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Molecular diagnostics, PCR, DNA amplification

Question Answer
3 anticoagulants used for specimen collection in molecular
EDTA (preffered) ACD Heparin (inhibits some enzymes used in molecular essay)
diagnostic lab
Whole blood Bone marrow PBSC (phoresis product) Serum/plasma Buccal cells Cultured cells Blood
Types of Specimens for the Molecular Diagnostics
spots Body fluids *CSF *Bronchial lavage *Amniotic *Semen *Urine Tissue samples Fresh/frozen
Paraffin-embedded Hair (shaft/root)
The effect of tissue fixatives on the purification of nucleic
high mol. weigh nucleic acid fixation time
acid Formaldehyde
The effect of tissue fixatives on the purification of nucleic
good or excellent nucleic acid yelds
acid. Alcohol
Paraffin-embedded Tissue Sections Is formalin-fixed tissue
Paraffin-embedded Tissue Sections Are mercury or other
heavy metal fixatives acceptable?
Specimen Storage Requirements - DNA 22-25 C Not reccomended (<24 hours)
Specimen Storage Requirements - DNA 2-8 C suitable for up to 72 hours
not reccomended NOTE: Do not freeze blood or bone marrow before lysing red blood cells (RBCs).
Specimen Storage Requirements - DNA -20 C
Leukocyte pellet can be frozen for up to 1 year.
not reccomended NOTE: Do not freeze blood or bone marrow before lysing red blood cells (RBCs).
Specimen Storage Requirements - DNA -70 C
Leukocyte pellet can be frozen for >1 year.
Specimen Storage Requirements RNA; 22-25 C Not recommended within 2 hours
Specimen Storage Requirements RNA 2-8 C Not recommended within 2 hours
Not recommended 24 weeks NOTE: Do not freeze blood or bone marrow before lysing red blood cells
Specimen Storage Requirements RNA -20 C
Not recommended 24 weeks NOTE: Do not freeze blood or bone marrow before lysing red blood cells
Specimen Storage Requirements RNA -70 C
1 Amplification methods (PCR, LCR) 2 Restriction enzyme digest 3 Hybridization methods (Southern
DNA 4 Preparation Applications
analysis) 4 Sequencing
RNA 2 Preparation Applications 1 Amplification methods (RT-PCR) 2 Hybridization methods (Northern analysis)
1. Processing speed 2. Ease of use 3. Yield of DNA or RNA 4. Quality of DNA and RNA prepared
Nucleic Acid Preparation Choosing an Isolation Method. 7
(amplification performance) 5. Shelf life/storage conditions 6. Quality assurance criteria 7. Cost of
Important factors are:
1. Separate WBCs from RBCs, if necessary 2. Lyse WBCs or other nucleated cells 3. Denature/digest
6 Basic Steps in Isolating
proteins 4. Separate contaminants (e.g., proteins, heme) from DNA 5. Precipitate DNA if necessary 6.
DNA from Clinical Specimens
Resuspend DNA in final buffe
Phenol (50):chloroform/isoamyl alcohol (50:49:1) Lysed samples mixed with above; two layers are
DNA Isolation Methods; Liquid Phase Organic Extraction formed. Proteins remain at interface. DNA is removed with top aqueous layer. DNA is precipitated with
alcohol and rehydrated.
DNA Isolation Methods: Liquid Phase Nonorganic Salt Cell membranes are lysed and proteins are denatured by detergent (such as SDS). RNA is removed with
Precipitation RNase. Proteins are precipitated with salt solution. DNA is precipitated with alcohol and rehydrated.
1. Fast and easy method 2. Uses nontoxic materials, no fume hood required, no hazardous materials
3 Advantages of NONORGANIC Salt Precipitation
disposal issues 3. Produces high-quality DNA
1. Slow, labor-intensive, toxic (phenol, chloroform) 2. Fume hood required, disposal of hazardous
2 disadvantages of Liquid Phase ORGANIC Extraction
materials required
1. solid support columns (Fibrous or silica matrices bind DNA allowing separation from other
DNA Isolation Methods SOLID PHASE; 3 solid supports contaminants), 2. magnetic beads (DNA binds to beads; beads are separated from other contaminants
with magnet); 3. chelating agents
An advantage of SOLID PHASE isolation Fast and easy, no precipitation required
1. Lyse RBCs 2. Protein digestion-ProK 3. Separate proteins from DNA 4. Precipitate DNA-alcohol 5.
5 steps in LIQUID Phase DNA Purification
Rehydrate DNA
4 steps in SOLID Phase DNA Purification 1. re-lyse cells 2. Apply sample 3. Wash 4. Elute DNA
1. Separate WBCs from RBCs, 2. Lyse WBCs or other nucleated cells with protein denaturants, RNase
6 Basic Steps in Isolating
inhibitors 3. Denature/digest proteins 4. Separate proteins, DNA, and contaminants from RNA 5.
RNA from Clinical Specimens
Precipitate RNA 6. Resuspend RNA in final buff
RNA Isolation Methods Cesium Chloride Gradient. Advantage: high quality ---------------------------------------Disadvantages: extremely time-consuming,
Advantage and Disadvantages. hazardous materials disposal issues
Cesium Chloride Gradient method is used for.. RNA Isolation
1. Nonorganic Salt Precipitation.................. 2. Guanidinium-based Organic Isolation.......... 3.Cesium
3 RNA Isolation Methods
Chloride Gradient
RNA Isolation Methods. Guanidinium-based Organic Advantage: faster than CsCl method......... Disadvantages: fume hood required, hazardous waste disposal
Isolation.Advantage/disadvantages issues
RNA Isolation Methods. Nonorganic Salt Precipitation. 2
1. Fast and easy, nontoxic...... 2. Produces high quality RNA
4 methods for assessing quantity, quality, and molecular size 1. UV spectrophotometry..... 2. Agarose gel electrophoresis..... 3. Fluorometry........ 4. Colorimetric
of DNA or RNA. blotting
Absorption wavelength: 1. DNA/RNA ....2. Proteins....3.
1. DNA/RNA at 260nm......2. Proteins at 280 nm.....3. Background scatter at 320nm
Backgroung scatter
A260/A280 = 1.7 2.0 What does this resut mean? Good DNA or RNA
A260/A280 < 1.7 What does this resut mean? Too much protein or other contaminant
Agarose Gel Electrophoresis. What does smearing indicate? DNA degradation or too much DNA loaded
High-quality RNA has these 2 characteristics: 1. 28S rRNA band : 18S rRNA band = 2:1 intensity .... 2. Little to no genomic DNA (high MW band)
DNA storage conditions Store DNA in TE buffer at 4 C for weeks or at 20 C to 80 C for long term.
RNA storage conditions Store RNA in RNase-free ultra pure water at 70 C.
DNA Gel electrophoresis Larger fragments migrate faster.
True or false?
Agarose Electrophoresis High % of agarose gives better
resolution of larger DNA fragments. True or False?
What is the main advantage PAGE over Agarose? Higher resolution
Capillary DNA Electrophoresis. What DNA fragments
migrate faster?
3 types of DNA/RNA electrophoresis 1. Horizontal (agarose) 2. Vertical (PAGE) 3. Pulse Field (uses more than one alternating electric field)
What's the purpose of adding urea into gel for DNA
To keep DNA long, strait and unpaired to avoid DNA hybridisation (folding) interferences.
What type of gel is also named as submarine gel? Horisontal
What gel (agarose or PAGE) separates larger DNA
PAGE provides (high/low) resolution of (high/low) mol.
PAGE provides high resolution of low mol. weight nucleic acids.
weight nucleic acids (500bp)
With what can sticky ends be converted to blunt ends? With nuclease or polymerase.
How can blunt ends be converted to sticky ends? By ligating to synthetic adaptors.
Restriction enzyme mapping. What does the number of
The number of restriction sites
bands indicate?
Southern blots. What is immobilized on solid support? DNA
Northern blots. What is immobilized on solid support? RNA
Western blots. What is immobilized on solid support? Proteins
Restriction Enzyme Mapping. What does the size of the
The distance between restriction sites.
bands indicate?
1.Extract DNA from cells, etc 2. Cut with RE 3. Run on gel (usually agarose) 4. Denature DNA with
Southern blot. 6 steps.
alkali 5. Transfer to nylon or nitrocellulose(usually capillary action) 6. Detection with lables.
Southern blot. 3 types of transfer. 1. Capillary 2. Electrophoretic 3. Vacuum
Southern blot. What is stringency? A combination of conditions in which the target is exposed to the probe.
2 enzymatic labels 1. peroxidase, 2. alkaline phosphatase
2 luminescence labels 1. Adamantyl Phosphate derivatives, 2. Lumi-Phos"
Tm in Solution is a Function of 4 parameters: 1. Length of DNA 2. GC content (%GC) 3. Salt concentration (M) 4. Formamide concentration
Tm in Solution. Formula Tm = 81.5C + 16.6 logM + 0.41 (%G + C) - 0.61 (%formamide) - 600/n (DNA:DNA)
1. Prehybridization to block non-specific binding 2. Hybridization under appropriate conditions 3. Post-
Three steps of hybridization reaction
hybridization to remove unbound probe
High Stringency for well matched hybrids (blank)
Low Stringency 1. Low temp, 2. Low formamide 3. Washing with high salt
What increases stringency(4 conditions)? 1. High Formamide concentration 2. Low salt 3. Heat 4. High G+C
1. Genetics, oncology (translocations, gene rearrangements) 2. Typing/classification of organisms 3.
4Southern Blot Applications
Cloning/verification of cloned DNA 4. Forensic, parentage testing (RFLP, VNTR)
2 types of probes in western blot 1. Specific binding proteins 2. Antibodies
Nucleic acid (NA) amplification methods fall into 3
1. Target amplification systems 2. Probe amplification systems 3. Signal amplification
1. PCR 2. NASBA - Nucleic Acid Sequence-Based Amplification 3. TMA Transcription Mediated
4 Target Amplification Methods
Amplification 4. SDA - Strand Displacement Amplification
2 Signal Amplification methods 1. bDNA Branched DNA probes 2. Hybrid Capture Anti-DNA-RNA hybrid antibody
2 Probe Amplification methods 1. LCR Ligase Chain Reaction 2. Cleavase Invader FEN-1 DNA polymerase (cleavase)
3 steps in PCR 1. Denaturation of target (template) 2. Annealing of primers 3. Extension (synthesis) of new strand
PCR Denaturation temperature 95 C
1.primers 2. dATP, dCTP, dGTP, dTTP 3. KCl 4. Tris, pH 8.4 5. MgCl2 6. polymerase 7. 100 - 10000
7 components of a Standard PCR Reaction Mix
copies of template
PCR Denaturation temp. and time 90 - 96 C 20 sec
PCR Annealing temp. and time 40 - 68 C 20 sec
PCR Extension temp. and time 70 - 75 C 30 sec
PCR. 3 controls 1. Blank 2. Negative 3. Positive
PCR Blank control Controls for contamination Contains all reagents except DNA template
Controls for specificity of the amplification reaction Contains all reagents and a DNA template lacking
PCR. Negative control
the target sequence
PCR. Positive control Controls for sensitivity Contains all reagents and a known target-containing DNA template
Describe the relationship between A length of a log phase
The length of the lag phase is inversely proportional to the amount of starting material.
and the amount of starting material
1. Specific 2. Simple, rapid, relatively inexpensive 3. Amplifies from low quantities 4. Works on
6 PCR advantages
damaged DNA 5. Sensitive 6. Flexible
1. Contamination risk 2. Primer complexities 3. Primer-binding site complexities 4. Amplifies rare
5 PCR limitations
species 5. Detection methods