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Department
Microbiology
Course
MIC 115
Professor
Lifeng Xu
Semester
Fall

Description
MIC 115 Notes 9-26-2013 Email: [email protected] Methods of molecular biology and microbiology: Gel Electrophoresis: uses the molecular mass of DNA, RNA and protein to separate the samples. It also involves a power source. Gels can be agarose or acrylamide gels. When applied to the gel and allowing a current to flow through, the samples will move to the oppositely charged end. So DNA and RNA will move to the positive side (the anode). Migration rates of macromolecules through an electric field influenced by: charge shape and size of the molecules. DNA uniform charge per base. So the charge to mass ratio is the same. DNA can be in 3 different general shapes or “forms:” 1. Linear, 2. Circular – nicked, 3. Superhelical. Superhelical form moves the fastest while circular is the slowest. Linear is in between. Rate of migration is inversely proportional to the log of Molecular Weight of DNA. For DNA from 200 BP to 20 kb we need larger pores so this uses agarose gel. For DNA less than 200 bp we use smaller pores and require acrylamide gel, so as to reduce friction with unnecessarily large pores. Acrylamide gel uses two components: acrylamide monomer which polymerizes to a larger molecule and bis-acrylamide. Pulse field gel electrophoresis: The electrical field alternates to pull the DNA through and “snake” them through the pores. Larger DNA strands take longer to respond to the change in the field. This is used for DNA larger than 20 kb. However, the DNA no longer moves in a straight line with this method, so it can be difficult to determine which sample came from which original well. CHEF: contour-clamped homogenous electric field manages to force the net directions to be vertical so that this is corrected. RNA Secondary structures. RNA is single stranded, but it can base pair upon itself (dsRNA). How to get rid of RNA secondary structures? 1. Use denaturant which forces RNA to adopt the single-stranded form. One denaturant is called glyoxal. Another is formamide/formaldehyde. Glyoxal prevents base pairing upon itself while the formamide and formaldehyde prevent hydrogen bonding. Essentially if we eliminate all secondary structures, then charge and shape become uniform, thus mass is the only determining factor of the speed of RNA moving across a gel. Staining: DNA can be stained by EtBR (ethidium bromide, very common) or by SYBR Green. The normal form of EtBr fluoresces under UV but it can intercalate with DNA and fluoresce more intensely. SYBR Green is more expensive but also more sensitive to staining DNA. So more complex DNA mixtures may require SYBR Green. Especially if the DNA samples are rare or limited in quantity. For genomic staining however, neither of these will be very effective since there are so many strands of DNA. Staining will only yield giant smears. Detecting DNA/RNA by Hybridization: 1. Run labeled RNA or DNA on a gel. 2. Transfer the material to a special kind of paper (nitrocellulose paper). 3. Remove the paper with tightly bound nucleic acids. 4. Radiolabel the probe hybridized to separate DNA. 5. Read the positions of labeled markers and bands. Often read by an autoradiograph. Southern blotting is for DNA and Northern blotting is for RNA. Detection of Protein. Structures: primary, secondary (alpha helix and beta pleated sheets), tertiary and quaternary (interactions of different subunits). There isn’t a uniform charge per mass ratio with protein! Many amino acids are polar, some nonpolar, others are charged negatively or positively, and others still are aromatic. Again, we want to denature the proteins. We use SDS (sodium dodecyl sulfate) to swamp out the negative charges on the amino acids so that we have uniform charge/mass ratio again. This also destroys the secondary structure of the protein and forces it into the primary structure form. 1 SDS binds to 2 amino acids. Binding w/ SDS protein has uniform charge/mass ratio. 10-1- 2013 Molecular Techniques 2 Again migration rates of macromolecules through an electric field is influenced by: 1. charge of the molecule 2. shape of the molecule 3. size of the molecule For SDS, it will not destroy the disulfide bonds of protein. A reducing agent is required for that such as beta-mercaptanethanol. We still need a way to see the proteins since they are colorless like nucleic acids. There are 2 ways: staining and western blotting. For RNA we can actually stain it with EtBr or SYBR green but it must be done after separating them through the gel and removing the denaturants since it requires some secondary structure (ds-R NA) to bind and fluoresce. So DNA can be stained on the gel directly while separating but RNA must be stained after the gel separation and after removing the denaturants. For protein staining we can use Coommassie Blue or Silver Staining. Silver staining is more than 100 times more sensitive than Coommassie Blue. GAPDH is a housekeeping protein that is very complex and is nearly impossible to differentiate the bands by staining. In these cases we use Western Blotting. DNA uses southern blotting and RNA uses northern blotting. Conceptually it is the same idea but the procedure is very different. We use antibodies on our proteins to distinguish them. We have one or many primary antibodies that may be detected by many more secondary antibodies that are polyclonal. We use a substrate with horse radish peroxidase which produces a chemiluminescent product. If we then expose the protein on the membrane to a special piece of film it will yield a stronger visualization. - PCR: Used to Amplify DNA. We need the following: 1. Template, 2. Primers, 3. dNTP, 4. Polymerase (TAG), 5. Buffer (ion) solution. Two types of quthtitative PCR (also called real time PCR): SYBR Green-based PCR. We add SYBR as the 6 component to the above reagents. SYBR green fluoresces more intensely during the extension steps. Thus as the amount of product increases so does the amount of fluorescence. The second type is TaqMan probe-based qPCR. 10-3-2013 SYBR green based qPCR is lower cost and easier to optimize than TaqMan probe-based qPCR. However, SYBR green also has a relatively lower specificity as well. SYBR Green has a single amplification reaction, which is also a downside. QPCR has 3 stages: baseline, exponential and plateau. Afterwards we do a melting curve analysis. PRC product (dsNA) -> 95degC -> ssDNA -> 55degC -> dsDNA -> 0.1degC/s -> 95degC TaqMan probe-based qPCR: Different from SYBR green in 2 respects. 1. SYBR Green is not used. 2. We use a specific probe Taq DNA polymerase has a 5’ -> 3’ polymerase activity and 5’ -> 3’ exonuclease activity. The DNA probe degrades as the polymerization from the primer moves forward. So as the DNA probe degrades it releases a reporter dye which gives a visualization. Thus if you use different reporter dyes for different probes you can have multiple products run in the same tube. This is called a multiplex product. This also leads to higher specificity. Unfortunately, TaqMan probe is harder to optimize and is more expensive. So far we have discussed how to separate big molecules by gel electrophoresis. This includes DNA RNA and protein. PCR is a way we have used to amplify DNA and RNA by converting the RNA to cDNA. PCR only works for bp of at least 200 or greater though. Typically for 200 bp to several kb. So what to do for when we use for less than 200 bp? Chemical synthesis is the answer. And for fragments larger than several kb we use molecular cloning. Molecular cloning: we conjugate the DNA of interest. Column chromatography: We apply a sample to a solid matrix. We then apply solvent continuously to the top of the column. The different proteins then travel through the matrix at different rates and each can be eluted and collected in test tubes separately. 3 types of column chromatography: 1. Ion exchange – uses positively or negatively charged beads to bind to the different proteins. More negative proteins attract more positive beads and more positive proteins attract more negative beads. Neutral proteins will move fastest since they are unbound to the column. 2. Gel filtration (or size exclusion) – uses porous beads. Smaller proteins move through the pores of the beads and thus take a longer path and move slower. Larger proteins can’t fit through the pores of the beads and thus take a shorter time to move through the column. 3. Affinity – Uses beads to covalently bond with the proteins. Those that bind with a covalently attached substrate will stay attached to the column while the other unbound proteins will pass through. Changing the ionic strength of the ionic buffer can then help wash the bound protein away from the affinity column. Affinity may not be the most cost-effective necessarily, but it can easily yield the highest level of purification when compared exclusively to gel filtration or ion exchange. 10-8-2013 Gel-shift analysis also called gel shift assay or Electrophoretic Mobility Shift assay. Works for nucleic acids and protein by a native gel. Nucleic acid: larger charge/mass ratio than proteins. DNAse 1 footprinting analysis/assay. DNAse 1 lightly cuts DNA by chance at any phosphodiester bond. But DNA is labeled on one end only so we can see where the cut is. However, we can bind the DNA to a specific region or nucleotide sequence which prevents DNAse 1 from cutting that section. If we then separate the protein from the nucleic acid we can then observe a lack of bands since the protein protected that segment of DNA from the digestion. Methylation interference assay. Very similar where we radioactively label one end of the DNA. Then we treat with DMS: dimethyl sulfate which methylates any bases indiscriminately. But it is light so only one base per molecule is methylated. However, when a protein of interest is added the methylation prevents the protein from binding to it. Then we remove the protein and treat DNA with piperidine to cleave at the methylated sites. Then denature with the PAGE analysis, and the readout will show that the DNA bands where the protein tried to bind with a methylated site will be gone. So in DNAse footprinting we shield the DNA by the protein from the enzyme. In methylation interference we protect the DNA from the protein with methylation by DMS. Chromatin immune-precipitation: cross link cells with formaldehyde (HCOH). Isolate genomic DNA and sonicate to shear chromatin. The cross-linking is used to stabilize the DNA since it will be subjected to harsh conditions. Then we add an antibody specific to the protein of interest. We then perform an immunoprecipitation to isolate the DNA bound by the protein of interest. Reverse cross-link and isolate the DNA. Sequences can be very complex so they would be subjected to microarray analysis or high throughput sequencing instead of a gel electrophoresis to read them. LECTURE 4: Restriction Nucleases – Introduction Recombinant DNA. Can be separated by various methods. 1. Chemical method. 2. Physical method. 10-10-2013 Bacterial Phage Restriction – take bacterial strain B which has a much lower rate of infection efficiency than strain A. But if you infect strain B with strain A and then grow the next generation, then their infection efficiencies will reverse! Restriction endonucleases recognize a sequence within foreign DNA and cleave it. Restriction enzyme (system) – associated methylases protect the bacterial strains from their own restriction endonucleases from cleaving their own DNA. Hemi-methylated DNA. Restriction Enzymes are divided into 3 types by: 1. cofactor requirements, 2.sequence specificity, 3. cleavage position and 4. subunit composition. Type I restriction Enzymes: 1. Cofactor requirements – require ATP and S-adenosyl methionine as cofactors. 2. Sequence specificity – recognition sequences have no recognizable features. 3. Cleavage position – cut DNA1-5 kb from the recognition sequence – create a gap on one strand of DNA 4. Subunit composition – methylase and endonuclease in one multi-subunit protein complex Type III restriction Enzymes: 1. Cofactor requirements – require ATP 2. Sequence specificity – recognition sequences have no recognizable features. 3. Cleavage position – cleavage site generally 24 – 26 base pairs off to one side of recognition site 4. Subunit composition – methylase and endonuclease in one multi-subunit protein complex Type II restriction Enzymes: 1. Cofactor requirements - only requires Mg++ 2. Sequence specificity – recognition sequences often have a twofold axis of rotational symmetry 3. Cleavage position – cleave at defined sites close or within recognition sequence 4. Subunit composition – methylase and endonuclease are physically separate entities. This makes type 2 restriction enzymes very useful for molecular cloning purposes What is the chance a restriction enzyme can cut a random piece of DNA? See OneNote for how to calculate. Dam methylase helps protect restriction sequences from restriction enzyme digestion. How do we get around this? 1. We can use isoschizomers. 2. We can use a different bacterial strain that does not have methylation protection at the desired sites. Efficiency of restriction digestion is affected by: 1. supercoil of DNA 2. the nature of the flanking DNA 3. Mg++ Type II restriction enzymes only cleave dsDNA, not ssDNA or ssRNA Bring a UCD 1000 scantron for the quiz! Lecture 6 – Other DNA/RNA modifying enzymes 10-15-2013 DNA ligases • Require proximal 3’-OH and 5’P groups • Cannot fill gaps • Do not work on single stranded nucleic acids 1. T4 DNA ligase • Uses ATP as energy source • Ligate nicked DNA, sticky ends and blunt ends 2. E Coli DNA ligase • Uses NAD as energy source • Ligate nicked DNA, sticky ends • Do not ligate blunt ends RNA ligases 1. T4 RNA ligase • Requires 3’-OH and 5’-P ends • Ligate single-stranded RNA or DNA • Uses ATP as energy source E. Coli DNA Polymerase I 1. DNA-dependent DNA polymerase 2. one polypeptide chain, three enzymatic activities • 5’ to 3’ polymerase activity – requires a DNA template, a DNA or RNA primer with a 3’ OH, Mg++ and dNTP • 5’ to 3’ exonuclease activity: requires dsDNA, Mg++ - nick translation – the 5’ to 3’ exonuclease activity can be separated from the polymerase activity by protease cleavage or by genetic engineering to generate Klenow fragment. • 3’ to 5’ exonuclease activity – proofreading activity 10-17-2013 Nick translation: a nick is made in the DNA first. Then DNA pol 1 synthesizes a new strand of DNA and also incorporates exonuclease activity to remove any preexisting DNA in front of it. T4 DNA polymerase 1. DNA-dependent DNA polymerase 2. T4 DNA polymerase has: • 5’ to 3’ polymerase activity • No 5’ to 3’ exonuclease activity • Strong 3’ to 5’ exonuclease activity Taq DNA polymerase 1. DNA-dependent polymerase 2. Taq-DNA Polymerase has: • 5’ to 3’ polymerase activity • 5’ to 3’ exonuclease activity • No 3’ to 5’ exonuclease activity – no proofreading – higher error rate • Heat stable Reverse Transcriptase • RNA dependent DNA polymerase • Uses single-stranded RNA or DNA templates • Polymerizes 5’ to 3’ • Requires RNA or DNA primer • No exonuclease activity Common features of DNA polymerases • Polymerizes 5’ to 3’ to synthesize new strand of DNA • Uses single-stranded RNA or DNA templates • Requires RNA or DNA primer with 3’–OH group RNA Polymerases • DNA-dependent RNA polymerases • Do not need a primer • Requires appropriate promoter on DNA template for activity Terminal Transferase • Template-independent polymerase • Can work on ss or dsDNA • Prefers protruding 3’ end as a substrate • Adds new bases depending on what dNTP is used. If using a 1:1:1:1 mixture of all 4 types of nucleotides then the newly added sequence will be random. S1 Nuclease • Degrade single-stranded nucleic acids (DNA or RNA) • Also degrade any single stranded region of dsDNA or RNA Exo III Nuclease • 3’ to 5’ exonuclease activity • Cleaves nucleic acids in a duplex with a complementary strand • Not active on single stranded nucleic acids DNase I • Endonuclease with little sequence specificity • In the presence of Mg++ nicks each strand of dsDNA independently in a random fashion –creates sticky ends. Also used for nick translation in this case. • In the presence of Mn++ cleaves both strands of dsDNA at approximately the same site – creates blunt ends RNase H • Endonuclease with little sequence specificity • Degrade RNA in a duplex with a complementary strand of DNA – only degrades RNA half • Does not digest single or double-stranded DNA or RNA Alkaline Phosphatase • Removes terminal monophosphates from DNA, RNA, and dNTPs • Does not remove phosphate from phosphodiester bonds. • Leaves OH groups after Phosphate groups are removed T4 polynucleotide kinase • Transfers gamma phosphate from rATP to ss or dsDNA or RNA cointaining 5’ –OH group • Exchanges gamma phosphate from rATP with 5’-P group on ss or dsDNA or RNA 10-22-2013 Lecture 8 – Plasmid Biology Common Properties for Cloning Vectors • Ability to promote autonomous replication • Contain a selectable marker gene for selection • Unique restriction sites to facilitate cloning of insert DNA • Minimum amount of non-essential DNA to optimize cloning Any DNA -> Replicon: functional unit of replication In E. coli this could be a chromosome, or even the entire genome, or just a gene. Its genome is 4 x 10^6 bases or 4 MB. Typically only 0.1% or less is your gene of interest for E. coli. Naturally Occurring replicons in E. coli 1. E. coli chromosome 2. Bacteriophage – Lambda, M13 phage 3. Plasmid – F plasmids, drug resistance plasmids, ColE1 plasmids First widely used and customly designed plasmid • Amp from RS 2124 (a drug resistance plasmid) R • Tet from pSC101 (another drug resistance plasmid) • ColE1 origin of replication • Minimum amount of nonessential DNA ColE1-Type plasmids • Segregation of daughter strands – xer protein and cer sequence • Partitioning of ColE1 plasmids • Plasmid incompatibility ROP = repressor of primer 10-24-2013 1. Origin of replication 2. Selectable marker. Remember that after transformation of a bacterial colony, some will have the acquired plasmid DNA, and all the rest will not. 3. Unique restriction sites 4. Minimum amount of non-essential DNA to optimize cloning (i.e. minimum amount of plasmid vector). We want goi/vector to be maximized. lacZ: beta-galactosidase pUC19: 5’ – fragment of lacZ –N-terminal of Beta-gal Multiple cloning sites. Polylinker region Typical Plasmid Cloning Steps • Choose an appropriate plasmid vector • Restriction digest target DNA and plasmid vector • Ligate target DNA with plasmid vector • Transform E. coli with the ligation products • Grow on agar plates with selection for antibiotic resistance • Identify bacterium clones carrying the correct plasmid-target DNA recombinants Choosing a plasmid vector • Understand the experiments for which the recombinant molecule will be used • Choose plasmid vectors with appropriate MCS (multiple cloning sites) • Choose plasmid vectors with suitable copy number • Choose plasmid vectors with the appropriate selection markers Restriction Digestion and Ligation of Target and Plasmid DNA • Restriction digestion to create compatible ends • Treatment of plasmid vector to avoid self-ligation • Ligation of target DNA with plasmid vector 10-29-2013 Transformation of E. coli • E. coli can be made competent for reception of exogenous DNA by chemical treatment – treatment with divalent cations e.g. Ca++, Rb++ - al
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