Visualizing DNA (and RNA, protein): non-specific detection methods Quantitation of DNA Electrophoresis Visualizing DNA (& protein) in gels

Quantitation of DNA by UV absorbance Measure absorbance of UV light by sample (the aromatic bases have a characteristic absorbance maximum at around 260 nanometers) 1.0 A260 = DNA concentration of 50 micrograms per ml (double stranded DNA) or 38 micrograms per ml (single-stranded DNA or RNA) the effective range for accurate measurement is rather narrow: A260 from 0.05 to 2.0 (DNA concentrations from 2.5 to 100 micrograms/ml) Sample must be very pure for accurate measurements (RNA, EDTA and phenol all absorb at 260 nm)

How can concentration be determined by absorbance? DNA has a characteristic “molar extinction coefficient”  The Beer-Lambert law: I = Io10- dc I = intensity of transmitted light Io = intensity of incident light  = molar extinction coefficient d = optical path length c = concentration of absorbing material How much light gets through a solution depends on what’s in it and how much of it there is

The Beer-Lambert law: I = Io10- dc Absorbance A measured by a spec is log I/Io When path length d = 1 cm, A is called the optical density OD If you know the , the absorbance of a solution will tell you the concentration: OD = c for nucleic acids: dsDNA: 6.6 ssDNA, RNA: 7.4 (but these values change with pH and salt concentration!)

A typical (good) “scan” (multiple wavelengths) of a DNA sample A260/A280: 1.8 is good (lower values indicate significant protein contamination) 0.5 A260 = 0.327 (1 cm path length) Absorbance 200 260 400 Wavelength (nm)

How does A260 give you the quantity of DNA? Example: sample of 250 base pair fragment of DNA has an A260 = .327 What is its molar concentration? Given: (1.0 A = 50 micrograms/ml DNA) DNA conc. = 0.327 x 50 = 16.35 micrograms/ml MW of an average bp. = 650 Daltons Therefore 250 bp. Fragment has a MW of 1.6 x 10 5 Daltons Solve for molarity: 1.02 x 10 -7 M, or 102 nanomolar (nM)

0.102 x 10-6 molar [0.1 micromolar (M)] What is the molarity of a 16.35 microgram/ml solution of a 250 base pair DNA fragment? 16.35 micrograms 1000 ml 1 gram 1 mole 1 ml 1 L 106 micrograms 1.6 x 105 grams 1.02 x 10 -7 molar 0.102 x 10-6 molar [0.1 micromolar (M)] 102 x 10-9 molar [102 nanomolar (nM)]

Fluorometry: another method for quantitation of DNA Hoechst 33258 (a fluorescent dye) Binds to DNA in the minor groove (without intercalation) Fluorescence increases following binding Good for quantitation of low concentrations of DNA (10-250 ng/ml) rRNA and protein do not interfere But you need a fluorometer

Another method for quantitation of DNA: Ethidium bromide (fluorescent dye) binding Compare sample DNA fluorescence to standards of known concentration (dilution series) In solution *or* using gel electrophoresis A commercially available quantitative DNA standard

Visualizing DNA: Electrophoresis Allows separation of biomolecules (DNA, RNA, protein) on basis of size A separation matrix, or gel (agarose or polyacrylamide), is saturated with an electrically conductive buffer Samples are loaded, an electric field is applied, and negatively charged biomolecules in the sample travel toward the cathode The larger the molecule, the slower the travel through the gel matrix Dyes allow a visual estimate of the rate of travel through the gel The choice of matrix depends mainly on the size of DNA being analyzed

Agarose gels Agarose: a polysaccharide polymer of alternating D- and L- galactose monomers, isolated from seaweed Pore size is defined by the agarose concentration (higher concentration, slower DNA migration overall) The conformation of the DNA (supercoiled, nicked circles, linear) affects the mobility of the DNA in gels Rate of DNA migration is affected by voltage (5 to 8 Volts/cm is close to optimal) Agarose comes in a myriad of types (variable melting temperatures, generated by differential hydroxyethylation of the agarose)

Agarose gels Standard gels can separate DNA fragments from 100 bp to about 20,000 bp Pulsed-field gels separate very large DNA fragments (up to 10,000,000 bp, or 10 Mb) This apparatus allows periodic shifts in the direction of DNA migration: 120° refers to the reorientation angle (difference between orientation of electric fields A and B

 - + Typical agarose gel Load samples in wells xylene bromophenol cyanol bromophenol blue - +  (the DNA fragments are not visible without some sort of staining) time of electrophoresis (progress monitored by marker dyes)

Polyacrylamide gels Acrylamide monomers (toxic!) polymerized to form gel matrix The gel structure is held together by the cross-linker-- usually N, N'-methylenebisacrylamide ("bis" for short) Pore size defined by concentration of gel (total percentage) and concentration of the crosslinker (bis) relative to acrylamide monomer Very high resolution (better than agarose) Suitable for separation of nucleic acids from 6 to 1000 base pairs in length

Polyacrylamide gels Native gels (DNA stays double-stranded) Denaturing gels--run in the presence of high concentrations of denaturant (usually urea) and at high temperature: DNA is single stranded (sequencing gels) (also useful in separation of proteins, when proteins are treated with SDS, which denatures proteins and gives a uniformly negative surface charge)

Recipe for a polyacrylamide gel: Acrylamide (anywhere from 4 to 20 %, depending size of nucleic acids or proteins in the gel) Bis-acrylamide (the ratio of Bis to regular acrylamide is important) Water Buffer To initiate polymerization, add APS: Ammonium persulfate -- generates free radicals needed for polymerization TEMED: N,N,N’,N’ - tetramethylethylenediamine -- accelerates free radical generation by APS

More about gels There has to be a buffer (for carrying current) TAE (Tris-acetate-EDTA): good resolution of DNA, but buffering capacity is quickly depleted TBE (Tris-borate-EDTA): High buffering capacity, resolution is pretty good Use gel loading “buffers” (relatively simple) Dense material to carry sample to bottom of wells (sucrose, glycerol, or ficoll) Dyes for tracking progress of electrophoresis Bromophenol blue: fast migration Xylene cyanol: slow migration Occasionally denaturant is present (formamide) for denaturing gels (e.g. sequencing gels)

Protein electrophoresis Almost always polyacrylamide based The anionic detergent SDS (sodium dodecyl sulfate) is used to denature the proteins, giving each protein a “uniform” negative charge Protein separation occurs as a function of size Discontinous Tris-Cl/glycine buffer system: Stacking gel: pH 6.8, low polyacrylamide concentration, focuses proteins into thin layer (gives higher resolution upon separation) Separating gel: pH 8.8, separates proteins on the basis of size

Polyacrylamide gel set up (protein gels) Stacking gel: at low pH, glycine is protonated (no neg. charge), Cl- ions at the leading edge, glycine trailing, steep voltage gradient in between, that’s where the proteins get “focused” into a thin band Separating gel: at higher pH, glycine deprotonates, runs with the Cl- at the leading edge, and the proteins separate based on size

Staining nucleic acids ethidium bromide, an anti-trypanosomal drug for cattle Stain works by intercalating in stacked base pairs, elongates DNA helix Fluorescence increases upon DNA binding Stained bands visualized by UV illumination (302 or 260 nm) G-C base pair Ethidium bromide

Example of an agarose-DNA gel, Stained with ethidium bromide Fragments of bacteriophage  genomic DNA (48 kb) cut with the restriction enzyme Hind III The fragments are equimolar--why is the band intensity different? Direction of electrophoresis

Another ethidium bromide-stained agarose gel samples The marker lane (M) gives size standards for comparison with the sample lanes

Other methods for staining DNA SYBR gold (Molecular Probes, Eugene, OR), more than 10-fold more sensitive than ethidium bromide for detecting DNA, but expensive! methylene blue: not toxic, but the staining protocol is time consuming, and sensitivity somewhat lower than ethidium bromide silver staining: high degree of sensitivity, but the protocols are time consuming, and proteins are also stained by silver

Protein detection in gels Coomassie Brilliant Blue R-250: dye from the textile industry that has a high affinity for proteins Proteins in gels must be “fixed” (rendered insoluble) first with acetic acid/methanol Dye probably interacts with NH3- groups of the proteins, also through van der Waals forces http://www.galab.de/laboratories/services/biopharma/img/sds.jpg

Protein detection in gels Silver staining: 100 to 1000-fold more sensitive than Coomassie stain for detecting proteins (need far less sample to see it on a gel) Process relies on differential reduction of silver ions bound to amino acid side chains (like the photographic process) There is protein-to-protein variability of staining ugly (keratin) good bad (overstained)

Protein detection in gels Sypro Ruby (Molecular Probes inc, proprietary compound) As sensitive as silver staining, less variability Fast protocol Expensive 1 nanogram of protein Standard gel 2-D gel

Visualizing DNA (and RNA, protein): non-specific methods Quantitation of DNA (Course Reading 4) Electrophoresis (Course Reading 5 ) Visualizing DNA (& protein) in gels (Course Reading 6)

Methods for detecting specific biomolecules Southern blots (DNA-DNA hybridization)(Methods for labeling “probe” DNA) CR7, MC 6.33 - 6.38 B. Northern blots (DNA-RNA hybridization) CR8, MC 7.21 - 7.26, MC 7.82 - 7.84 Western blots (detection of proteins with specific antibodies) CR9, MC A9.28, MC A8.52-A8.55

Visualizing DNA, RNA and Protein: detecting specific sequences or proteins Techniques allow one to distinguish specific sequences or proteins in a large, mixed population, e.g. in cell extracts or genomic DNA preparations For DNA and RNA, specific sequence detection is based on DNA and RNA complementarity and base-pairing For proteins, the specific detection is based on antibodies that recognize the protein of interest (or based on a specific assay for activity of the protein)

Detecting specific DNA sequences: the Southern blot

Immobilization of nucleic acids nitrocellulose or nylon membrane boundary: DNA binds to it Agarose or Polyacrylamide gel A typical capillary blotting apparatus. Electroblotting is also commonly used

Immobilization of target DNA and detection Southern blotting: Immobilization of target DNA and detection DNA is fixed to the nylon membrane by: Baking, 80°C UV crosslinking (links thymines in DNA to + charged amine groups in membrane), DNA only Probe to detect sequence of interest by base-pairing (hybridization) Obtain probe DNA: synthetic oligonucleotide or cloned gene (single stranded) Label probe for later detection Radioactivity Non-radioactive label

Radioactive probes: 32P labeling Use T4 polynucleotide kinase --catalyzes the transfer of the gamma phosphate of 32P ATP to the 5’ end of DNA fragment to be used as a probe 32P is a high energy beta particle emitter, and provides good sensitivity for detection of hybridization between the probe DNA and the target (blot) DNA Detect radiolabel with --autoradiography (X ray film) --phosphorimager (phosphor coated plates store the energy of the radioactive particle, laser excitation releases photons of light that are collected and represented as a picture, greater dynamic range than film, and faster too

Non-radioactive labels …e.g. horseradish peroxidase oxidation

Non-radioactive labels …or digoxygenin/antibody-conjugated HRP oxidation can also use biotinylated DNA probe

Hybridize probes to membranes blocking agents (e.g. milk, SDS) prevent non-specific interactions between probes and membrane Volume exclusion agents (eg. dextran sulfate) increase rate and level of hybridization Wash blot with increasing stringency… Low stringency: high salt, low temperature, probe binds to sequences with mismatches High stringency: low salt, higher temp., probe binds only to fully complementary sequences

Southern Blot--one example (or PCR fragment) (RFLPs)

Northern blots: Same basic technique as Southern blots, but RNA is run on the initial gel and is transferred to the membrane. Use this method to measure levels of gene transcription in vivo (detecting changes in the levels of RNA transcript under differing conditions) Microarrays for measuring mRNA abundance are based on this principle, but many probes are immobilized in a regular array -- reverse transcribed (and fluorescently labelled) RNA “lights up” the probes on the microarray

Western blots: proteins Proteins are transferred to membranes using the same principle as Southern blots Specific proteins detected by probing blot with antibodies to protein of interest Antibody binding is detected by antibody to the original antibody that has enzyme (horseradish peroxidase, alkaline phosphatase) or radioactivity (125I) conjugated to it

Methods for detecting specific biomolecules Separate DNA, RNA, or proteins on the basis of size (gel electrophoresis) Immobilize the separated DNA, RNA, or protein “Probe” the blot with something that will specifically interact with a target DNA and RNA: complementary nucleic acid Protein: antibody to that protein