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Reminder: All molecular techniques are based on the chemical “personality” (or chemical properties) of the DNA molecule (or nucleic acids) Studies of cell -Fractionation -Purification/ Identification -Structure/ Function Organelle level Cell fractionation -Nucleus -Mitochondria -RER, cell membrane -SER -Cytosol Cellular level Microscope Molecular level: Macromolecules Proteins Carbohydrates Lipids Nucleic acids Atomic level C, H, O, N, S, P CONTENTS Electrophoresis Blotting and Hybridization Polymerase Chain Reaction DNA Sequences Negatively-charged phosphate-sugar backbone Various lengths - - - Specificity of nucleotides Hydrogen bonds DNA GEL ELECTROPHORESIS 1. For separating DNA strands of any size/length 2. Uses a gel to separate DNA strands 3. Uses electricity Electrophoresis Molecules are separated by electric force F = qE : where q is net charge, E is electric field strength The velocity is encountered by friction qE = fv : where f is frictional force, v is velocity Therefore, mobility per unit field (U) = v/q = q/f = q/6pr : where is viscosity of supporting medium, r is radius of sphere molecule E v F - f q+ +--- + Electrophoresis Factors affected the mobility of molecules - 1. Molecular factors • Charge • Size • Shape 2. Environment factors • Electric field strength • Supporting media (pore: sieving effect) • Running buffer + Electrophoresis Electrophoresis Types of supporting media Paper Agarose gel (Agarose gel electrophoresis) Polyacrylamide gel (PAGE) pH gradient (Isoelectric focusing electrophoresis) Cellulose acetate Agarose Gel Agarose: purified large MW polysaccharide (from agar) very open (large pore) gel used frequently for large DNA molecules Agarose gel staining Ethidium bromide Electrophoresis Polyacrylamide Gels Acrylamide polymer; very stable gel can be made at a wide variety of concentrations gradient of concentrations: large variety of pore sizes (powerful sieving effect) SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) Sodium Dodecyl Sulfate = Sodium Lauryl Sulfate: CH3(CH2)11SO3- Na+ Amphipathic molecule Strong detergent to denature proteins Binding ratio: 1.4 gm SDS/gm protein Charge and shape normalization Isoelectric Focusing Electrophoresis (IFE) - Separate molecules according to their isoelectric point (pI) - At isoelectric point (pI) molecule has no charge (q=0), hence molecule ceases - pH gradient medium 2-dimensional Gel Electrophoresis - First dimension is IFE (separated by charge) - Second dimension is SDSPAGE (separated by size) - So called 2D-PAGE - High throughput electrophoresis, high resolution 2-dimensional Gel Electrophoresis Spot coordination pH MW Hybridization and Blotting Hybridization Pairing of complementary DNA and/or RNA Hybridization It can be DNA:DNA, DNA:RNA, or RNA:RNA (RNA is easily degraded) It depended on the extent of complementation It depended on temperature, salt concentration, and solvents Small changes in the above factors can be used to discriminate between different sequences (e.g. small mutations can be detected) Probes can be labeled with radioactivity, fluorescent dyes, enzymes. Probes can be isolated or synthesized sequences Oligonucleotide probes Single stranded DNA (usually 15-40 bp) Degenerate oligonucleotide probes can be used to identify genes encoding characterized proteins • Use amino acid sequence to predict possible DNA sequences • Hybridize with a combination of probes • TT(T/C) - TGG - ATG - GA(T/C) - TG(T/C) could be used for FWMDC amino acid sequence Can specifically detect single nucleotide changes Detection of Probes Probes can be labeled with radioactivity, fluorescent dyes, enzymes. Radioactivity is often detected by X-ray film (autoradiography) Fluorescent dyes can be detected by fluorometers, scanners Enzymatic activities are often detected by the production of dyes or light (x-ray film) RNA Blotting (Northerns) RNA is separated by size on a denaturing agarose gel and then transferred onto a membrane (blot) Probe is hybridized to complementary sequences on the blot and excess probe is washed away Location of probe is determined by detection method (e.g., film, fluorometer) Western Blot Protein blotting Highly specific qualitative test Can determine if above or below threshold Typically used for research Use denaturing SDS-PAGE Solubilizes, removes aggregates & adventitious proteins are eliminated Components of the gel are then transferred to a solid support or transfer membrane weight Transfer membrane Paper towel Paper towel Wet filter paper Western Blot Block membrane e.g. dried nonfat milk Rinse with ddH2O Add monoclonal antibodies Rinse again Antibodies will bind to specified protein Add antibody against yours with a marker (becomes the antigen) Stain the bound antibody for colour development It should look like the gel you started with if a positive reaction occurred Polymerase Chain Reaction (PCR) PCR A simple rapid, sensitive and versatile in vitro method for selectively amplifying defined sequences/regions of DNA/RNA from an initial complex source of nucleic acid - generates sufficient for subsequent analysis and/or manipulation Amplification of a small amount of DNA using specific DNA primers (a common method of creating copies of specific fragments of DNA) DNA fragments are synthesized in vitro by repeated reactions of DNA synthesis (It rapidly amplifies a single DNA molecule into many billions of molecules) In one application of the technology, small samples of DNA, such as those found in a strand of hair at a crime scene, can produce sufficient copies to carry out forensic tests. Each cycle the amount of DNA doubles Background on PCR The Ability to generate identical high copy number DNAs made possible in the 1970s by recombinant DNA technology (i.e., cloning). Cloning DNA is time consuming and expensive Probing libraries can be like hunting for a needle in a haystack. Requires only simple, inexpensive ingredients and a couple hours PCR, “discovered” in 1983 by Kary Mullis, Nobel Prize for Chemistry (1993). It can be performed by hand or in a machine called a thermal cycler. Three Steps Separation Double Stranded DNA is denatured by heat into single strands. Short Primers for DNA replication are added to the mixture. Priming DNA polymerase catalyzes the production of complementary new strands. Copying The process is repeated for each new strand created All three steps are carried out in the same vial but at different temperatures Step 1: Separation Combine Target Sequence, DNA primers template, dNTPs, Taq Polymerase Target Sequence 1. Usually fewer than 3000 bp 2. Identified by a specific pair of DNA primers- usually oligonucleotides that are about 20 nucleotides Heat to 95°C to separate strands (for 0.5-2 minutes) • Longer times increase denaturation but decrease enzyme and template Magnesium as a Cofactor Mg stabilizes the reaction between: • oligonucleotides and template DNA • DNA Polymerase and template DNA Heat Denatures DNA by uncoiling the Double Helix strands. Step 2: Priming Decrease temperature by 15-25 °C Primers anneal to the end of the strand 0.5-2 minutes Shorter time increases specificity but decreases yield Requires knowledge of the base sequences of the 3’ end Selecting a Primer Primer length Melting Temperature (Tm) Specificity Complementary Primer Sequences G/C content and Polypyrimidine (T, C) or polypurine (A, G) stretches 3’-end Sequence Single-stranded DNA Step 3: Polymerization Since the Taq polymerase works best at around 75 ° C (the temperature of the hot springs where the bacterium was discovered), the temperature of the vial is raised to 72-75 °C The DNA polymerase recognizes the primer and makes a complementary copy of the template which is now single stranded. Approximately 150 nucleotides/sec Potential Problems with Taq Lack of proof-reading of newly synthesized DNA. Potentially can include di-Nucleotriphosphates (dNTPs) that are not complementary to the original strand. Errors in coding result Recently discovered thermostable DNA polymerases, Tth and Pfu, are less efficient, yet highly accurate. How PCR works 1. Begins with DNA containing a sequence to be amplified and a pair of synthetic oligonucleotide primers that flank the sequence. 2. Next, denature the DNA at 94˚C. 3. Rapidly cool the DNA (37-65˚C) and anneal primers to complementary s.s. sequences flanking the target DNA. 4. Extend primers at 70-75˚C using a heat-resistant DNA polymerase (e.g., Taq polymerase derived from Thermus aquaticus). 5. Repeat the cycle of denaturing, annealing, and extension 20-45 times to produce 1 million (220) to 35 trillion copies (245) of the target DNA. 6. Extend the primers at 70-75˚C once more to allow incomplete extension products in the reaction mixture to extend completely. 7. Cool to 4˚C and store or use amplified PCR product for analysis. Thermal cycler protocol Example Step 1 7 min at 94˚C Step 2 45 cycles of: 20 sec at 94˚C 20 sec at 64˚C 1 min at 72˚C Step 3 7 min at 72˚C Step 4 Infinite hold at 4˚C Initial Denature Denature Anneal Extension Final Extension Storage The Polymerase Chain Reaction PCR amplification Each cycle the oligo-nucleotide primers bind most all templates due to the high primer concentration The generation of mg quantities of DNA can be achieved in ~30 cycles (~ 4 hrs) OPTIMISING PCR THE REACTION COMPONENTS Starting nucleic acid - DNA/RNA Tissue, cells, blood, hair root, semen Thermo-stable DNA polymerase e.g. Taq polymerase Oligonucleotides Design them well! Buffer Tris-HCl (pH 7.6-8.0) Mg2+ dNTPs (dATP, dCTP, dGTP, dTTP) RAW MATERIAL Organims, Organ, Tissue, cells ( blood, hair root, semen, callus, leaves, root, seed) Obtain the best starting material. Some can contain inhibitors of PCR, so they must be removed e.g. Haem in blood Good quality genomic DNA if possible Empirically determine the amount to add POLYMERASE Number of options available Taq polymerase Pfu polymerase Tth polymerase How big is the product? 100bp 40-50kb What is end purpose of PCR? 1. Sequencing - mutation detection -. Need high fidelity polymerase -. integral 3’ 5' proofreading exonuclease activity 2. Cloning 3. Marker development PRIMER DESIGN Length ~ 10-30 nucleotides (21 nucleotides for gene isolation) Base composition: 50 - 60% GC rich, pairs should have equivalent Tms Tm = [(number of A+T residues) x 2 °C] + [(number of G+C residues) x 4 °C] Initial use Tm–5°C Avoid internal hairpin structures No secondary structure Avoid a T at the 3’ end Avoid overlapping 3’ ends – will form primer dimers Can modify 5’ ends to add restriction sites PRIMER DESIGN Use specific programs OLIGO Medprobe PRIMER DESIGNER Sci. Ed software Also available on the internet http://www.hgmp.mrc.ac.uk/GenomeWeb/nuc-primer.html Mg2+ CONCENTRATION 1 1.5 2 2.5 3 3.5 4 mM Normally, 1.5mM MgCl2 is optimal Best supplied as separate tube Always vortex thawed MgCl2 Mg2+ concentration will be affected by the amount of DNA, primers and nucleotides USE MASTERMIXES WHERE POSSIBLE How Powerful is PCR? PCR can amplify a usable amount of DNA (visible by gel electrophoresis) in ~2 hours. The template DNA need not be highly purified — a boiled bacterial colony. The PCR product can be digested with restriction enzymes, sequenced or cloned. PCR can amplify a single DNA molecule, e.g. from a single sperm. Applications of PCR Amplify specific DNA sequences (genomic DNA, cDNA, recombinant DNA, etc.) for analysis 1. Gene isolation 2. Fingerprint development Introduce sequence changes at the ends of fragments Rapidly detect differences in DNA sequences (e.g., length) for identifying diseases or individuals Identify and isolate genes using degenerate oligonucleotide primers • Design mixture of primers to bind DNA encoding conserved protein motifs Genetic diagnosis - Mutation detection The basis for many techniques to detect gene mutations (sequencing) 1/6 X 10-9 bp Applications of PCR Paternity testing Mutagenesis to investigate protein function Quantify differences in gene expression → Reverse transcription (RT)-PCR Identify changes in expression of unknown genes → Differential display (DD)-PCR Forensic analysis at scene of crime Industrial quality control DNA sequencing DNA Sequencing DNA sequencing Determination of nucleotide sequence the determination of the precise sequence of nucleotides in a sample of DNA Two similar methods: 1. Maxam and Gilbert method 2. Sanger method They depend on the production of a mixture of oligonucleotides labeled either radioactively or fluorescein, with one common end and differing in length by a single nucleotide at the other end This mixture of oligonucleotides is separated by high resolution electrophoresis on polyacrilamide gels and the position of the bands determined The Maxam-Gilbert Technique Principle: Chemical Degradation of Purines • Purines (A, G) damaged by dimethylsulfate • Methylation of base • Heat releases base • Alkali cleaves G • Dilute acid cleave A>G Maxam-Gilbert Technique • Pyrimidines (C, T) are damaged by hydrazine • Piperidine cleaves the backbone • 2 M NaCl inhibits the reaction with T Maxam and Gilbert Method Chemical degradation of purified fragments (chemical degradation) The single stranded DNA fragment to be sequenced is end-labeled by treatment with alkaline phosphatase to remove the 5’phosphate It is then followed by reaction with P-labeled ATP in the presence of polynucleotide kinase, which attaches P labeled to the 5’terminal The labeled DNA fragment is then divided into four aliquots, each of which is treated with a reagent which modifies a specific base 1. Aliquot A + dimethyl sulphate, which methylates guanine residue 2. Aliquot B + formic acid, which modifies adenine and guanine residues 3. Aliquot C + Hydrazine, which modifies thymine + cytosine residues 4. Aliquot D + Hydrazine + 5 mol/l NaCl, which makes the reaction specific for cytosine The four are incubated with piperidine which cleaves the sugar phosphate backbone of DNA next to the residue that has been modified Maxam-Gilbert sequencing - modifications Maxam-Gilbert sequencing: Summary Advantages/disadvantages Maxam-Gilbert sequencing Requires lots of purified DNA, and many intermediate purification steps Relatively short readings Automation not available (sequencers) Remaining use for ‘footprinting’ (partial protection against DNA modification when proteins bind to specific regions, and that produce ‘holes’ in the sequence ladder) In contrast, the Sanger sequencing methodology requires little if any DNA purification, no restriction digests, and no labeling of the DNA sequencing template Sanger Fred Sanger, 1958 • Was originally a protein chemist • Made his first mark in sequencing proteins • Made his second mark in sequencing RNA 1980 dideoxy sequencing Original Sanger Method Random incorporation of a dideoxynucleoside triphosphate into a growing strand of DNA Requires DNA polymerase I Requires a cloning vector with initial primer (M13, high yield bacteriophage, modified by adding: betagalactosidase screening, polylinker) Uses 32P-deoxynucleoside triphosphates Sanger Method in-vitro DNA synthesis using ‘terminators’, use of dideoxi- nucleotides that do not permit chain elongation after their integration DNA synthesis using deoxy- and dideoxynucleotides that results in termination of synthesis at specific nucleotides Requires a primer, DNA polymerase, a template, a mixture of nucleotides, and detection system Incorporation of di-deoxynucleotides into growing strand terminates synthesis Synthesized strand sizes are determined for each dideoxynucleotide by using gel or capillary electrophoresis Enzymatic methods Dideoxynucleotide PPP O 5’ CH2 O BASE 3’ no hydroxyl group at 3’ end prevents strand extension The principles Partial copies of DNA fragments made with DNA polymerase Collection of DNA fragments that terminate with A,C,G or T using ddNTP Separate by gel electrophoresis Read DNA sequence 3’ primer 5’ CCGTAC 5’ 3’ dNTP ddATP ddTTP GGCA ddCTP ddGTP GGCAT A T C GGC G G GG GGCATG Chain Terminator Basics Target Template-Primer TGCA ddA Extend ddA AddC AC ddG ACG ddT ddT ddC ddG Labeled Terminators dN : ddN 100 : 1 Electrophoresis Sanger Method Sequencing Gel Sequencing of DNA by the Sanger method Comparison Sanger Method • Enzymatic • Requires DNA synthesis • Termination of chain elongation Maxam Gilbert Method • Chemical • Requires DNA • Requires long stretches of DNA • Breaks DNA at different nucleotides