Purpose
To release nucleic acid from the cell for use in other procedures
Must be free from contamination with protein, carbohydrate, lipids or other nucleic acids.
Used pure nucleic acids for testing.
Principles for Handling ofAll Clinical Specimens Observe universal precautions for
biohazards. Use protective gowns, gloves, face
and eye shields. Decontaminate all spills and work
areas with 10% bleach. Dispose of all waste in appropriate
biologic waste containers. Use gloves. Your RNA depends
on it!
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 Amniotic Semen Urine
Tissue samples Fresh/frozen Paraffin-embedded
Hair
Fundamentals of Specimen Handling: Specimen Labeling Patient name, date of birth, and medical record number
Ordering physician Type of specimen Accession number Date and time of collection Laboratory technician identification
(initials) Requested test(s)
Blood and Bone Marrow
Isolation of nucleic acids Genomic DNA RNA
Collection: Collect in an anticoagulant, mix
well but gently to avoid disruption of cells
Anticoagulants
EDTA Preferred specimen
ACD (Acid Citrate Dextrose) Yellow-top Vacutainer
Heparin Green-top Vacutainer Inhibits several enzymes used in
molecular assays
Heparina compound occurring in the liver and other tissues which inhibits blood coagulation. A sulphur-containing polysaccharide, it is used as an anticoagulant in the treatment of thrombosis.
Specimen Packaging and Shipping:
DO NOT FREEZE!!! Protect from temperature extremes Packaging must comply with shipping
rules for bloodborne pathogens Protective container Absorbent material in packing Sealed container in plastic bag Labeled as Biohazard
Nucleic Acid PreparationApplication? DNA
Amplification methods (PCR) Restriction enzyme digest Hybridization methods (Southern
analysis) Sequencing
2–25 °C 2–8 °C –20 °C –70 °C
Recommendedfor long-term
storage
<4 Months1–3 Years <7 Years >7 Years
Nucleic Acid Storage Requirements: Storage of DNA Specimens
Nucleic Acid PreparationApplication?
RNA Amplification methods (RT-PCR)
Hybridization methods (Northern analysis)
Nucleic Acid PreparationOther Considerations
What is the size or volume of each sample? Amount of DNA or RNA required Equipment and tube sizes required
How many samples are being processed? Capacity of the centrifuge Isolation method speed
Is an automated system available? 96-well plate methods Walk-away or semi-automation
Nucleic Acid PreparationChoosing an Isolation Method Important factors are:
Processing speed Ease of use Yield of DNA or RNA Quality of DNA and RNA prepared
(amplification performance) Shelf life/storage conditions Cost of preparation
Isolation
Routinely isolated from human, fungal, bacterial and viral sources.
Pretreat to make nucleated cells available, whole blood Tissue samples Microorganisms
Need sufficient sample for adequate yield.
Organic Isolation
Must purify DNA by removing contaminants.
Accomplished by using combination of high salt and an organic mixture of phenol and chloroform.
To avoid RNA contamination add RNAse, enzyme that degrades RNA.
Phenol/Chloroform
Biphasic Hydrophobic layer on bottom has cell
debris. Hydrophilic layer on top has
dissolved DNA Remove top layer, add cold ethanol, DNA precipitates out.
Inorganic Isolation Methods Also called “salting out”.1. Uses low pH and high salt condition
to selectively precipitate proteins.DNA is left in solution (picture on left).
2. Precipitate out DNA with isopropanol (right side picture).
1
2
Solid Phase Isolation More rapid and effective Use solid matrix to bind the DNA. Wash away contaminants. Elute DNA from column
Solid Phase Isolation cont….. The diagram below explains the attractive properties
of solid phase for DNA and RNA.DNA/RNA hydrophilic……..AbsorbLipids/protiens……. Hydrophobic……wash away
Isolation of Mitochondrial DNA Mitochondrial DNA is passed from
generation to generation along the maternal lineage.
Centrifugation to separate out Lyse Precipitate with cold ethanol.
Nuclear DNA
Present in almost every cell
Combination from both parents; 23 chromosomes from each parent
Mitochondrial DNA
Each cell contains thousands of mt, each containing copies of its DNA
Mt DNA is in larger quantities in a cell
Nuclear DNA is larger in size
Mt DNA is 16,569 bases in length and consists of 2 different regions Coding Region Produces
13 proteins, 22 tRNAs 2 rRNAs
This region has very little variability
So everyone’s DNA in this region will be nearly the same sequence of TGCAs
Human Mitochondrial GenomeHuman Mitochondrial Genome
Human mtDNA composes of a control region (CR)
Control RegionThis region is highly variable within the human population
Consists of 2 subregions
HV1 = 342 bp HV2 = 268
Mutations occur in the control region of mt
We can compare DNA from the controlling region to other living humans See how related to you are to each other
Compare to prehistoric remains of human fossils Identify where you DNA originated Identify ancestral relationships between modern
populations
Compare your highly variable regions to other species
1. Isolate DNA from cheek cells2. Complete a PCR reaction
Produce millions of extra copies of HV1 on the control region of mtDNA
3. Send amplified DNA away to be sequenced (Identify the exact sequence of TGCAs in HV1 in your mtDNA)
4. Compare your sequence …… prehistoric DNA
Isolation of RNA
Requires STRICT precautions to avoid sample degradation.
RNAses RNases are naturally occurring
enzymes that degrade RNA Common laboratory
contaminant (from bacterial and human sources)
Also released from cellular compartments during isolation of RNA from biological samples
Can be difficult to inactivate
RNAses cont……
RNAses are enzymes which are small proteins that can renature and become active.
MUST be eliminated or inactivated BEFORE isolation.
CRITICAL to have a separate RNAse free area of lab.
Protecting Against RNAse Wear gloves at all times Use RNase-free tubes and pipet tips Use dedicated, RNase-free, chemicals Pre-treat materials with extended
heat (180 C for several hours), wash with DEPC-treated water (
Di ethyl pyro carbonate) Supplement reactions with RNase
inhibitors
Total RNA
80-90% of total RNA is ribosomal RNA.
2.5-5% is messenger RNA
Organic RNA Extraction1.Lyse/homogenize cells2.Add
phenol:chloroform:isoamyl alcohol to lysed sample, and centrifuge
3.Organic phase separates from aqueous phase Organic solvents on bottom Aqueous phase on top (contains total
RNA) Cellular debris and genomic DNA
appears as a “film” of debris at the interface of the two solutions
4.Remove RNA solution to a clean tube;
5. precipitate RNA and 6. wash with ethanol, then 7. resuspend RNA in water
Types of nucleic acid
Viral RNA Bacterial RNA Genomic RNA Viral DNA Bacterial DNA Genomic DNA Plasmid mitochondrial
TRI Reagent combines phenol and guanidine thiocyanate in a mono-phase
solution to facilitate the immediate and most effective inhibition of RNase activity.
A biological sample is homogenized or lysed in TRI Reagent . guanidinium isothiocyanate
powerful protein denaturant)
RNA is stable in trizol which deactivates RNases.
Chloroform causes proteins to become denatured and become soluble in the organic phase or interphase, while nucleic acids remain in the aqueous phase.
Isopropanol RNA is
insoluble in isopropanol so it will aggregate together, giving a pellet upon centrifugation.
This step also removes alcohol-soluble salt.
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