DNA and RNA isolation and purification (course readings 10 and 11) I. Genomic DNA preparation overview II. Plasmid DNA preparation III. DNA purification • Phenol extraction • Ethanol precipitation IV. RNA work What do we need DNA for? •Detect, enumerate, clone genes •Detect, enumerate species •Detect/sequence specific DNA regions •Create new DNA “constructs” (recombinant DNA) What about RNA? •Which genes are being transcribed? •When/where are genes being transcribed? •What is the level of transcription? DNA purification: overview cell harvest and lysis cell growth DNA concentration DNA purification Bacterial genomic DNA prep: cell extract Lysis: • Detergents • Organic solvent • Proteases (lysozyme) • Heat “cell extract” Genomic DNA prep: removing proteins and RNA chloroform Need to mix gently! (to avoid shearing breakage of the genomic DNA) Add the enzyme RNase to degrade RNA in the aqueous layer 2 ways to concentrate the genomic DNA 70% final conc. “spooling” Ethanol precipitation Genomic DNA prep in plants -how get rid of carbohydrates? CTAB: Cationic detergent CH3 CH3 (low ionic conditions) N+ CH3 Br- C16H33 (MC 6.61-6.62) 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 Plasmid purification: alkaline lysis Alkaline conditions denature DNA Neutralize: genomic DNA can’t renature (plasmids CAN because they never fully separate) DNA purification: silica binding Binding occurs in presence of high salt concentration, and is disrupted by elution with water DNA purification: phenol/chloroform extraction 1:1 phenol : chloroform or 25:24:1 phenol : chloroform : isoamyl alcohol Phenol: denatures proteins, precipitates form at interface between aqueous and organic layer Chloroform: increases density of organic layer Isoamyl alcohol: prevents foaming Phenol extraction 1. Aqueous volume (at least 200 microliters) 2. Add 2 volumes of phenol:chloroform, mix well 3. Spin in centrifuge, move aqueous phase to a new tube 4. Repeat steps 2 and 3 until there is no precipitate at phase interface 5. (extract aqueous layer with 2 volumes of chloroform) Ethanol precipitation (DNA concentration) Ethanol depletes the hydration shell surrounding DNA… • Allowing cations to interact with the DNA phosphates • Reducing repulsive forces between DNA strands • Causing aggregation and precipitation of DNA • Aqueous volume (example: 200 microliters) -- add 22 microliters sodium acetate 3M pH 5.2 -- add 1 microliter of glycogen (gives a visible pellet) -- add 2 volumes (446 microliters) 100% ethanol -- mix well, centrifuge at high speed, decant liquid -- wash pellet (70% ethanol), dry pellet, dissolve in appropriate volume (then determine DNA concentration) DNA purification: overview cell harvest and lysis cell growth DNA concentration DNA purification DNA --------------> mRNA --------------> protein Lots of information in mRNA: When is gene expressed? What is timing of gene expression? What is the level of gene expression? (but what does an mRNA measurement really say about expression of the protein?) Isolation of RNA -- Course reading 11 RNA in a typical eukaryotic cell: 10-5 micrograms RNA 80-85% is ribosomal RNA 15-20% is small RNA (tRNA, small nuclear RNAs) About 1-5% is mRNA -- variable in size -- but usually containing 3’ polyadenylation The 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 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 Top 10 sources of RNAse contamination (Ambion Scientific website) 1) Ungloved hands 2) Tips and tubes 3) Water and buffers 4) Lab surfaces 5) Endogenous cellular RNAses 6) RNA samples 7) Plasmid preps 8) RNA storage (slow action of small amounts of RNAse 9) Chemical nucleases (Mg++, Ca++ at 80°C for 5’ +) 10) Enzyme preparations 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) Inhibitors of Rnase Vanadyl ribonucleoside complexes competitive inhibitors of RNAses, but need to be removed from the final preparation of RNA Protein inhibitors of RNAse horseshoe-shaped, leucine rich protein, found in cytoplasm of most mammalian tissues must be replenished following phenol extraction steps Making and using mRNA (1) Making and using mRNA (2) 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 H20 (-80°C) in formamide (deionized) at -20°C 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 Other methods to capture mRNA Poly(U) sepharose chromatography Poly(U)-coated paper filters Streptavidin beads: •A biotinylated oligo dT is added to guanidiniumtreated cells, and it anneals to the polyA tail of mRNAs •Biotin/streptavidin interactions permit isolation of the mRNA/oligo dT complexes How good is the RNA prep? The rRNA should form 2 sharp bands in ethidium bromide-stained gels (but mRNA will not be visible Use radiolabelled poly dT in a pilot Northern hybridization--should get a smear from 0.6 to 5 kb on the blot Use a known, “standard” gene probe (e.g. GAPDH in mammalian cells) in Northern hybridization--there should be a sharp band with no degradation products In vitro amplification of DNA by PCR I. II. III. Theory of PCR Components of the PCR reaction A few advanced applications of PCR a) b) c) d) e) Reverse transcription PCR (for RNA measurements) Quantitative real-time PCR PCR of long DNA fragments Inverse PCR MOPAC (mixed oligonucleotide priming) Molecular Cloning, p. 8.1-8.24 What is PCR? • Polymerase Chain Reaction--first described in 1971 by Kleppe and Khorana, re-described and first successful use in 1985 • Allows massive amplification of specific sequences that have defined endpoints • Fast, powerful, adaptable, and simple* • Many many many applications * usually Why amplify specific sequences? • To obtain material for cloning and sequencing, or for in vitro studies • To verify the identity of engineered DNA constructs • To monitor gene expression • To diagnose a genetic disease • To reveal the presence of a micro-organism • To identify an individual • Etcetera, etcetera What you need for PCR: 1. Template DNA that contains the “target sequence” 2. Primers: short oligonucleotides that define the ends of the target sequence 3. Thermostable DNA polymerase 4. Buffer, dNTPs 5. A thermal cycler A typical PCR program: Denaturation: denature template strands (94°C for 2-5 minutes), can also add your DNA polymerase at this temp. for a “hot start” (adding DNA pol to a hot tube can prevent false priming in the initial round of DNA replication) Annealing: The default is usually 55°C. This temperature variable is the most critical one for getting a successful PCR reaction. This is the best variable to start with when trying to optimize a PCR reaction for a specific set of primers. Annealing temperatures can be dropped as low as 40-45°C, but non-specific annealing can be a problem A typical PCR program: Extension: generally 72°C, this is the operating temperature for many thermostable DNA polymerases. Number of cycles: Depends on the number of copies of template DNA and the desired amount of PCR product. Generally 20-30 cycles is sufficient. How it works: a simple PCR reaction, first cycle (Can also be Single-stranded) 94°C 50°C 72°C Cycles of denaturation, primer annealing, and primer extension by DNA polymerase a simple PCR reaction, second cycle new reactions like first cycle a simple PCR reaction, third cycle PCR animation: http://www.dnai.org/b/index.html http://www.dnalc.org/ddnalc/resources/shockwave/pcran whole.html Choosing primers: • Should be 18-25 (17-30?) nucleotides in length (giving specificity) • Calculated melting temperature varies depending on the method used (55-65°C using the Wallace Rule, eg. see MC), but should be nearly identical for both primers • Avoid inverted repeat sequences and self-complementary sequences in the primers, avoid complementarity between primers (‘primer dimers’) • Have a G or C at the 3’ end (a G/C “clamp”) • Many computer programs exist for helping meet these criteria (ex: Biology Workbench, workbench.sdsc.edu) Thermostable DNA polymerases (See Molecular Cloning table 8-1) • Isolated from thermophilic bacteria and archaea (T. aquaticus is a bacterium, not an archaeon) • Bacterial enzymes (e.g. Taq) good for routine reactions and small PCR products, fidelity of replication is somewhat low • Archaeal enzymes (e.g. Pfu) also good for routine reactions and best for cloning: 3’--5’ exonuclease activity provides very high fidelity, and enzymes are very stable to heat Thermal cyclers Standard: heat block, “ramp” times fairly long (10 -20 seconds to change temperature), 30 cycle PCR lasts 2-3 hours. Advantage: easily automated, heat blocks can PCR up to 384 samples at a time Disadvantage: relatively slow New: reactions are being sped up significantly --capillary tubes heated and cooled by blasts of air-30 cycle-PCR done in >30 minutes (harder to scale up) --fluid flow cells: channels force liquid through temperature gradients, very fast (but still not widely available) Sources of problems in PCR • Inhibitors of the reaction from the the template DNA preparation (protease, phenol, EDTA, etc) • Cross-contamination by DNA from sources other than the template added – if this becomes a problem: • Work in a laminar flow hood (decontaminate using UV light 254 nm) • Use PCR dedicated pipettors (with barrier tips), PCR dedicated reagents • Centrifuge tubes before opening them to prevent spattering, pipet contamination Controls to include in difficult PCRs: Bystander DNA template DNA Target DNA Specific primers Positive controls 1 + - + + 2 - - + + 3 - - - + 4 + - - + Negative controls Bystander DNA: not recognized by primers Target DNA: known to contain primer recognition sequences Hot Start of PCR reactions • • Witholding some component of the reaction until the denaturing temperature is reached (94°C) This helps prevent non-specific priming, which may occur at the low temperatures (room temp.) -- the non-specific priming could give artifactual amplification as PCR block temperature rises A) Wait until 94°C to add enzyme --or-B) Use enzyme bound to an inactivating enzyme antibody that releases at high temperature --or-C) Use wax beads containing Mg++ that can only be released at high temp. Touchdown PCR • Useful if your primers are not 100% complementary to your template DNA (e.g. degenerate oligos), or when there are multiple members of the gene family you are amplifying • Allows you to selectively amplify only the best sequences (with the least mismatches) while minimizing non-specific PCR products • Start with 2 cycles at an annealing temperature about 3°C higher than the calculated primer melting temperatures. • Progressively reduce the annealing temperature by 1°C at 2 cycle intervals Trouble-shooting: -- Very little product -- No PCR product -- Multiple bands Etc. (see Molecular cloning, tables 8-4 and 8-5) III. Special applications for PCR A. Reverse transcription PCR (for RNA measurements) B. Quantitative (real-time) PCR C. PCR of long DNA fragments D. Whole genome amplification E. Inverse PCR E. MOPAC (mixed oligonucleotide priming) Amplification of RNA (monitor gene expression): reverse transcription PCR (RT-PCR) Step 1: generate a 1st strand cDNA using reverse transcriptase (catalyzes synthesis of DNA from an RNA template) A) B) C) Step 2: normal PCR (from cDNA) using gene-specific primers Quantitative (real time) PCR The more target DNA there is, the more probe anneals, the more it is cleaved (by Taq’s 5’-3’ exonuclease activity) Fluorescence measurements are done simultaneously with PCR cycles, yields an instantaneous measurement of product levels Quantitative Real Time (QRT) PCR Position of the steep part of the curve varies depending on the amount of template DNA or RNA, can measure variation over 5 or 6 orders of magnitude more template less template Another quantitative measure of double stranded DNA in a PCR reaction: binding of SYBR Green Dye Non-fluorescing SYBR green dye Fluorescing SYBR green dye From the Molecular Probes website (www.probes.com) Use of a quenching dye to reduce measurement of “primer dimer” artifacts in QRT-PCR QSY quencher dye: it absorbs fluorescence from sybr green dyes in the vicinity--prevents accumulation of signal from primer dimers Always do your controls! QRT-PCR using Sybr green dye fluorescence Standard curve: what is “threshold” for specific number of DNA molecules? (From the Invitrogen website) PCR of long sequences (>2 kb) Long DNAs are difficult to amplify – Breakage of the DNA – Non-processive behavior of DNA polymerase – Misincorporation by error prone DNA polymerases PCR of long sequences (>2 kb) Changes to protocol that assist in long PCR – Make sure DNA is exceedingly clean – Use DNA polymerase “cocktail”: Taq for it’s high activity, and Pfu for its proofreading activity (it can actually correct Taq’s mistakes) – Increase time of extension reaction (5-20 minutes, compared to the standard 1 minute for short PCRs) •Amplified product longer than 3 kilobases with high fidelity •10 times fewer mutations than with conventional PCR •Taq DNA pol (no proofreading) plus an archaeal DNA pol (does proofreading) •Betaine (amino acid analogue with several small tetraalkyammonium ions)--reduces non-specific amplification products--reduces non-complementary primer-template interactions? (unknown how it works) Whole genome amplification: multiple displacement amplification (MDA) Applications: forensics, embryonic disease diagnosis, microbial diversity surveys, etc. How it works: Strand-displacement amplification used by rollingcircle replication systems. Phi29 DNA polymerase (very low error rate) and random hexamer primers, low temperature! (30°C) Whole genome amplification : multiple displacement amplification (MDA) 20-30 micrograms human DNA can be recovered from 1-10 copies of the human genome Distribution of products appears to be random sampling of the available template (and this is good!) Inverse PCR: sequencing “out” from known sequence “Vectorette” PCR First primer: known sequence Vectorette primer: only in vectorette-ligated sequence--it cannot anneal until there is a single round of primer extension from the specific primer http://www.bio.psu.edu/People/Faculty/Akashi/vectPCR.html MOPAC: Mixed oligonucleotide primed amplification of cDNA If you only have a protein sequence, and you want to clone the gene for the protein: 1. Design oligonucleotides based on deduced mRNA (and DNA) sequence (but since multiple codons can encode the same amino acid, this gets complicated quickly) 2. program your oligo synthesizer to make primer sets that are randomized for the degenerate positions of each codon 3. use universal nucleotides like inosine, which base pairs with C, T, and A (limits degeneracy) 4. Do your PCR and hope for the best