY TRANSFER
SOUTHERN BLOTS TRANSFER METHODS:
- uses ELECTRIC CURRENT to move the DNA transversely through the gel to the membrane.
- Used mostly for small fragments or proteins.
ELECTROPHORETIC TRANSFER
SOUTHERN BLOTS TRANSFER METHODS:
- uses SUCTION and buffer recirculation to move the DNA out of the gel and onto the membrane;
- faster than capillary transfer for large DNA fragments
- Requires specialized equipment.
VACUUM TRANSFER
SOUTHERN BLOTS
TYPES OF MEMBRANE?
- Nitrocellulose
- Nylon
- Cellulose modified with a dimethyl aminoethyl
- Carboxymethyl (CM) chemical group
- Polyvinyl difluoride (PVDF)
SOUTHERN BLOTS (TYPES OF MEMBRANE)
- bind 70-150 µg of nucleic acids per cm squared
- High bonding capacity to nucleic acids & proteins
NITROCELLULOSE
- prevent the probe from binding to the nonspecific sites on the membrane surface
PREHYBRIDIZATION
PREHYBRIDIZATION BUFFER COMPOSITION
Denhardt solution (ficoll, polyvinyl pyrrolidone, bovine serum albumin)
Salmon sperm DNA
SDS sodium dodecyl acetate) , 0.01% may also be included with formamide
o Membrane is exposed to the buffer at the optimal hybridization temperature for ?
30 mins several hours
SOUTHERN BLOT APPLICATION
- ID of the single gene in a pool of DNA fragments.
- Gene mapping
- Analysis of genetic patterns of DNA
- Detection of specific DNA sequences in a genome
- Study of gene deletions, duplications, and mutations that cause various diseases
- Detection of genetic disease and cancer
- Detects the presence of a gene family in a genome
- DNA fingerprinting such as forensic tests, paternity testing, and sex determination
- Designed to investigate RNA structure and quantity:
1. Levels of gene expression (transcription from DNA) and stability
2. RNA structural abnormalities resulting from aberrations in synthesis/processing (alternative splicing)
NORTHERN BLOT APPLICATION
- 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 and anion (DEAE) or cation (CM) exchange cellulose.
- SIGNALS: chemiluminescent or color signals
WESTERN BLOT
Combination of conditions under which the target is exposed to the probe
HYBRIDIZATION CONDITIONS, STRINGENCY
o High hybridization temp + low concentration of salt in the buffers
o Probe will NOT BIND to its TARGET.
HIGH STRINGENCY CONDITIONS
o Low hybridization temp + a high concentration of salt in the buffer
o Probe will NOT BIND to UNRELATED TARGETS.
LOW STRINGENCY CONDITIONS
FACTORS AFFECTING STRINGENCY
o TEMP. of hybridization.
o SALT CONCENTRATION of the hybridization buffer.
o CONCENTRATION OF DENATURAT (formamide) in buffer.
o LENGTH AND NATURE OF THE PROBE SEQUENCE.
Long probes with the higher percent of G and C bases WILL BIND under more stringent conditions than shorter probes with higher percent of A and G bases.
T OR F?
TRUE
HOW TO ESTIMATE THE IDEAL HYBRIDIZATION CONDITIONS?
Calculate the melting temperature (T”m)” of the probe sequence.
Hybridization are generally performed in
hybridization bags/glass cylinders
RECOMMENDED VOLUME FOR HYBRIDIZATION BUFFER
approx. 10 mL/100 cm² of membrane surface area.
Shorts probes (less than 20 bases) can hybridize in
1- 2 HOURS
Long probes (greater than 1000 bases) can hybridize in
16 HOURS OR MORE
___________ - Whether the probe has bound to the immobilized target.
DETECTION SYSTEM
DETECTION SYSTEM
- Unbound probe is washed off and the blot is EXPOSED TO LIGHT- SENSITIVE FILM to detect the fragments that are hybridized to the radioactive probe.
DNA/ RNA Probe Labeled with Radioactive Phosphorus Atoms
DETECTION SYSTEM
- Unbound probe is washed away and anti-digoxigenin antibody/streptavidin is added to bind labeled probe target complex
Indirect Nonradioactive Detection System
Relies on microarrays, which are solid supports (e.g. glass slides/chips) containing an array of DNA & RNA probes that can hybridized with complementary nucleic acid sequences
ARRAY - BASED HYBRIDIZATION
is a glass slide carrying hundreds to thousands of probes
MICROARRAY/DNA CHIP
- Applied to expression, mutation and amplification/deletion analyses.
- Target DNA/RNA is deposited directly on the membrane by means of various devices (vacuum systems and pipette for few samples).
- Performed on cloned plasmids, PCR products, selected mRNA selections
DOT/SLOT BLOTS
- Target is deposited in a CIRCLE or DOT.
- Useful for MULTIPLE QUANTITATIVE ANALYSES where many targets are being compared (mutational screening)
DOT OR SLOT BLOT?
DOT BLOT
- Target is deposited in a OBLONG BAR
- Useful for ACCURATE QUANTIFICATION by densitometry scanning.
DOT OR SLOT BLOT?
SLOT BLOT
- Type of hybridization analysis allows simultaneous study of a large number of targets.
- APPROACHES:
a) Macroassays
b) Microassays
c) High-density oligonucleotide arrays
d) Microelectronic arrays
GENOMIC ARRAY TECHNOLOGY
GENOMIC ARRAY TECHNOLOGY TYPE OF APPROACHES
- REVERSE DOT BLOT: immobilized probe is now effectively the target and the labeled specimen
o DNA, RNA, proteins are actually the probes
- DETECTION OF HYBRIDIZATION: radioactive /chemiluminescent signals
- READING OF HYBRIDIZATION: by eye or phosphorimager
- ANALYSIS: signal intensity from test “spots” was compared to control samples spotted on duplicate membranes.
MACROASSAY
GENOMIC ARRAY TECHNOLOGY TYPE OF APPROACHES
- Glass substrates are used for the production of arrays.
- Improved spotting and has the ability to deposit very small target spots.
- Automated deposing systems (arrayers).
o Can place less than 80,000 spots on the glass substrate.
o Pen-type and ink-jet
MICROASSAYS
GENOMIC ARRAY TECHNOLOGY TYPE OF APPROACHES
- High-density oligonucleotide array
- Deposit target by DNA synthesis directly on the glass/silicon support.
- Uses sequence information to design oligonucleotides at designated positions on the chip.
- Used for mutation and polymorphism analysis, DNA methylation analysis, and sequencing.
PHOTOLITHOGRAPHY TECHNIQUE
PHOTOLITHOGRAPHY TECHNIQUE
- indicates amplification of the test sample locus.
EXCESS PURPLE LABEL
PHOTOLITHOGRAPHY TECHNIQUE
- indicates deletion of the test sample locus.
EXCESS BLACK LABEL
PHOTOLITHOGRAPHY TECHNIQUE
- indicates equal test and reference DNA.
NEUTRAL OR GRAY
- Probes are immobilized on beads, allowing hybridization of the targets in the bead suspension.
- Multiple suspensions can be tested simultaneously.
- Beads are color-coded with a particular shade of red fluorescent dye.
- Sample is labeled with a green dye.
- Used for protein and nucleic acid targets.
- Available for infectious diseases and tissue typing.
BEAD ARRAY TECHNOLOGY
- Neither the probe nor the target is immobilized.
- Probes and targets bind in solution, followed by resolution of the bound products.
- To measure mRNA expression
SOLUTION HYBRIDIZATION
SOLUTION HYBRIDIZATION VERSION
- labeled probe probe is hybridized to the target sample in solution.
RNAseprotection (S1 mapping)
SOLUTION HYBRIDIZATION VERSION
- uses 2 probes (capture probe and detection probe.
Capture of DNA probe: RNA target hybrids on a solid support/beads
