Unit 6 Nucleic Acid Extraction Methods

1. Unit 6 Nucleic Acid Extraction Methods Terry Kotrla, MS, MT(ASCP)BB Fall 2007

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. 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.

4. Organic Isolation • Must purify DNA by removing contaminants. • Accomplished by using combination of high salt, low pH and an organic mixture of phenol and chloroform. • To avoid RNA contamination add RNAse, enzyme that degrades RNA.

5. Phenol/Chloroform • Biphasic emulsion forms • Hydrophobic layer on bottom has cell debris. • Hydrophilic layer on top has dissolved DNA • Remove top layer, add cold ethanol, DNA precipitates out.

6. Inorganic Isolation Methods • Also called “salting out”. • Uses low pH and high salt condition to selectively precipitate proteins. • DNA is left in solution (picture on far left). • Precipitate out DNA with isoproproanol (middle and right side pictures). • I know this is not scientific but the precipitated out DNA is usually referred to as “snotty”.

7. Solid Phase Isolation • More rapid and effective • Use solid matrix to bind the DNA. • Wash away contaminants. • Elute DNA from column • Textbook page 70, Figure 4.3

8. Solid Phase Isolation • The diagram below explains the attractive properties of solid phase for DNA and RNA.

9. Crude Lysis • Used for: • Screening large numbers of samples • Isolation of DNA in limited amounts • Isolation from challenging samples, ie, paraffin embedded tissue • Usually not done in clinical laboratory. • Textbook page 71, Figure 4-4

10. 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.

11. Isolation of RNA • Requires STRICT precautions to avoid sample degradation. • RNA especially labile.

12. 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

13. RNAses • 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.

14. 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, NaOH or H2O2 • Supplement reactions with RNase inhibitors

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

16. 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

17. Affinity Purification of RNA • Lyse cells, and spin to remove large particulates/cell debris • Apply lysate (containing nucleic acids and cellular contaminants) to column with glass membrane • Wash with alcohol to remove contaminants; nucleic acids stick to glass membrane while contaminants wash through. Treat with DNase enzyme to remove contaminating DNA. • Apply water to the column; purified RNA washes off the glass and is collected

18. Isolation of PolyA (messenger) RNA • Only 2.5-5% • mRNA molecules have a tail of A’s at the 3’ end (polyA tail) • Oligo(dT) probes can be used to purify mRNA from other RNAs • mRNA can be eluted from oligo(dT) matrix using water or low-salt buffer • Textbook page 75, Figure 4.7

19. Electrophoresis • Analyze DNA and RNA for quality by electrophoresis. • Fluorescent dyes • Ethidium bromide • SybreGreen • Appearance depends on type of DNA isolated. • Will be covered in more detail in unit 7.

20. Staining with Ethidium Bromide and Sybr Green • DNA is electrophoresed and stained. • Left – Ethidium Bromide Right – Sybr Green • The lane in the left side of the picture on the left is a “ladder” which has fragments of known base pair (bp) sizes. This allows determination of the size of isolated fragments.

21. Spectrophotometry • Sample absorbances are determined on the spectrophotometer at 260nm and 280nm • nucleic acid (DNA, RNA, nucleotides) absorb light at 260nm • protein absorbs light at 280 nm 230nm: guanidine • A260/A280 ratio is a measure of DNA purity • The absorbance wavelength is directly proportional to the concentration of nucleic acid.

22. Determining Concentration • Formula Concentration = 260 Reading * Absorbance unit * Dilution factor • One optical density or absorbance unit at 260 nm is equal to • 50 ug/mL for DNA • 40 ug/mL for RNA. • Textbook page 77 has sample calculations.

23. Determining Purity • The OD at 260nm should be 1.6-2.00 times more than the absorbance at 280nm. • Divide the OD at 260nm by the OD at 280nm to get the ratio. • If the 260nm/280nm ratio is less than 1.6 for DNA, 2.0-2.3 for RNA this indicates contamination, usually with protein. • DNA -If the OD ratio is higher than 2.0 it may be contaminated with RNA. • Ratio of the readings : O.D.260/O.D.280 is a measure of purity. • Pure preparations of DNA and RNA have O.D 260/280 of 1.8 and 2.0 respectively.

24. Fluorometry • Fluorometry utilizes fluorescent dyes which specifically bind DNA or RNA. • It requires a negative control (to set the zero point on the fluorometer) and a standard of known concentration. • The fluorometer shines light on the sample (excitation) and then measures level of fluorescent light being emitted to the side (at a 90º angle) of the excitation light beam. • The fluorescent dyes are relatively specific to nucleic acids as opposed to protein and other cellular components. • The fluorescence of the dyes increases when they bind nucleic acids.

25. Fluorometry • Fluorometry is about 1,000x more sensitive than spectrophotometric absorbance (i.e. measurement of A260) and less susceptible to protein and RNA contamination. • However it also does not give a crude measurement of purity (like an A260/A280 ratio) nor does it assure that the DNA or RNA is not degraded (e.g. like size determination by gel electrophoesis). • Do not use glass (spectrophotmetry) cuvettes in a fluorometer because the frosted glass on the side of the cuvette interferes with detection of fluorescent light.