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 1. Lyse/homogenize cells 2. 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; precipitate RNA and wash with ethanol, then resuspend RNA in water

17. Affinity Purification of RNA 1. Lyse cells, and spin to remove large particulates/cell debris 2. Apply lysate (containing nucleic acids and cellular contaminants) to column with glass membrane 3. Wash with alcohol to remove contaminants; nucleic acids stick to glass membrane while contaminants wash through. Treat with DNase enzyme to remove contaminating DNA. 4. 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.