Nucleic Acid Extraction Methods

1. Nucleic Acid Extraction Methods

2. 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.

3. 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!

4. 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

5. 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)

6. 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

7. Anticoagulants • EDTA • Preferred specimen • ACD (Acid Citrate Dextrose) • Yellow-top Vacutainer • Heparin • Green-top Vacutainer • Inhibits several enzymes used in molecular assays Heparin a 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.

8. 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

9. Nucleic Acid PreparationApplication? • DNA • Amplification methods (PCR) • Restriction enzyme digest • Hybridization methods (Southern analysis) • Sequencing

10. Nucleic Acid Storage Requirements: Storage of DNA Specimens <4 Months 1–3 Years <7 Years >7 Years 2–25 °C 2–8 °C –20 °C –70 °C Recommended for long-term storage

11. Nucleic Acid PreparationApplication? • RNA • Amplification methods (RT-PCR) • Hybridization methods (Northern analysis)

12. 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

13. 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

14. 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.

15. 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.

16. 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.

17. 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). 2 1

18. Solid Phase Isolation • More rapid and effective • Use solid matrix to bind the DNA. • Wash away contaminants. • Elute DNA from column

19. Solid Phase Isolation cont….. • The diagram below explains the attractive properties of solid phase for DNA and RNA. • DNA/RNA hydrophilic……..Absorb • Lipids/protiens……. Hydrophobic……wash away

20. Isolation of Mitochondrial DNA • Mitochondrial DNA is passed from generation to generationalong the maternal lineage. • Centrifugation to separate out • Lyse • Precipitate with cold ethanol.

21. Nuclear DNA • Present in almost every cell • Combination from both parents; 23 chromosomes from each parent

22. Mitochondrial DNA • Each cell contains thousands of mt, each containing copies of its DNA • Mt DNA is in larger quantitiesin a cell • Nuclear DNA is larger in size

23. 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

24. Human Mitochondrial Genome Human mtDNA composes of a control region (CR)

25. Control Region This region is highly variable within the human population Consists of 2 subregions HV1 = 342 bp HV2 = 268 610 bp with a lot of variability

26. Mutations occur in the control region of mt

27. How do we use this information? • 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

28. How is mtDNA isolated? • Isolate DNA from cheek cells • Complete a PCR reaction • Produce millions of extra copies of HV1 on the control region of mtDNA • Send amplified DNA away to be sequenced (Identify the exact sequence of TGCAs in HV1 in your mtDNA) • Compare your sequence …… prehistoric DNA

30. Isolation of RNA • Requires STRICT precautions to avoid sample degradation.

31. 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

32. RNAses cont…… • RNAses are enzymes which are small proteins that can renature and become active. • MUST be eliminated or inactivatedBEFORE isolation. • CRITICAL to have a separate RNAse free area of lab.

33. 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

34. Total RNA • 80-90% of total RNA is ribosomal RNA. • 2.5-5% is messenger RNA

35. Organic RNA Extraction • Lyse/homogenize cells • Add phenol:chloroform:isoamyl alcohol to lysed sample, and centrifuge • 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 • Remove RNA solution to a clean tube; • precipitate RNA and • wash with ethanol, then • resuspend RNA in water

36. Types of nucleic acid • Viral RNA • Bacterial RNA • Genomic RNA • Viral DNA • Bacterial DNA • Genomic DNA • Plasmid • mitochondrial

38. 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. • Chloroformcauses • proteins to become denatured and • become soluble in the organic phase or interphase, • while nucleic acids remain in the aqueous phase.

39. Isopropanol • RNA is • insoluble in isopropanol so it will aggregate together, giving a pellet upon centrifugation. • This step also removes alcohol-soluble salt.