Molecular diagnostics, PCR, DNA amplification

Question 3 anticoagulants used for specimen collection in molecular diagnostic lab

Answer EDTA (preffered)ACDHeparin (inhibits some enzymes used in molecular essay) Whole bloodBone marrowPBSC (phoresis product)Serum/plasmaBuccal cellsCultured cellsBlood spotsBody fluids*CSF*Bronchial lavage*Amniotic*Semen*UrineTissu e samplesFresh/frozenParaffinembeddedHair (shaft/root) high mol. weigh nucleic acidfixation time

Types of Specimens for the Molecular Diagnostics Laboratory

The effect of tissue fixatives on the purification of nucleic acidFormaldehyde The effect of tissue fixatives on the purification of nucleic acid. Alcohol Paraffin-embedded Tissue SectionsIs formalin-fixed tissue suitable? Paraffin-embedded Tissue SectionsAre mercury or other heavy metal fixatives acceptable?

good or excellent nucleic acid yelds

Yes

Not

Specimen Storage Requirements Not reccomended (<24 hours) DNA 22-25 C Specimen Storage Requirements suitable for up to 72 hours DNA 2-8 C

not reccomendedNOTE: Do not freeze blood or bone marrow before Specimen Storage Requirements lysing red blood cells (RBCs). DNA -20 C Leukocyte pellet can be frozen for up to 1 year. not reccomendedNOTE: Do not freeze blood or bone marrow before Specimen Storage Requirements lysing red blood cells (RBCs). DNA -70 C Leukocyte pellet can be frozen for >1 year. Specimen Storage Requirements RNA; 22-25 C Specimen Storage Requirements RNA 2-8 C Specimen Storage Requirements RNA -20 C Not recommended within 2 hours Not recommended within 2 hours Not recommended 24 weeksNOTE: Do not freeze blood or bone marrow before lysing red blood cells (RBCs). Not recommended 24 weeksNOTE: Do not freeze blood or bone marrow before lysing red blood cells (RBCs). 1 Amplification methods (PCR, LCR) 2 Restriction enzyme digest 3 Hybridization methods (Southern analysis) 4 Sequencing 1 Amplification methods (RT-PCR) 2 Hybridization methods (Northern analysis) 1. Processing speed2. Ease of use 3. Yield of DNA or RNA4. Quality of DNA and RNA prepared

Specimen Storage Requirements RNA -70 C

DNA 4 Preparation Applications

RNA 2 Preparation Applications Nucleic Acid Preparation Choosing an Isolation Method. 7 Important factors are:

(amplification performance)5. Shelf life/storage conditions6. Quality assurance criteria 7. Cost of preparation 1. Separate WBCs from RBCs, if necessary2. Lyse WBCs or other nucleated cells3. Denature/digest proteins4. Separate contaminants (e.g., proteins, heme)from DNA5. Precipitate DNA if necessary6. Resuspend DNA in final buffe 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. 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. 1. Fast and easy method2. Uses nontoxic materials, no fume hood required, no hazardous materials disposal issues3. Produces highquality DNA 1. Slow, labor-intensive, toxic (phenol, chloroform)2. Fume hood required, disposal of hazardous materials required

6 Basic Steps in Isolating DNA from Clinical Specimens

DNA Isolation Methods; Liquid Phase Organic Extraction

DNA Isolation Methods: Liquid Phase Nonorganic Salt Precipitation

3 Advantages of NONORGANIC Salt Precipitation

2 disadvantages of Liquid Phase ORGANIC Extraction

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 Fast and easy, no precipitation required 1. Lyse RBCs2. Protein digestionProK3. Separate proteins from DNA4. Precipitate DNA-alcohol5. Rehydrate DNA 1. re-lyse cells2. Apply sample 3. Wash4. Elute DNA 1. Separate WBCs from RBCs,2. Lyse WBCs or other nucleated cells with protein denaturants, RNase inhibitors3. Denature/digest proteins 4. Separate proteins, DNA, and contaminantsfrom RNA5. Precipitate RNA6. Resuspend RNA in final buff Advantage: high quality --------------------------------------Disadvantages: extremely time-consuming, hazardous materials disposal issues RNA Isolation 1. Nonorganic Salt Precipitation..................2. Guanidinium-based Organic Isolation..........3.Cesium Chloride Gradient

An advantage of SOLID PHASE isolation 5 steps in LIQUID Phase DNA Purification 4 steps in SOLID Phase DNA Purification

6 Basic Steps in Isolating RNA from Clinical Specimens

RNA Isolation Methods Cesium Chloride Gradient.Advantage and Disadvantages. Cesium Chloride Gradient method is used for..

3 RNA Isolation Methods

RNA Isolation Methods. Guanidinium-based Organic Isolation.Advantage/disadvantage s RNA Isolation Methods. Nonorganic Salt Precipitation. 2 Advantages

Advantage: faster than CsCl method.........Disadvantages: fume hood required, hazardous waste disposal issues 1. Fast and easy, nontoxic......2. Produces high quality RNA

1. UV spectrophotometry.....2. 4 methods for assessing quantity, Agarose gel electrophoresis.....3. quality, and molecular size of DNA Fluorometry........4. Colorimetric or RNA. blotting Absorption wavelength: 1. DNA/RNA ....2. Proteins....3. Backgroung scatter A260/A280 = 1.7 2.0 What does this resut mean? A260/A280 < 1.7 What does this resut mean? Agarose Gel Electrophoresis. What does smearing indicate? High-quality RNA has these 2 characteristics: 1. DNA/RNA at 260nm......2. Proteins at 280 nm.....3. Background scatter at 320nm Good DNA or RNA Too much protein or other contaminant DNA degradation or too much DNA loaded 1. 28S rRNA band : 18S rRNA band = 2:1 intensity ....2. Little to no genomic DNA (high MW band) Store DNA in TE buffer at 4 C for weeks or at 20 C to 80 C for long term. Store RNA in RNase-free ultra pure water at 70 C. False

DNA storage conditions

RNA storage conditions DNA Gel electrophoresisLarger fragments migrate faster.True or

false? Agarose ElectrophoresisHigh % of agarose gives better resolution of False larger DNA fragments.True or False? What is the main advantage PAGE over Agarose? Capillary DNA Electrophoresis.What DNA fragments migrate faster? 3 types of DNA/RNA electrophoresis Higher resolution

Small. 1. Horizontal (agarose)2. Vertical (PAGE)3. Pulse Field (uses more than one alternating electric field)

To keep DNA long, strait and What's the purpose of adding urea unpaired to avoid DNA hybridisation into gel for DNA electrophoresis? (folding) interferences. What type of gel is also named as submarine gel? What gel (agarose or PAGE) separates larger DNA fragments? PAGE provides (high/low) resolution of (high/low) mol. weight nucleic acids (500bp) With what can sticky ends be converted to blunt ends? How can blunt ends be converted to sticky ends? Restriction enzyme mapping. What does the number of bands Horisontal agarose PAGE provides high resolution of low mol. weight nucleic acids. With nuclease or polymerase. By ligating to synthetic adaptors. The number of restriction sites

indicate? Southern blots. What is immobilized on solid support? Northern blots. What is immobilized on solid support? Western blots. What is immobilized on solid support? Restriction Enzyme Mapping. What does the size of the bands indicate? DNA RNA Proteins The distance between restriction sites. 1.Extract DNA from cells, etc2. Cut with RE3. Run on gel (usually agarose)4. Denature DNA with alkali5. Transfer to nylon or nitrocellulose(usually capillary action)6. Detection with lables. 1. Capillary2. Electrophoretic3. Vacuum A combination of conditions in which the target is exposed to the probe. 1. peroxidase, 2. alkaline phosphatase 1. Adamantyl Phosphate derivatives, 2. Lumi-Phos" 1. Length of DNA2. GC content (%GC)3. Salt concentration (M)4. Formamide concentration Tm = 81.5C + 16.6 logM + 0.41 (%G + C) - 0.61 (%formamide) -

Southern blot. 6 steps.

Southern blot. 3 types of transfer. Southern blot. What is stringency? 2 enzymatic labels 2 luminescence labels Tm in Solution is a Function of 4 parameters: Tm in Solution. Formula

600/n (DNA NA) 1. Prehybridization to block nonspecific binding2. Hybridization under appropriate conditions3. Posthybridization to remove unbound probe (blank) 1. Low temp, 2. Low formamide3. Washing with high salt 1. High Formamide concentration2. Low salt 3. Heat 4. High G+C 1. Genetics, oncology (translocations, gene rearrangements)2. Typing/classification of organisms3. Cloning/verification of cloned DNA 4. Forensic, parentage testing (RFLP, VNTR) 1. Specific binding proteins2. Antibodies 1. Target amplification systems2. Probe amplification systems3. Signal amplification 1. PCR2. NASBA - Nucleic Acid Sequence-Based Amplification3. TMA Transcription Mediated Amplification4. SDA - Strand Displacement Amplification 1. bDNA Branched DNA probes2. Hybrid Capture Anti-DNA-RNA

Three steps of hybridization reaction

High Stringency for well matched hybrids Low Stringency What increases stringency(4 conditions)?

4Southern Blot Applications

2 types of probes in western blot Nucleic acid (NA) amplification methods fall into 3 categories:

4 Target Amplification Methods

2 Signal Amplification methods

hybrid antibody 2 Probe Amplification methods 1. LCR Ligase Chain Reaction2. Cleavase Invader FEN-1 DNA polymerase (cleavase) 1. Denaturation of target (template)2. Annealing of primers3. Extension (synthesis) of new strand 95 C 1.primers2. dATP, dCTP, dGTP, dTTP3. KCl4. Tris, pH 8.45. MgCl26. polymerase7. 100 - 10000 copies of template 90 - 96 C20 sec 40 - 68 C20 sec 70 - 75 C30 sec 1. Blank2. Negative3. Positive Controls for contaminationContains all reagents except DNA template Controls for specificity of the amplification reactionContains all reagents and a DNA template lacking the target sequence Controls for sensitivityContains all reagents and a known targetcontaining DNA template The length of the lag phase is inversely proportional to the amount of starting material.

3 steps in PCR PCR Denaturation temperature 7 components of a Standard PCR Reaction Mix PCR Denaturation temp. and time PCR Annealing temp. and time PCR Extension temp. and time PCR. 3 controls PCR Blank control

PCR. Negative control

PCR. Positive control Describe the relationship between A length of a log phase and the amount of starting material

6 PCR advantages

1. Specific2. Simple, rapid, relatively inexpensive3. Amplifies from low quantities4. Works on damaged DNA5. Sensitive6. Flexible 1. Contamination risk2. Primer complexities3. Primer-binding site complexities4. Amplifies rare species5. Detection methods

5 PCR limitations