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Molecular diagn.

Molecular diagnostics, PCR, DNA amplification

3 anticoagulants used for specimen collection in molecular diagnostic lab EDTA (preffered) ACD Heparin (inhibits some enzymes used in molecular essay)
Types of Specimens for the Molecular Diagnostics Laboratory Whole blood Bone marrow PBSC (phoresis product) Serum/plasma Buccal cells Cultured cells Blood 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 acid Formaldehyde ↓high mol. weigh nucleic acid ↑fixation time
The effect of tissue fixatives on the purification of nucleic acid. Alcohol good or excellent nucleic acid yelds
Paraffin-embedded Tissue Sections Is formalin-fixed tissue suitable? Yes
Paraffin-embedded Tissue Sections Are mercury or other heavy metal fixatives acceptable? Not
Specimen Storage Requirements - DNA 22-25 C Not reccomended (<24 hours)
Specimen Storage Requirements - DNA 2-8 C suitable for up to 72 hours
Specimen Storage Requirements - DNA -20 C not reccomended NOTE: Do not freeze blood or bone marrow before lysing red blood cells (RBCs). Leukocyte pellet can be frozen for up to 1 year.
Specimen Storage Requirements - DNA -70 C not reccomended NOTE: Do not freeze blood or bone marrow before lysing red blood cells (RBCs). 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
Specimen Storage Requirements — RNA -20 C Not recommended 2–4 weeks NOTE: Do not freeze blood or bone marrow before lysing red blood cells (RBCs).
Specimen Storage Requirements — RNA -70 C Not recommended 2–4 weeks NOTE: Do not freeze blood or bone marrow before lysing red blood cells (RBCs).
DNA 4 Preparation Applications 1 Amplification methods (PCR, LCR) 2 Restriction enzyme digest 3 Hybridization methods (Southern analysis) 4 Sequencing
RNA 2 Preparation Applications 1 Amplification methods (RT-PCR) 2 Hybridization methods (Northern analysis)
Nucleic Acid Preparation Choosing an Isolation Method. 7 Important factors are: 1. Processing speed 2. Ease of use 3. Yield of DNA or RNA 4. Quality of DNA and RNA prepared (amplification performance) 5. Shelf life/storage conditions 6. Quality assurance criteria 7. Cost of preparation
6 Basic Steps in Isolating DNA from Clinical Specimens 1. Separate WBCs from RBCs, if necessary 2. Lyse WBCs or other nucleated cells 3. Denature/digest proteins 4. Separate contaminants (e.g., proteins, heme) from DNA 5. Precipitate DNA if necessary 6. Resuspend DNA in final buffe
DNA Isolation Methods; Liquid Phase Organic Extraction Phenol (50):chloroform/isoamyl alcohol (50:49:1) Lysed samples mixed with above; two layers are 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 Precipitation Cell membranes are lysed and proteins are denatured by detergent (such as SDS). RNA is removed with RNase. Proteins are precipitated with salt solution. DNA is precipitated with alcohol and rehydrated.
3 Advantages of NONORGANIC Salt Precipitation 1. Fast and easy method 2. Uses nontoxic materials, no fume hood required, no hazardous materials disposal issues 3. Produces high-quality DNA
2 disadvantages of Liquid Phase ORGANIC Extraction 1. Slow, labor-intensive, toxic (phenol, chloroform) 2. Fume hood required, disposal of hazardous materials required
DNA Isolation Methods SOLID PHASE; 3 solid supports 1. solid support columns (Fibrous or silica matrices bind DNA allowing separation from other 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
5 steps in LIQUID Phase DNA Purification 1. Lyse RBCs 2. Protein digestion-ProK 3. Separate proteins from DNA 4. Precipitate DNA-alcohol 5. Rehydrate DNA
4 steps in SOLID Phase DNA Purification 1. re-lyse cells 2. Apply sample 3. Wash 4. Elute DNA
6 Basic Steps in Isolating RNA from Clinical Specimens 1. Separate WBCs from RBCs, 2. Lyse WBCs or other nucleated cells with protein denaturants, RNase inhibitors 3. Denature/digest proteins 4. Separate proteins, DNA, and contaminants from RNA 5. Precipitate RNA 6. Resuspend RNA in final buff
RNA Isolation Methods Cesium Chloride Gradient. Advantage and Disadvantages. Advantage: high quality ---------------------------------------Disadvantages: extremely time-consuming, hazardous materials disposal issues
Cesium Chloride Gradient method is used for.. RNA Isolation
3 RNA Isolation Methods 1. Nonorganic Salt Precipitation.................. 2. Guanidinium-based Organic Isolation.......... 3.Cesium Chloride Gradient
RNA Isolation Methods. Guanidinium-based Organic Isolation.Advantage/disadvantages Advantage: faster than CsCl method......... Disadvantages: fume hood required, hazardous waste disposal issues
RNA Isolation Methods. Nonorganic Salt Precipitation. 2 Advantages 1. Fast and easy, nontoxic...... 2. Produces high quality RNA
4 methods for assessing quantity, quality, and molecular size of DNA or RNA. 1. UV spectrophotometry..... 2. Agarose gel electrophoresis..... 3. Fluorometry........ 4. Colorimetric blotting
Absorption wavelength: 1. DNA/RNA ....2. Proteins....3. Backgroung scatter 1. DNA/RNA at 260nm......2. Proteins at 280 nm.....3. Background scatter at 320nm
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? False
Agarose Electrophoresis High % of agarose gives better resolution of larger DNA fragments. True or False? False
What is the main advantage PAGE over Agarose? Higher resolution
Capillary DNA Electrophoresis. What DNA fragments migrate faster? Small.
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 electrophoresis? 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 fragments? agarose
PAGE provides (high/low) resolution of (high/low) mol. weight nucleic acids (500bp) PAGE provides high resolution of low mol. weight nucleic acids.
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 bands indicate? The number of restriction sites
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 bands indicate? The distance between restriction sites.
Southern blot. 6 steps. 1.Extract DNA from cells, etc 2. Cut with RE 3. Run on gel (usually agarose) 4. Denature DNA with 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 = 81.5°C + 16.6 logM + 0.41 (%G + C) - 0.61 (%formamide) - 600/n (DNA:DNA)
Three steps of hybridization reaction 1. Prehybridization to block non-specific binding 2. Hybridization under appropriate conditions 3. Post-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
4Southern Blot Applications 1. Genetics, oncology (translocations, gene rearrangements) 2. Typing/classification of organisms 3. 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 categories: 1. Target amplification systems 2. Probe amplification systems 3. Signal amplification
4 Target Amplification Methods 1. PCR 2. NASBA - Nucleic Acid Sequence-Based Amplification 3. TMA – Transcription Mediated 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
7 components of a Standard PCR Reaction Mix 1.primers 2. dATP, dCTP, dGTP, dTTP 3. KCl 4. Tris, pH 8.4 5. MgCl2 6. polymerase 7. 100 - 10000 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
PCR. Negative control Controls for specificity of the amplification reaction Contains all reagents and a DNA template lacking 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 and the amount of starting material The length of the lag phase is inversely proportional to the amount of starting material.
6 PCR advantages 1. Specific 2. Simple, rapid, relatively inexpensive 3. Amplifies from low quantities 4. Works on damaged DNA 5. Sensitive 6. Flexible
5 PCR limitations 1. Contamination risk 2. Primer complexities 3. Primer-binding site complexities 4. Amplifies rare species 5. Detection methods
Created by: Teresa_indigo