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.