Techniques of Molecular Biology PDF

Techniques of Molecular Biology Chapter 7 Outline DNA and RNA Gel Electrophoresis. Restriction Endonucleases (Restriction Enzymes). Nucleic Acid Hybridization (Southern and Northern Blots). DNA Cloning Polymerase Chain Reaction (PCR) Immunoblot Protein Purification Peptide Analysis by Tandem Mass Sp...

Techniques of Molecular Biology Chapter 7 Outline DNA and RNA Gel Electrophoresis. Restriction Endonucleases (Restriction Enzymes). Nucleic Acid Hybridization (Southern and Northern Blots). DNA Cloning Polymerase Chain Reaction (PCR) Immunoblot Protein Purification Peptide Analysis by Tandem Mass Spectrometry Slides are from: Molecular Biology of the Gene 7th edition 1 Figure 7-1 DNA/RNA Separation by Gel Electrophoresis Agarose Gel System 2 DNA/RNA Separation by Gel Electrophoresis Polyacrylamide Gel System 3 DNA Migration in Electrophoresis System as a Function of the Number of Base Pairs 4 Figure 7-4 Examples of Restriction Enzymes and Types of Cuts Blunt ends Staggered ends with 5’ overhangs Staggered ends with 5’ overhangs Staggered ends with 3’ overhangs Recognition site generally 4 to 8 bp long and often palindromic around a center of symmetry. Go and read the wiki webpage on restriction enzymes for information on where they come from and the different types. You can also visit 5 “Rebase” for more information on RE specificities and other properties. Figure 7-5 Example of a Restriction Site leaving a 5’ Overhang aka “Cohesive” By convention the EcoR1 site can be simply represented by G/AATTC. 6 Table 7-1 Length of a Restriction Site Recognition Sequence and Frequency of Occurrence in a Random Genome Is the formula given in this Table of your textbook accurate? 7 Figure 7-3 DNA Migration in Electrophoresis System as a Function of the Number of Base Pairs 8 Plasmids Circular extrachromosomal double-stranded DNA found naturally in many bacteria and single-cell eukaryotes. They can occur as single or multiple copies inside the cell. Modified plasmids are used as vectors in DNA cloning. 9 Plasmids as Cloning Vectors: 3 Characteristics Multiple Cloning Site (MCS) Cloning vector: Size ≈ 3.0 kb Origin of Replication + Controlling Elements (Replicon) Selectable Marker Gene or Resistance Gene 10 Figure 4-26 Forms of Plasmid DNA as they appear on an Ethidium Bromide-Containing Gel after Electrophoresis Open Circular Linear Supercoiled 11 Figure 7-8 DNA Cloning 12 Figure 7-10 cDNA Library RT-PCR 13 Figure 4-14 Reannealing and Hybridization of DNA (DNA-RNA Hybrids) 14 Southern (DNA) or Northern (RNA) Blots 15 Figure 7-6 Southern Blot: Hybridization Probes identifies Single Genes from a Whole Genome 16 Figure 7-9 Screening for Colonies of Interest cDNA population Same principle as Southern blot method Isolate a single cDNA-containing bacterial colony from the population of initial cloned 17 cDNAs. Figure 7-7 Microarrays: A Reverse Northern Blot on a Genome-Wide Scale 18 Microarrays: A Reverse Northern Blot on a Genome-Wide Scale 19 Figure 7-12 The Polymerase Chain Reaction (PCR) PCR requires: 1)A template DNA 2)A pair of primers 3)A thermostable DNA polymerase 4)The 4 dNTP + Mg2+ ions 5)A buffer 6)A thermocycler The amplification is exponential, doubling the amount of DNA at every cycle. After 20 to 35 cycles, a single DNA band may be visible on an agarose gel. A single copy gene may be amplified and detected from a whole genome. A variant is called RT-PCR and involves the Reverse Transcription of RNA to DNA before PCR amplification. 20 Phases of a PCR Reaction in Log View Area of detection for Real-Time PCR 21 Phases of a PCR Reaction in Linear View 22 Figure 7-13 Dideoxynucleotides used in DNA Sequencing 23 Figure 7-14 Chain Termination of DNA Replication in the Presence of Dideoxynucleotides 24 Figure 7-15 DNA Sequencing by the Chain-Termination Method 25 Figure 7-16 DNA-Sequencing Gel (A Thin Polyacrylamide Gel) ddTTP not ddATP 26 Protein Separation by Polyacrylamide Gel Electrophoresis (PAGE) 27 =O Separation of Proteins by SDS-PAGE =O Na+ -O-S-O-(CH2)11-CH3 Sodium Dodecyl Sulfate (Detergent) SDS denatures proteins by binding to them with a ratio of about 1 SDS for every 2 amino acids. This gives proteins a constant charge-to-mass ratio allowing a separation based on molecular mass. A reducing agent (2-Mercaptoethanol) is also added to protein samples to break disulfide bridges. 28 Separation of Proteins by SDS-PAGE How will the protein profile differ if the sample is heated without 2mercaptoethanol? 29 Separation of Proteins by SDS-PAGE 30 Detection of a Single Protein from a Crude Extract. The Western Blot or Immunoblot. Primary Ab Secondary Ab Based on the high specificity of antibody-antigen interactions 31 Detection of a Single Protein from a Crude Extract. The Western Blot or Immunoblot. 32 General Structure of Antibodies (IgG) Secondary antibodies are raised against the conserved regions 2 and 3 of the primary antibody. Fc domain is conserved for all antibodies of a given species, but differs between species. CH2 33 General Structure of Antibodies CDR CDR: ComplementarityDetermining Region. This is the site of binding of the antigen. An antibody has 2 antigen binding sites. The amino acids of the antigen recognized by the antibody is called an epitope. 34 Separation of Proteins by Chromatography (aka size exclusion) 35 Figure 7-26b Separation of Proteins by Chromatography 36 Separation of Proteins by Chromatography (can be anion- or cation-exchange) 1. Load sample. Proteins that don’t bind come out in the flow-through. 2. Wash to further remove unbound proteins. 3. Elute bound proteins with a salt gradient. 37 Separation of Proteins by Chromatography 38 Protein Preparation for Mass Spectrometry (MS) Prepare protein cell-extract Separate proteins by SDS-PAGE Stain gel to visualize proteins Cut protein band of interest Digest protein with a specific protease (eg Trypsin, which cuts after K or R) Mass Spectrometry (MS) and Tandem Mass Spectrometry (MS/MS) 39 MS/MS for Peptide Sequencing Once ionized, peptides are generally positively charged (singly or multiply). Peptides are in the gas phase and completely desolvated. 40 A Typical Peptide MS Spectrum (at MS1) Notice: mass over charge 41 MS/MS for Peptide Sequencing Once ionized, peptides are generally positively charged (singly or multiply). Peptides are in the gas phase and completely desolvated. 42 Precursor Ion Selection for MS/MS 43 Peptide Fragmentation after MS1 44 Nominal Structures of Fragment Ions The additional proton is abstracted from the precursor ion. 45 Peptide Fragmentation-Interpretation of MS/MS Spectra MW = 1166 Da 46 Peptide Fragmentation-Typical MS/MS Spectrum (MS2) 47 Peptide Fragmentation – y Ions 48 Peptide Fragmentation – b Ions 49