Molecular Diagnostics

Molecular diagnostics, PCR, DNA amplification Question

Answer

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 (1 year.

Specimen Storage Requirements —  Requirements — RNA; RNA; 22-25 C

Not recommended within 2 hours

Specimen Storage Requirements —  Requirements — RNA RNA 2-8 C

Not recommended within 2 hours

Specimen Storage Requirements —  Requirements —  RNA  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 —  Requirements —  RNA  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., (e.g., proteins, heme) from 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 la yers 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 mat rices 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 nucleat ed 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: --Disadvantages: extremely time-consuming, hazardous materials disposal issues

Cesium Chloride Gradient method is used for..

RNA Isolation

3 RNA Isolation Methods

1. Nonorganic Salt Precipita tion.................. tion.................. 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 1. UV spectrophotometry..... 2. Agarose gel electrophoresis..... 3. Fluorometry........ 4. Colorimetric of DNA or RNA.  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 –  1.7  – 2.0 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. Electrophoresis. What does smearing 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 bett er 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 f or 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 la rger 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. Posthybridization 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