Lecture 1-2 (DNA & PCR).pdf

Fall 2024 BIOD21 - Advanced Molecular Biology Lab Instructor: Prof. Sonia Gazzarrini ScienceMag.org  2 Goal 1: Learning how to work in a molecular biology research lab A. Planning and conducting e...

Fall 2024 BIOD21 - Advanced Molecular Biology Lab Instructor: Prof. Sonia Gazzarrini ScienceMag.org  2 Goal 1: Learning how to work in a molecular biology research lab A. Planning and conducting experiments Experimental plan (lab protocols/handouts) Workflow B. Data acquisition, interpretation, troubleshooting Careful description of each experimental step Data discussion Preparation of high-quality Figures C. Data presentation and discussion Written lab Report (research paper format) Oral presentation (similar to lab meeting or conference presentations)  3 Goal 2: Learning molecular techniques to study gene regulation and function A. Transcriptional regulation in response to stress RNA isolation from control and treatment (stress) plants cDNA synthesis and RT-qPCR B. Generation and characterization of mutants Genome editing by CRISPR/CAS9 Phenotypic analysis of wild type and mutants  4 Goal 2: Learning molecular techniques to study gene function C. Protein localization Subcellular localization of proteins using a translational reporter (YFP fusion protein) & confocal microscopy D. Gene cloning & sequencing Cloning strategies (Gateway, Golden Gate) Bacterial transformation, plasmid isolation and colony screening by PCR Sequencing data analysis  5 E. Bioinformatics analysis Navigating molecular biology databases and repositories of genome sequencing data (NCBI; TAIR) Using web-based tools for DNA and protein sequence analysis, alignment, primer design, etc. In silico gene expression analysis using available transcriptomic data (microarray and RNAseq) Primer and guide-RNA design in-silico PCR BAR eFP Browser  6 Useful links for this course Background reading Alberts et al. 2002, Mol. Biol. of the Cell. (Available at NCBI, Bookshelf) Chapter 8, section “Studying Gene Expression and Function”, available at NCBI, Bookshelf: https://www-ncbi-nlm-nih-gov.myaccess.library.utoronto.ca/books/NBK26818/ Chapter 8, section “Isolating, Cloning, and Sequencing DNA” https://www-ncbi-nlm-nih-gov.myaccess.library.utoronto.ca/books/NBK26837/ Lodish et al. 2000. Molecular Cell Biology. Section 7 “DNA cloning with plasmid vectors”. (Available at NCBI, Bookshelf) https://www-ncbi-nlm-nih-gov.myaccess.library.utoronto.ca/books/NBK21498/ Background explanation ThermoFisher Scientific https://www.thermofisher.com/ca/en/home/technical-resources/technical-reference-library.html Videos Addgene https://www.addgene.org/protocols/; in-house videos will be posted on Quercus.  7 Overview of WEEKs 1-5 LABS Studying gene regulation: transcriptional regulation by abiotic stress In the first part of the course, you will study the transcriptional regulation of the CBF4 gene by abiotic stress using quantitative PCR (RT-qPCR). The model organism of this course is Arabidopsis thaliana, a small plant from the mustard family (Brassicaceae). First, you will test in silico primers that will be used in qPCR (Bioinformatics Lab 1). Then, you will test the primers on genomic DNA using regular PCR (Genomic DNA isolation and PCR). Next, you will expose WT plants to control and abiotic stress conditions and determine if CBF4 is transcriptionally regulated (up- or down-) by stress (RNA isolation, RT-qPCR). You will also conduct in silico gene expression analysis using available transcriptomic data (microarray and RNAseq) (Bioinformatics Lab 2)  8 Week 1: Bioinformatics Lab 1 DNA sequence analysis and in silico PCR (NCBI) CBF4 genomic, cDNA, and CDS sequences Sequence alignment (Clustal Omega) Primer design and In silico PCR (Primer Blast)  9 In the Bioinformatics lab 1 you will learn to: ✓ navigate databases relevant to biotechnology and biomedicine ✓ use databases and bioinformatics tools to analyze your sequence of interest. Does your gene of interest have introns, exons, UTRs? How/where can I find their sequences? ✓ design primers that amplify a fragment of your gene Also, test primers that you will use in RT-qPCR ✓ find where the primers anneal on the gDNA and cDNA of your gene of interest and predict the PCR amplicon size.  10 Questions 1. Gene structure Promoter Exons Introns 5’UTR 3’UTR CDS Zien et al., 2004  11 Polymerase Chain Reaction (PCR) https://www.enzolifesciences.com  12 Polymerase Chain Reaction (PCR): Summary 1. PCR overview 2. Comparison between PCR and DNA replication 3. Thermostable DNA Polymerases 4. Advantages and Disadvantages of PCR 5. Setting up a PCR reaction: PCR components, master-mix, controls 6. Primer design 7. PCR cycle  13 Polymerase Chain Reaction (PCR): Overview PCR is a techniques that allows the exponential amplification of a short DNA stretch by repeated cycles of in vitro DNA polymerization. PCR mimics the in vivo process catalysed by the DNA polymerase during DNA replication, in that a new strand of DNA is DNA Gyrase synthesized from a template strand DNA replication - Image Credit: Designua / Shutterstock  14 Uses a thermostable DNA polymerase isolated from thermophilic bacteria/archea PCR is SIMPLE, it is all done by a PCR machine! Developed by Kary Mullis in 1987 (1993 Nobel Prize). It has revolutionized molecular biology, and it is used in many applications T. aquaticus from a hot PCR Machine spring at Yellowstone (BioRad) National Park  15 A. Basic Research PCR-based Cloning Genotyping Gene expression studies Sequencing Site-directed mutagenesis Applications of PCR Classification of organisms Many other applications ….. B. Applied research Genetic matching Detection of pathogens Prenatal diagnosis DNA fingerprinting Gene therapy Etc ….. www.goldbio.com 16 Template DNA Denaturation 94 ºC The PCR cycle Primer Primer Annealing ~50-60 ºC 30-35 cycles dNTPs Extension 68-72 ºC Taq DNA Polymerase New DNA strands NEB video: https://international.neb.com/tools-and-resources/video-library/overview-of-pcr?autoplay=1  17 Exponential amplification Rev primer For primer ThermoFisher Scientific Figure 1. Three steps of PCR ─denaturation, annealing, and extension─ as shown in the first cycle, and the exponential amplification of target DNA with repeated cycling.  18 DNA replication PCR mimics the in vivo process catalysed by the DNA polymerase during DNA replication. Let’s look at what is required for DNA replication : DNA Helicases stimulate the separation of the two strands. DNA Gyrase DNA Gyrase – this topoisomerase aids with the unwinding process. Primase - Synthesizes many RNA primers that bind throughout the genome. DNA Polymerase binds to the free 3'OH of the primers and synthesize a new strand of DNA in direction 5’ to 3’ (continuous synthesis on the leading strand). DNA replication - Image Credit: Designua / Shutterstock DNA Ligase - Nicks occur in the developing molecule because the RNA primer is removed (by the exonuclease enzyme), and synthesis proceeds in a discontinuous manner on the lagging strand. DNA ligase forms a covalent phosphodiester linkage between 3'-hydroxyl (OH-) and 5'-phosphate groups (PO4-). https://www.yourgenome.org/video/dna-replication  19 Differences and similarities between PCR & DNA replication Function PCR DNA replication DNA strands Heat (>90ºC to DNA Helicase, separation denature DNA) DNA Gyrase DNA synthesis Thermostable DNA DNA pol Pol (eg. Taq Pol) Primers 2 Primers added to Primase reaction mix Nick joining Not necessary, as DNA Ligase fragments are short  20 Power of PCR: thermostable DNA polymerase The Temperature required to denature double stranded DNA (94-95°C) inactivates the enzymes of most organisms including E. coli DNA polymerase I. Therefore, PCR uses a heat-stable DNA polymerases from a thermophilic bacteria, eg. Taq DNA polymerase from Thermus aquaticus or from archaebacteria, e.g. Pfu DNA polymerase from Pyrococcus furiosus. Many other DNA polymerases are now available. These thermostable DNA polymerases allow repeated synthesis of new DNA strands, and the exponential amplification of a defined region of the starting material in one tube.  21 Advantages and disadvantages of PCR ADVANTAGES Very Sensitive: a single DNA molecule can be amplified! PCR can amplify DNA that is of low quantity AND poor quality. If impurities present in DNA samples (phenol, EDTA, Proteinase K), just dilute your DNA DNA does not have to be intact, because smaller fragments are typically amplified by PCR (just the region between the primers, ~0.5-2Kb). Other techniques, such as Southern analysis, require the isolation of intact genomic DNA. Rapid: It takes 2-3h to complete PCR, all done by the PCR machine! PCR Machine (BioRad)  22 DISADVANTAGES Because PCR is so sensitive, DNA contamination can be a major problem. For example, if your primer or Taq enzyme stock contain genomic DNA contamination (introduced by mistake by yourself or lab mates), it will affect your results. Can you explain why? To confirm absence of contamination, a good practice is to add a negative control reaction without template DNA. Can you explain why? PCR can produce errors, depending on the length of the gene to be amplified and the polymerase used. Thus, PCR products need to be sequenced to verify correct nucleotides sequences.  23 Setting up a PCR reaction: PCR components 1. DNA 2. Enzyme 3. dNTPs 4. Buffer 5. Cofactors (Mg2+) 6. Primers www.goldbio.com  24 1. DNA. 0.1-1 µg for genomic DNA 0.01-1 ng plasmid DNA Too much template DNA usually increases the yield of nonspecific PCR products When amplifying a gene or DNA fragment, higher amount of genomic DNA is required compared to plasmid DNA. Why? Figure 1. Comparison of PCR results with plasmid vs. human gDNA template. The same DNA polymerase was used to amplify a 2 kb target sequence from varying amounts of input DNA under the recommended conditions. From ThermoFisher Sci  25 2. Enzyme DNA polymerase from thermophilic bacteria, such as Taq polymerase, which can withstand high temperatures, are typically used in PCR. In the lab, recombinant Taq DNA polymerase is typically used. The gene encoding the Taq DNA pol. is first isolated from Thermus aquaticus. The native enzyme is cloned into a vector and transformed into E. coli (much easier to grow). The recombinant Taq DNA pol is then expressed and purified from E. coli and used in PCR. Better-performing DNA polymerases, which are better suitable for the many applications in which PCR is used, are continually being developed. Temperature: optimum of 68-80°C for DNA synthesis (extension step)  26 Non-proofreading DNA polymerases: Taq polymerase Taq polymerase is a highly thermostable DNA polymerase from the thermophilic bacterium Thermus aquaticus. Catalyzes the polymerization of nucleotides into duplex DNA in the 5’=>3’ direction. Doesn’t have 3’=>5’ exonuclease activity (non-proofreading) Like other DNA polymerases without 3’=>5’ exonuclease activity, Taq DNA Thomas Brock discovered T. aquaticus from a hot spring at Polymerase exhibits terminal transferase activity, which frequently results Yellowstone National Park in the addition of extra adenines (poly-A tail) at the 3’end of PCR products (an advantage for cloning PCR products: TA cloning). Routinely used in PCR amplifications, such as colony PCR. PCR products up to 3-5Kb. Error rate in PCR: 2.2x10-5 errors/ per nt/ per cycle Accuracy of PCR: 4.5x104. NB: Accuracy is the inverse of the error rate and shows an average number of correct nucleotides incorporated before an error occurs.  27 Proofreading DNA polymerases: Pfu and Vent High-fidelity DNA polymerases are used for applications requiring high accuracy during DNA amplification, such as cloning, sequencing or mutagenesis. They include: Pfu DNA polymerase, isolated from the hyper-thermophilic archaeum, Pyrococcus furiosus Vent Polymerase, from the Archeum Thermococcus litoralis which grows at 110 degrees C in hydrothermal vent ecosystems in the deep ocean. These enzymes catalyzes the polymerization of nucleotides in the 5’=>3’ direction. Also exhibits 3’=>5’ exonuclease activity (proofreading), that enables the polymerase to correct nucleotide incorporation errors. The proofreading domain also enables a polymerase to remove unpaired 3´ overhanging nucleotides to create blunt-end PCR products. 8 times more accurate than Taq DNA polymerase.  28 Proofreading DNA polymerase: Phusion Phusion High-Fidelity DNA Polymerase was engineered by fusing a thermophilic, non specific, dsDNA-binding domain to a Pyrococcus-like proofreading polymerase. This allows extremely low error rates and high speed, and are considered a gold standard for high-fidelity PCR. Accuracy: 6x more accurate than Pfu, 52x more accurate than Taq https://www.thermofisher.com/ca  29 3. dNTPs deoxy-riboNucleoside TriPhosphate (dNTP) dNTPs: dATP (deoxyAdenosine TriPhosphate), dCTP (cytosine), dGTP (guanosine), dTTP (thymine). Usually at a final concentration of 200 µM each. It is very important to have equal concentrations of each dNTP (dATP, dCTP, dGTP, dTTP), as inaccuracy in the concentration of even a single dNTP dramatically increases the misincorporation level.  30 4. Buffer The buffer provides a chemical environment and pH suitable for the activity of DNA polymerase. pH is usually between 8.0 and 9.5. A common component in the buffer is potassium (K+) from KCl, which promotes primer annealing to the template DNA. Ammonium sulphate (NH4SO4) can be used to prevent mismatch between primers and template DNA by destabilizing weak hydrogen bonds, thereby enhancing specificity. It is typically used to optimize PCR. Figure 9. PCR results from varying concentrations of MgCl 2 in two different buffer types, illustrating importance of buffer choice for PCR specificity. A 0.95 kb fragment was amplified from human gDNA with TaqDNA polymerase in these reactions. From ThermoFisher Sci.  31 5. Magnesium ions (Mg2+) Very important component! Mg2+ forms complexes with dNTPs, primers and DNA templates. Also, DNA polymerases require the presence of Mg2+ Optimal Mg2+ concentration used must be determined for each experiment. The recommended range of MgCl2 concentration is 1-4mM, under standard reaction conditions. Excess Mg 2+ stabilizes the DNA double strand and prevents proper denaturation. Also, promotes misincorporation of dNTPs. Thus, lower Mg2+ concentrations are desirable when fidelity of DNA synthesis is critical. Too low Mg 2+ results in a low yield of PCR product.  32 6. Primers A primer is a short synthetic oligonucleotide, used in many molecular techniques from PCR to DNA sequencing. These primers are designed to have a sequence which is complementary to a region of template or target DNA to which we wish the primer to anneal.  33 Primer concentration 0.1 to 1 µM If the primers concentrations are too high, there is a greater chance of mis-priming and therefore non-specific product accumulation High concentrations produce primer-dimers (primers annealing to each other) artefacts  34 Mis-priming Mis-priming: primer anneals to a sequence that is not 100% complementary Generally, lower primer concentration and increased temperature will increase specificity (only the desired gene will be amplified). At high temperatures, primers that are not 100% complementary to the sequence will not be stable and dissociate. Higher primer concentration and lower temperature will promote mis-priming 5’AATCGTAACC 3’ 5’ AATCGTAACC 3 ::::::: ::: :: ::: :: 3’ TTAGCATTGG 5’ 3’ TTTGCAGGGG5’ perfect priming mis-priming  35 Primer-dimer and Hairpin The primer should not be self-complementary (score complementary to the template strand. This means the For primer has the same sequence as the coding strand. Reverse (Rev) primer --> reverse & complementary to the coding strand. This means the Rev primer has a sequence that is reverse and complementary to the sequence of the coding strand given in the database. 3’ Rev 5’ DNA coding strand DNA template strand 5’ For 3’ Video from Addgene ON PRIMER DESIGN: https://www.addgene.org/protocols/primer-design/  39 Reverse primer (Rev): 5’- ACC ATA GCA … … … -3’ Rev 3’ 5’ ACG ATA CCA DNA coding strand DNA template strand TCA GAG GTG … 5’ 3’ For Forward primer (For): 5’- TCA GAG GTG … … … -3’  40 Practice exercise 1: Design (5’ → 3’) For and Rev primers annealing to the sequence shown below: 3’ Rev 5’ 5’- ATA TCA CCC GAA GTT ACG … TTT GAG CCT TTA GGG CCT -3’ DNA coding strand 5’ For 3’ 3’- TAT AGT GGG CTT CAA TGC … AAA CTC GGA AAT CCC GGA -5’ DNA template strand Forward primer (For): 5’- -3’ Reverse primer (Rev): 5’- -3’  41 Guidelines for primer selection Length. PCR primers are usually 18-24 nucleotides in length (no longer that 30-35nt). This length is long enough for adequate specificity and short enough for primers to bind easily to the template at the annealing temperature. Longer primers provide higher specificity. GC content. Ideally it should be 40-60%. The C and G nucleotides should be distributed uniformly throughout the primer. Primers 3’ end. Ideally, primers should end with a GC clamp: a G or C, or CG or GC at their 3': this increases efficiency and specificity of priming, due to the stronger bonding of G and C bases. NB: More than three G or C nucleotides at the 3'-end of the primer should be avoided, as nonspecific priming may occur.  42 The Melting temperature (Tm) Tm is the temperature at which 50% of the oligonucleotide and its perfect complement are in duplex. It indicates primer stability. The melting temperature of a primer can be estimated: using the following formula (Wallace Rule): Tm= 4 (G + C) + 2 (A + T) However, sophisticated computer programs are now routinely used to calculate Tm, where the interactions of adjacent bases, the influence of salt concentration, etc. are evaluated.  43 The melting temperature of a couple of primers (forward and reverse) should not differ by more than 5°C, so the GC content and length must be chosen accordingly. Usually, Tm are between 55 to 65°C (and max of 80°C). Primers with melting temperatures above 65oC have a tendency for secondary annealing. There are several computer programs that can calculate Tm, GC content, secondary structure of a primer, etc. In the D21 you will use Primer3 (when using Primer Blast).  44 The Annealing Temperature The primer melting temperature is critical in determining the annealing temperature (Ta). Annealing temperature should be approx. 5°C lower than the melting temperature. Ta = (Tm(for)+Tm(rev)/2) - 5C Ta that is too high will produce insufficient primer-template hybridization resulting in low PCR product yield. Ta that is too low may possibly lead to non-specific products caused by a high number of base-pair mismatches. Questions: Tm (For primer) = 58 Tm (Rev primer) = 62 Ta= ? Is this a good primer set? Why?  45 Step 1: Setting up a PCR reaction: the master mix PCR master mix When setting up a PCR reaction, there are a few things to consider: Since there are many reagents to include in a PCR reaction, it is best practice to prepare a ‘master mix’ with all common reagents that go in each PCR tube: Buffer, cofactors, dNTPs, enzyme and water. You will then pipette an equal volume of the master mix into each Step 2: PCR tube. DNA Then, you will add the template DNA and the primers. Note: If you are using the same template in all reactions (for example when amplifying different genes from the same genomic DNA template), you can include also the genomic DNA in the master mix. If you are using the same primers in all reactions (for example when genotyping different plants as in todays’ lab exercise) you can include also Step 3: the primers in the master mix. primers The goal is to include as many reagents as possible in the master mix, to minimize pipetting mistakes (forgetting to add a primer in a tube, etc.),which may result in PCR failure  46 Setting up a PCR reaction: the controls M (-) (+) S A) Negative control (-): include all reagents, but one. For example: No template control. The PCR reaction should yield no products. Why is this step important? PCR is very sensitive, any DNA contamination in your reagents will results in PCR amplification. How can this happen? Several scientists may be working in the same lab and using the same reagents. Therefore, there is the possibility that one researcher may contaminate a common reagent. How can you contaminate a reagent? Forget to change the tips between samples (-) negative control (ddH20) (+) positive control (plasmid DNA) Let’s say you find a band in the negative (no template) control. How can (S) Genomic DNA you find out which reagent is contaminated? B) Positive control (+). If your PCR fails to amplify any DNA, even after optimization of the PCR conditions, a positive control should be used. For example?  47 PCR cycle Initial Denaturation 1. Denaturation 2. Primer Annealing 30-35 cycles 3. Extension Final extension  48 Initial Denaturation Step Very important: incomplete denaturation of DNA results in the inefficient utilization of template in the first amplification cycle and in a poor yield of PCR product. The initial denaturation should be performed over an interval of 1-3min at 95°C if the GC content is 50% or less. This interval should be extended up to 10min for GC- rich templates. If the initial denaturation is no longer than 3 min at 94-95°C, Taq DNA Polymerase can be added into the initial reaction mixture. If longer initial denaturation or a higher temperature is necessary, Taq DNA Polymerase should be added only after the initial denaturation, as the stability of the enzyme dramatically decreases at temperatures over 95°C.  49 The PCR cycle 1. Denaturation Step 0.5-2min at 94-95°C. The PCR product synthesized in the first amplification cycles is significantly shorter than the template DNA. Denaturation NB: For DNA with very high GC content, increase denaturation time up 94-95 ºC to 3-4min. 2. Primer Annealing Step 0.5-2min at 5°C lower than the Tm of the primers (~50-60°C) Primer Primer Annealing NB: If you obtain non-specific PCR products, optimize annealing ~50-60 ºC temperature by increasing it stepwise by 1-2°C. NB: If you do not obtain any PCR product, how would you optimize the annealing temp? dNTPs Extension 3. Extending Step Temp: ~68-72°C. The rate of DNA synthesis by Taq DNA Polymerase is Taq DNA Polymerase 68-72 ºC highest at this temperature. Other DNA polymerases my have different annealing temp. Time: 15 sec per kb (plasmids template); 30 sec per kb (genomic DNA template).  50 Final Extension Step A post-PCR final incubation step of 5–10 min at 72°C is often recommended to promote complete synthesis of all PCR products. This step can be shortened to 30–60 sec for small PCR products of 100–1,000 bp See link to video from Addgene: https://www.addgene.org/protocols/pcr/  51 Practice exercise 2: Fill in the Table and answer the questions Reagent Stock [ ] Working [ ] Final volume Final volume (1 sample) (10 sample) Buffer (KCl, Mg2Cl) 10x 1x ? ? dNTPs 10 mM 0.2 mM ? ? FW Primer 10 μM 0.4 μM ? ? REV Primer 10 μM 0.4 μM ? ? BP Primer 10 μM 0.4 μM ? ? Template 100 ng/μL 100 ng ? ? (genomic DNA) Taq DNA Pol 2 U/μL 1U ? ? Water - - ? ? Total - - ? ? Question: You have extracted genomic DNA (gDNA) from Arabidopsis WT plants and adjusted its concentration to 100 ng/μL. You want to amplify 6 different genes from gDNA. To avoid pipetting mistakes, you prepare a PCR master mix with all necessary reagents that you will aliquot in 6 PCR tubes. 1. Which reagents go in the master mix? 2. How much volume of the master mix will you add in each PCR tube? 3. What else do you need to add in each PCR tube? 4. Why are you preparing a master mix for 8 samples, if you need to amplify 8 genes?  52 Questions?  53 Week 2: gDNA isolation and PCR 1. Genomic DNA Isolation 2. DNA Analysis 3. DNA Amplification by PCR 4. Analysis of PCR  54 Genomic DNA Crush samples Isolation Lyse in extraction buffer Transfer supernatant Precipitate DNA in isopropanol Wash DNA in ethanol Resuspend DNA in TE buffer  55 Genomic DNA (gDNA) Analysis Gel electrophoresis Preparing and running standard agarose DNA Gels Staining and Visualization of DNA Migration and mobility of DNA fragments in agarose Agarose Gel Electrophoresis VIDEO: https://www.addgene.org/protocols/gel-electrophoresis/ 55 From: Drabick and Silberring. Chapter 7 Gel electrophoresis  56 Preparing and running agarose DNA gels Agarose gels are made by adding agarose to Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE) buffer, microwaving the solution and pouring it into a gel cast with comb. The gel is then submerged with running buffer (TAE or TBE). DNA samples are mixed with loading buffer, which contains something dense (e.g. glycerol) to allow the sample to "fall" into the wells and one or two tracking dyes (such as bromephenol blue or xylene cyanol), and then pipetted into the wells. The lid and power leads are placed on the apparatus, and a current is applied. DNA is negatively charged and moves towards the anode (usually colored in red). You can confirm that current is flowing by observing bubbles coming off the electrodes.  Migration and mobility of DNA fragments in agarose 57 High and low MW DNA fragments are separated based on their size. The loading buffer allow visual monitoring of how far the electrophoresis has proceeded. The distance DNA has migrated in the gel can be judged by visually monitoring migration of the tracking dyes. Bromophenol blue (BPB) and xylene cyanol (XC) dyes have a slight negative charge and migrate through agarose gels at roughly the same rate as double-stranded DNA fragments of 300bp and 4000 bp, respectively.  58 Staining of DNA DNA fragments are visualized by staining with a fluorescent dye, which is often incorporated into the gel so that staining occurs during electrophoresis. The gel can also be stained after electrophoresis by soaking in a dilute solution of the fluorescent dye. Ethidium bromide (EtBr) intercalates between bases of DNA (and RNA) and emits red/orange fluorescence when exposed to UV light (stronger when it is bound to DNA). NOTE: Ethidium bromide is a known mutagen and should be handled as a hazardous chemical → wear gloves while handling. RedSafe binds DNA. RedSafe emits green fluorescence when exposed to UV light (and bound to nucleic acids). RedSafe is as sensitive as EtBr and it’s not toxic, not mutagenic. In the lab we will use RedSafe. https://www.bulldog-bio.com/product/redsafe-nucleic-acid-staining-solution/  59 Visualization of DNA UV transilluminator UV light is used to visualize DNA (or RNA) stained with the fluorescent dyes. After gel electrophoresis, the gel is placed on an ultraviolet (UV) transilluminator or in a gel documentation system. Be aware that DNA will diffuse within the gel over time, and examination or photography should take place shortly after cessation of electrophoresis. Gel documentation system UV Transilluminator (an ultraviolet light box) is used to visualize stained DNA in gels. NOTE: always wear protective eyewear when observing DNA on a transilluminator to prevent damage to the eyes from UV light. If using a Gel documentation system, the gel is placed within a box, connected to a computer with no exposure to UV light.  60 Factors affecting the mobility of DNA fragments in agarose gels 1. Agarose Concentration: Higher concentrations of agarose facilitate separation of small DNA fragments, while low agarose concentrations allow resolution of larger DNA fragments. The image shows migration of DNA fragments in three concentrations of agarose, all of which were in the same gel tray and electrophoresed at the same voltage and for identical times. Larger fragments are much better resolved in the 0.7% gel The smaller fragments separated best in 1.5% agarose. The 1000 bp fragment is indicated in each lane.  61 2. Electrophoresis Buffer: The most commonly used for duplex DNA are TAE (Tris- acetate-EDTA) and TBE (Tris-borate-EDTA). DNA fragments will migrate at somewhat different rates in these two buffers due to differences in ionic strength. Buffers not only establish a pH, but provide ions to support conductivity. If you mistakenly use water instead of buffer, there will be essentially no migration of DNA in the gel! Conversely, if you use concentrated buffer (e.g. a 10X stock solution), enough heat may be generated in the gel to melt it. 3. Voltage: the higher the voltage, the faster the fragments will migrate.  62 Genomic DNA analysis on agarose gel Genomic DNA isolated using a crude DNA extraction protocol. Samples were loaded on a 1% agarose gel. High MW Genomic DNA bands What do these bands represent? Questions Why do we quantify genomic DNA isolated with this crude extraction method by running it on a gel and not with the Spectrophotometer? What else have you extracted along with genomic DNA? If you want to quantify the DNA by spectrophotometer, which additional step would you need to perform?  63 Quantitation of DNA and RNA Gel electrophoresis Spectrophotometer  64 Quantitation of DNA (and RNA) Gel electrophoresis. You can quantitate DNA by using nucleic acid intercalating dyes, such as ethidium bromide, RedSafe, etc. Upon UV irradiation, compare the fluorescence emitted by your sample with that of a standard. Quantitation is less accurate, but you can quantify low amount of DNA, as low as 1ng DNA. Simply run the DNA on a gel and compare the intensity (brightness) of your DNA sample to that of a series of DNA standard concentrations. Often, the approx. DNA concentration is measured by comparing the intensity of your DNA sample with that of the DNA mass ladder. Remember: the amount of fluorescence is proportional to the mass of your DNA. So compare the intensity of a similar mass band (bp) in the ladder. Alternatively, calculate the intensity of your band in relation to the band in the ladder that shows similar intensity and than relate to the mass of your DNA.  65 Quantitation of DNA (and RNA) Spectrophotometer: The absorbance of UV light at 260 nm wavelength by nucleic acids gives an estimate of their concentration. The absorbance of UV light at 280 and 230 nm determine the purity of DNA. Absorbance at 260 nm will determine DNA or RNA concentration Absorbance at 280 nm will measure the amount of protein or contaminants (phenol) Absorbance at 230 nm will measure the amount of carbohydrates or contaminants (EDTA, salts, phenol, etc.) A ratio of readings at 260nm and 280nm (OD260/OD280) will determine the purity of your DNA sample. Optimal OD260/OD280 of 1.8 indicates a pure DNA preparation. Optimal OD260/OD280 of 2 indicates a pure RNA preparation. Contaminants will decrease the OD260/OD280 and OD260/OD230 ratios. dsDNA: OD 260 1 = 50 µg/ml RNA: OD 260 1 = 40 µg/ml To ensure the numbers are useful, the A260 reading should be within the Spectrophotometer linear range (generally 0.1–1.0). Limitations: Regular Spectrophotometers cannot quantitate low amount of DNA (less than 250 ng/mL) or heavily contaminated DNA (with proteins, phenol). However, Nanodrop Spectrophotometers, can estimate (pure) nucleic acid concentrations in 1 µL samples.  66 Questions?