NUCLEIC ACID ISOLATION PROTOCOLS

1. CENTRAL DOGMA OF MOLECULAR BIOLOGY
2. NUCLEIC ACID PREPARATION & APPLICATIONS
3. FUNDAMENTAL STEPS IN DNA PURIFICATION
4. ANALYSIS OF NUCLEIC ACIDS
5. STORAGE CONDITIONS

1. NUCLEIC ACID ISOLATION

2. The Central Dogma of Molecular Biology Replication Transcription Translation mRNA non-coding RNA (rRNA, tRNA, siRNA, etc.)

3. Deoxyribonucleotide Ribonucleotide

4. NUCLEIC ACID PREPARATION Application? Amplification methods (PCR, LCR) Restriction enzyme digest Hybridization methods (Southern analysis) Sequencing RNA Amplification methods (RT-PCR) Hybridization methods (Northern analysis) DNA The very first DNA isolation was done by a Swiss physician, Friedrich Miescher in 1869

5. FUNDAMENTAL STEPS OF DNA PURIFICATION Sample Lysis Removal contaminants from Nucleic acids Concentration of Nucleic acids Measurement of purity and concentration of Nucleic acids.

6. DNA purification: overview cell growth cell harvest and lysis DNA purification DNA concentration

7. CELL LYSIS Mechanical Method Grinding in Liq N2 Sonication Homogenization Heat Chemical method SDS Triton X-100 CTAB Enzymatic Method Lysozyme Zymolase & Murienase Proteinsae K

8. Separation of Nucleic acids from the contaminants Organic (Phenol-Chloroform) Extraction Non-Organic ( Proteinase K and Salting out) Chelex (Ion Exchange Resin) Extraction Silica Based FTA Paper (Collection, Storage, and Isolation)

9. ORGANIC SOLVENT EXTRACTION METHOD

10. Adsorption Chromatography Method Step 1: Prepare crude lysate Silica-gel membrane Apply to column Step 2: Adsorb to silica surface Centrifuge Flow through (discard) Nucleic acids Surface silanol groups are weakly acidic, and will repel nucleic acids at near neutral or high pH due to their negative charge Extraction Buffer composition favors DNA and RNA adsorption to silica: • Low pH • High ionic strength • Chaotropic salt Nucleic acids bind to the membrane, while contaminants pass through the column.

11. Adsorption Chromatography Method Centrifuge Nucleic acids Step 3: Wash away residual contaminants Wash buffer Nucleic acids Flow through (discard) Nucleic acids Elution buffer Elution Buffer composition is unfavorable to surface binding: High pH Low ionic strength Step 4: Elute nucleic acids Centrifuge Nucleic acids

12. FTA TM Paper Extraction Cellulose based storage paper Extraction/storage of nucleid acids from blood, buccal cells, tissue, cultured cells, microorganisms, plant tissue and other.. DNA is stable at room temperature for years

13. Fast and efficient DNA preparation: punch and purify Apply sample on FTA paper and let dry Cells are lysed on contact Use punch to add sample to tube Wash to remove PCR inhibitors Add „punch“ directly to PCR reaction.

14. FTA purification reagent (whatmann): For purification of nucleic acids stored on FTA cards Ensures superior quality dna for pcr Removes heme, pcr inhibitors and other potential contaminants. Non-toxic, hypoallergenic aqueous solution.

15. FTA PAPER Exposes nucleic acids - lyses cells and organelle membranes - physically entraps nucleic acids Preserves and protects nucleic acids - prevents damage by UV/ free radicals - prevents enzymatic damage - inhibits fungi and microbial growth Provides user safety - inactivates potentially harmful viruses http://www.dnasafestorage.com/httpdocs/FTA%20Elute%20card.JPG

16. CONCENTRATION OF THE GENOMIC DNA 70% final conc. “spooling” Ethanol precipitation Ethanol depletes the hydration shell surrounding DNA and Reduces repulsive forces between DNA strands which Causes aggregation and precipitation of DNA

17. Plasmids: vehicles of recombinant DNA Bacterial cell genomic DNA plasmids Non-chromosomal DNA Replication: independent of the chromosome Many copies per cell Easy to isolate Easy to manipulate

18. Plasmid purification: alkaline lysis Alkaline conditions denature DNA Neutralize: genomic DNA can’t renature (plasmids can because they never fully separate)

19. Problem(s) with RNA: RNA is chemically unstable -- spontaneous cleavage of phosphodiester backbone via intramolecular transesterification RNA is susceptible to nearly ubiquitous RNA-degrading enzymes ( RNases ) RNases are released upon cell lysis RNases are present on the skin RNases are very difficult to inactivate -- disulfide bridges conferring stability -- no requirement for divalent cations for activity

20. Common sources of RNase and how to avoid them Contaminated solutions/buffers Use good sterile technique Treat solutions with depc (when possible) Make small batches of solutions Contaminated equipment Use “ rna -only” pipets , glassware, gel rigs Bake glassware, 300°c, 4 hours Use “ rnase -free” pipet tips Treat equipment with depc

21. Top 10 sources of RNAse contamination ( Ambion Scientific website) Ungloved hands Tips and tubes Water and buffers Lab surfaces Endogenous cellular RNAses RNA samples Plasmid preps RNA storage (slow action of small amounts of RNAse Chemical nucleases (Mg++, Ca++ at 80°C for 5’ +) Enzyme preparations

22. Inhibitors of Rnase DEPC: Diethylpyrocarbonate alkylating agent, modifying proteins and nucleic acids fill glassware with 0.1% DEPC, let stand overnight at room temp. Solutions may be treated with depc -- add depc to 0.1%, then autoclave (DEPC breaks down to CO2 and ethanol).

23. Making and using mRNA (1)

24. Making and using mRNA (2)

25. Purifying RNA: the key is speed Break the cells/ solubilize components/inactivate RNAses by the addition of guanidinium thiocyanate (very powerful denaturant) Extract RNA using phenol/chloroform (at low pH) Precipitate the RNA using ethanol/ LiCl Store RNA: in DEPC-treated H 2 0 (-80°C) in formamide ( deionized ) at -20°C.

26. Selective capture of mRNA: oligo dT -cellulose Oligo dT is linked to cellulose matrix RNA is washed through matrix at high salt concentration Non- polyadenylated RNAs are washed through polyA RNA is removed under low-salt conditions (not all of the non- polyadenylated RNA gets removed).

27. Other methods to capture mRNA Poly(U) sepharose chromatography Poly(U)-coated paper filters Streptavidin beads: A biotinylated oligo dT is added to guanidinium -treated cells, and it anneals to the polyA tail of mRNAs Biotin/ streptavidin interactions permit isolation of the mRNA/ oligo dT complexes

28. NUCLEIC ACID ANALYSIS DNA or RNA is characterized using several different methods for assessing quantity, quality, and molecular size. UV spectrophotometry Agarose gel electrophoresis Fluorometry Colorimetric blotting.

29. Quantity from UV Spectrophotometry DNA and RNA absorb maximally at 260 nm. Proteins absorb at 280 nm. [ dsDNA ] = (A260) X dilution factor X 50 µg/ mL [ ssDNA ] = (A260) X dilution factor X 33 µg/ mL [RNA] = (A260) X dilution factor X 40 µg/ mL [ Oligonucleotides ] = (A260) X dilution factor X 20-30µg/ mL Quantity from UV Spectrophotometry

30. The A 260 /A 280 ratio is ~1.8 for dsDNA , and ~2.0 for ssRNA . Ratios lower than 1.7 usually indicate significant protein contamination. The A 260 /A 230 ratio of DNA and RNA should be roughly equal to its A 260 /A 280 ratio (and therefore ≥ 1.8). Lower ratios may indicate contamination by organic compounds (e.g. phenol, alcohol, or carbohydrates). Quality from UV Spectrophotometry

31. Quality from Agarose Gel Electrophoresis Genomic DNA: 0.6% to 1% gel, 0.125 µg/ mL ethidium bromide in gel and/or in running buffer Electrophorese at 70–80 volts, 45–90 minutes. Total RNA: 1% to 2% gel, 0.125 µg/ml ethidium bromide in gel and/or in running buffer Electrophorese at 80–100 volts, 20–40 minutes.

32. 100 bp ladder 1 kb ladder Lambda DNA cut with Hin d III Lambda DNA 48,500 bp (48.5 kb) 12,218 bp 23,130 bp 9,416 bp 6,557 bp 4,361 bp 2,322 bp 2,027 bp 517 bp 1,636 bp 3,054 bp 6,018 bp 100 bp 300 bp 600 bp 1,000 bp 1,500 bp 1,018 bp 2,036 bp DNA Size from Agarose Gel Electrophoresis: Compares unknown DNA to known size standards

33. Lambda DNA marker Human Whole Blood DNA Lambda DNA cut with Hin d III marker Whole blood genomic DNA DNA Quality from Agarose Gel Electrophoresis

34. Degraded RNA DNA 28S 18S 5S rRNA, tRNA, and other small RNA molecules mRNA = background smear high  low MW 100 50 25 ng Genomic DNA markers Cultured Cell RNA

35. Storage Conditions Store DNA in TE buffer at 4 °C for weeks or at –20 °C to –80 °C for long term. Store RNA in RNase -free ultra pure water at –70 °C.