BIOL 3500: Recombinant DNA Technology

How does colony hybridization work?

*Like a southern blot with a membrane, x-ray film, blotting but no gel instead has a "master plate" where all your colonies have already grown - each colony is a different cDNA 1. A nylon membrane is gently laid onto the master plate and lifted, yielding a replica of the master plate 2. The membrane is treated with detergent to permeabilize the bacteria, and the DNA is fixed to the membrane. 3. NaOH is added to denature the DNA (into ssDNA) and the membrane is submerged in a solution containing a radiolabeled probe that is complementary to the beta globin gene 4. The membrane is washed to remove unbound probe and then placed next to an x-ray film 5. Match the film up with the master plate so you know which ones contain your gene (those are the ones that show up on the x-ray film)

Describe the steps of construction a cDNA library

*Used if you want to learn more about a particular gene (that is too large for a plasmid or phage) 1. Start with mRNA and make ds cDNA 2. On cDNA put linker DNA - can fuse a sequence that has a particular restriction enzyme in it (make it that all your cDNA has this linker protein on the outside) *The linker DNA makes them all equal because they all have the same end 3. Have an enzyme there within the linker sequence that will cut it so all of the ends will now be the same 4. Put these cDNA fragments into a vector and put the vectors into bacteria (like with genomic libraries) 5. Grow these on cultures (each colony represents a unique cDNA) and get your desired sequence through colony hybridization

Describe how the western blot process works

1. 3 tubes with protein from different tissue types, add to SDS PAGE gel 2. Gel separates the protein sequences by size 3. Electrolyte mechanism - assembles together the cathode/anode plates stacked around the blotting paper, nitrocellulose filter, and gel - sits for about an hour for protein to be pulled through and has a dye to stain the membrane when protein is on it 4. Add primary antibody first (binds to the protein on the membrane) 5. Add secondary antibody which has a reporter gene on it - used to detect where the primary antibody is (binds to primary antibody and releases a color) *No prehybridization step but uses milk proteins to essentially do the same thing which is added to the solution and membrane sits in it so coated

What are the different types of restriction enzyme cutting? What problem can occur with blunt end cuts? How can this be fixed

1. Blunt ends: Cut both strands between same 2 base pairs 2. Sticky end 5' overhang: Staggered cuts that result in 5' overhangs 3. Sticky end 3' overhang: Staggered cuts that result in 3' overhangs Directionality - can potentially have a sequence cut at either side of enzyme and don't know if its the right way or backwards Directional cloning - using 2 enzymes each with their own cut site and then have a piece cut with both as well so it only goes in one way

What are the different steps in PCR? What changes and what stays the same when using PCR?

1. Denaturation 2. Annealing 3. Elongation (replication) Stays the same: - Temp for denaturation is 95 degrees C - Temp for Elongation is 72 degrees celsius - Time for denaturation and annealing (about 10-60 sec for denaturation and maybe 40-45 seconds for annealing?) What changes: - Temperature for annealing process - Time for elongation step (depends on how long the product is - about 30 seconds to 6 minutes)

What are the different transformation methods? In both methods what is essentially done?

1. Electroporation - cells + DNA in tube - expose to electric current - opens pores in membrane to allow DNA to enter *Used when cell can't go through chemical transformation ex. agrobacterium (transforms Arabidopsis) 2. Chemical transformation - Cells treated with calcium chloride *Whether chemical or electroporation, they will permeabalize the membrane so it opens up and the plasmid can go in (and plate Gus solution for selection looking for the transformed bacteria)

What are the included equipment parts of an agarose gel electrophoresis? Describe the process

1. Gel box (buffer on both sides) 2. Gel tray (gel made here slots for combs) 3. Gel box lid (attaches wires to box; stops current if lid is removed) 4. Gel combs (different sizes/number of wells) 1. Restriction enzymes cleave DNA into smaller segments of various sizes 2. DNA segments are loaded into wells in a porous gel. The gel floats in a buffer solution witin a chamber between two electrodes 3. When an eletric current is passed through the chamber, DNA fragments move towards the positively charges anode - "run to red" 4. Smaller DNA fragments move faster the farther than larger DNA fragments

What are the different types of microarrays?

1. Gene expression profiling (what we talked about so far) 2. Protein microarrays (similar to above, but shows the abundance of proteins with antibodies - they bind to specific proteins so you know which are active) 3. CGH - Comparative genomic hybridization microarrays (measure WT and mutant at once and compare them using different fluorescent markers )

What are the steps of dideoxy DNA sequencing?

1. Heat denature template 2. Primer annealing 3. DNA synthesis (dNTP and ddNTP incorporation) 4. ddNTP stops synthesis, adds fluorescent tag 5. Separate products on gel 6. Read with laser - Smallest products run fastest (closer to primer) *On the graph, each peak corresponds with a certain signal (sometimes can get multiple peaks and ambiguity because of heterozygotes)

What are the uses of the Southern Blot?

1. Identify mutations, deletions, and gene rearrangements 2. Used in prognosis of cancer and in prenatal diagnosis of genetic diseases 3. Leukemias 4. Diagnosis of HIV-1 and infectious disease

What are some practical uses of recombinant DNA? How can you do this?

1. Insulin production (use to have to rely on cows/pigs but now can use bacteria as factories 2. HGH (human growth hormone) To do this... 1. Drive expression of insulin mRNA with E. coli promoter 2. Make as much as needed 3. Purify

What are the basic DNA cloning steps?

1. Isolate DNA from organism 2. Cut DNA into pieces with restriction enzymes 3. Put individual pieces into cloning vector (bacteria, virus, etc. as long as DNA didn't come from that source it's considered recombinant DNA) *Result: recombinant DNA molecule 4. Transform into host (E. coli, yeast, animal or plant cell) - 1st put it in a something simple like E. coli the "gateway vector" for subcloning - Then put into your target host such as a eukaryotic cell

What are some essential characteristics for plasmid cloning? (What are needed)

1. Origin of replication (ORI). They must be able to replicate themselves or they are of no practical use as a vector. 2. Selectable marker. They must have a marker so you can select for cells that have your plasmids. (a way to know your plasmid is in the cell - you need a selectable marker per vector) - antibiotic - (plants) herbicide resistance 3. Restriction enzyme sites in non-essential regions. You don't want to be cutting your plasmid in necessary regions such as the ORI. - allows you to insert your gene into the vector! (must be a unique site in a nonessential region of the plasmid) 4. Small. - easier to isolate (you get more) - easier to transform into cells 5. Multiple UNIQUE restriction enzyme sites in multi-cloning site - more sites give you greater flexibility in cloning

How do you construct a genomic library? What are the applications?

1. Partial digestion of DNA by a restriction enzyme that generates a series of overlapping fragments, each with identical 5' GATC sticky ends 2. Clone each into vector - resulting fragments may be inserted into the multicloning site of the BAC/YAC *You put all the pieces together in a "contig" to get this overall sequence with redundancy in for backup Applications: 1. Sequencing a genome Overlap alike sequences 2. Identifying cloned gene of interest

What are applications of DNA fingerprinting?

1. Paternity and Maternity Testing 2. Criminal Identification and Forensics 3. Personal Identification

Explain the cloning example (how to put the DNA segment into the vector)

1. Restriction enzyme cuts through the LacZ+ region in a jagged manner 2. DNA fragments match the jagged part 3. When the pieces come together, this disrupts the LacZ coding sequence thats part of the multi-cloning sequence - these plasmids confer ampicillin resistance but cannot make functional Beta-galactosidase *An insertion mutation here means the LacZ gene doesn't work

What must you keep in mind about restriction site locations?

1. Restriction sites are SPECIFIC DNA sequences 2. Naturally & artificially in cloning vectors 3. Naturally in genome

Describe the steps of construction a genomic library

1. Start with vectors with a single restriction site (BACs and YACs) 2. Cut and opened up at that particular site 3. Chromosome is cut with the same restriction enzyme to get fragments of chromosomal DNA 4. Each piece of chromosome will go into vectors (mix vectors and DNA fragments under conditions that favor base pairing) 5. Add ligase to seal (covalently join pieces together *Technically your library is now complete 6. Transform the vector into a bacteria (each hybrid vector contains a different fragment of chromosomal DNA) to make many copies *library is stored one vector at a time in bacterial cells 7. Plate on petri plate containing the selected antibiotic to find the vectors that were transformed - sequence them

What is a human application for gene cloning?

1. Used to allow production of useful proteins, compounds - Microorganisms used as living factories to produce things such as: 1) Vaccines 2) Insulin 2. Insect, pesticide resistant crops 3. Without it, most modern cancer research NOT possible

What are the steps to make a southern blot?

1. You start with genomic DNA and it is digested with specific restriction enzymes 2. Use a gel eletrophoresis to separate the DNA fragments (agarose gel contains EtBr for visualization with UV light) 3. Denature DNA into ssDNA by soaking the gel into 0.5 M NaOH 4. Transfer ssDNA to a nylon membrane (positively charged) via capillary action with dry paper towels - buffer solution in bottom tray travels upward, moving DNA from gel into the membrane *DNA on membrane in same pattern as on gel 5. Pre-hybridization step to block non-specific sites on membrane so probe only binds to your DNA 6. Prepare the probe (make a radioactive copy of DNA fragment) 7. Hybridization step (membrane incubated with radioactive ssDNA probe which binds to complementary sites on membrane) 8. Wash off excess probe 9. Expose membrane to X-ray film to visualize which samples contain the desired gene

Describe the steps of Chemical Transformation. How does it work? What DNA works with this?

1. You treat the bacterial cell with calcium chloride so that the walls are more permable to the DNA 2. Add the plasmid DNA 3. The bacteria are now transformed and selection on bacterial growth medium to select for the transformed bacterial cells The calcium ions form coats around the negatively charged backbone of the DNA of the cell wall so that the negatively charged DNA being inserted is not repelled and instead can get through Only circular DNA can survive in cells (linear DNA will be degraded - means that any cut vectors that are not put back together are degraded by endonucleases because they are seen as foreign DNA)

Describe the steps of cDNA synthesis (for use in RT-PCR and in general)

1. dT primer is added to the poly A tail 2. Add reverse transcriptase and dNTPs to get a complementary strand of cDNA - essentially 1 round of PCR *This is where you can stop and use this cDNA for RT-PCR because the RNA will degrade anyway but may go on if you need double stranded cDNA 3. Add RNase to degrade RNA 4. Add DNA polymerase to get ds cDNA

What are the different types of vectors?

1. plasmid 2. phage - phage DNA engineered so that lysogeny is NOT possible - DNA inserted into restriction sites on chromosome in non-essential location - Bacteria infected with recombinant phage - As phage reproduces, so does DNA = transparent plaques as cells lyse 3. BAC ("backs") - Can clone large DNA fragments up to 200 kb - Can handle like regular plasmids 4. YAC ("yaks") - Linear vector - Clone fragments up to 500 kb

What are DNA libraries? What are the different types of DNA libraries (what determines which is which)?

A collection of thousands of different fragments of DNA, each of which is inserted into a vector 1. When the starting material is chromosomal DNA, the library is called a genomic library - Only 1 genomic library per organism (entire makeup of genome) 2. A cDNA library contains hybrid vectors with cDNA inserts - Should represent the genes expressed in the cells from which the RNA was isolated - Many options of cDNA libraries per organism - represent the active DNA at any given time during development or any specific tissue

What is a DNA fingerprint? Who has them? How do you find a particular VNTR? What can this be used for?

A pattern formed by repeated sequences of base pairs which are called Variable Number Tandem Repeats (VNTRs) Every person has VNTRs To find a particular VNTR we use a radioactive version of the one in question Can compare two individuals to see how similar their genome is, do this because of variable number of tandem repeats Radioactive probes, get a "finger print" of the individual and see that on film

What is dideoxy DNA sequencing?

An old technique used for sequencing DNA - uses dideoxy (which is a DNA with an extra H instead of the 3' OH which is needed to add on sequences - so it is used to stops sequencing) - If they have unique fluorescent patterns for each nucleotide, wherever these ddNTPs bind, will stop and label and this pattern can be read - You also add in dNTPs to keep sequencing the genome instead of stopping after the first few base pairs are bound with ddNTPs *The DNA is more likely to pick up a dNTP than a ddNTP so that you get varying lengths of products (each time ddNTP binds a new product is made) so you can run through the pieces through a gel to see their size and are fluorescently tagged by the ddNTP - Use a lazer to detect the fluorescent tags and can figure out what the original template sequence was for sequencing (plot signal and time)

What is a BAC? YAC? What are they used for?

BAC = bacterial artificial chromosome YAC = yeast artificial chromosome They are used for genome storage (have to do with getting large numbers of sequences stored)

Why is getting "white" colony not enough to confirm correct gene presentation in a Blue-White selection? What do you do after this to determine what was inserted?

Because no matter what happens you will get positive transformants, but you don't know what was inserted so you need to do an experiment to figure out what is inserted Once you have position transformants... 1. Grow small liquid cultures of several individual colonies (independent transformants to determine which one has your cDNA in it) 2. Isolate plasmid DNA (alkaline lysis) 3. Cut DNA with restriction enzymes 4. Run on agarose gel to separate fragments 5. Analyze to determine if correct insert present *If really important - SEQUENCE!!

What is the cloning experimental goal? How many hybrid vectors will contain this? What must you do to distinguish them? What process does this call for?

Cloning experimental goal: to clone a specific gene Small percentage of the library hybrid vectors would contain the gene Use probe to distinguish rare colonies from others Colony Hybridization

Describe how restriction enzymes are used in DNA cloning

Cloning involves putting two different pieces together from different DNA You use the same restriction enzyme (for example EcoRI) that make cuts on both the vector DNA and the DNA that you are wanting to insert in - both have the same type of break so the complementary ends (usually sticky ends) can anneal and gaps sealed by ligase to form recombinant DNA molecules

What does construction of a genomic library require? What are different scenarios for constructing a library and their outcomes?

Construction of a genomic library requires partial digestion Scenario 1: Digest DNA with restriction enzyme that recognizes 8 bp Result: One segment is too small to clone in a bacteriophage and yield a recombinant DNA molecule of aprroximately 49Kb, the other fragments are too large Scenario 2: Complete digestion of DNA with restriction enzyme that recognizes 4bp sites Results: Fragments too small to clone in a bacteriophage vector Scenario 3: Partial digestion of DNA with restriction enzyme that recognizes 4 bp site Result: fragments of proper size to clone into vector and yield 49Kb recombinant DNA; fragments too large to close are not used *Scenario 3 is the way to go - partial digestion of 4 cutter will get all different possibilities that overlap; the point is to get an enzyme that will cut the DNA so that its an optimal size to fit into the vector (not too big, not too small)

What is Ct? When is it reached? When do you start measuring product levels for quantification of Q-PCR? What do you normally do with these measurements? How will the graphs differ depending on the concentration of your starting template DNA?

Ct or CT value = cycle threshold Ct reached when fluorescence greater than background Exponential accumulation of products can be measured after Ct (after the Ct is reached is when you can take a measurements to get a numerical level and graph it) You plot the different measurements after each cycle to create a curve of amplification You will get more fluorescence faster the more DNA you have in the tube to start - takes longer to get over threshold if diluted (signal over time is different)

What does each dideoxy DNA sequencing reaction contain? What causes different fragment sizes to result?

Each sequencing reaction contains: 1. Fluorescently labeled dideoxy nucleotides (ddATP, ddGTP, ddCTP, ddTTP) 2. Normal deoxy nucleotides (dATP, dGTP, dCTP, dTTP) 3. DNA polymerase *ddNTP : dNTP ratio = 1:100 Different fragment sizes result - wherever ddNTP incorporated, synthesis halts - lack of 3'OH group inhibits phosphodiester bond formation with new dNTP

What does electrophoresis mean? What is agarose? How does an agarose gel electrophoresis work?

Electrophoresis = to carry with electricity Agarose = fine form of agar (a polysaccharide) - agar is made from seaweed - agarose + water-based buffer = jelly-like substance DNA molecules added to wells at one end, move through the polysaccharide matrix - matrix functions like a sieve to "catch" molecules as they are transported by current - Smaller molecules can navigate through faster than larger ones, so they make it further down the gel

How is DNA visualized in gel? What is this a type of? What does that make it?

EtBr is a fluorescent dye - added to gel before solidifying - use UV light to visualize Also an intercalating dye - EtBr inserts between bases that are stacked in the center of the DNA helix. - 1 EtBr molecule binds to 1 base - As each dye molecule binds to the bases the helix is unwound to accommodate the strain from the dye. *Intercalates between adjacent bases, distorts DNA, and causes insertions or deletions Mutagen!!! - During DNA replication, can cause insertion or deletion of base pairs! - EtBr is why we wear gloves during gel work!

How do you test to see if the MCS (multicloning site) is in the lacZ gene? What is this process called?

Expose the plasmid to the X-gal substrate - β-galactosidase also can use X-gal as a substrate (lactose analog; 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) - Product: deep blue color If the LacZ gene is functional, a blue color will form and the multicloning site is not in the plasmid; if there is not blue substrate, you know something was inserted into the lacZ region to make it non functional This is called Blue-White selection

What is complementation? What is this best used on? Describe the steps

Finding a gene that will "rescue" a mutant (so you can see what is causing the mutation - either don't know what is defective or do but want to learn more about it) Better odds with cDNA library (expressed genes) 1. DNA from mutant WT strain - make genomic library/cDNA library out of it 2. Pieces of yeast genome put into vectors 3. Transform into yeast - have mutant cells that won't grow on a specific medium 4. Add in genomic library vectors - any yeast cells that take up the vector with genome in it are able to survive *yeast colonies that grow have extra DNA in mutant strain 5. Take these colonies and see what is going on

What is subcloning? Why is it important

First putting your recombinant DNA into a "gateway vector" or a simple host before putting it in your target host Plasmids (or other vectors) are very useful subcloning vectors because they can be easily transfected into cells, amplified, and purified to yield large quantities of DNA.

What is an advantage of cDNA in the lab? What are the 2 ramifications of this?

From a research perspective, an important advantage of cDNA is that it lacks introns This has two ramifications 1. It allows researchers to focus their attention on the coding sequence of a gene (smaller product and only has exons) 2. It allows the expression of the encoded protein: Especially, in cells that would not splice out the introns properly (e.g., a bacterial cell) *Must use cDNA in bacterial cells because they don't have splicing machinery

What is another name for a microarray? What is it used for? What is the most common use? What are the basic steps?

Gene chips (think of a snapshot of gene expression usually for the whole genome at once) Functional genomics Analysis of many genes simultaneously *This gives a holisitc view of the genome at once rather than looking at specific genes Most common use - measure gene expression patterns - Measure all gene expression at a given time 1. Isolate RNA 2. Make cDNA 3. Add fluorescent probes to cDNA 4. Wash the fluorescently probed cDNA over the gene chip (there are thousands of little spaces where they synthesize nucleotides that are representative of certain genes - had to know the sequence of organism to do this and found what sequences are unique for each gene)

How do bacteria prevent cutting self DNA using restriction endonucleases? Where do restriction enzymes cut?

How do bacteria prevent cutting self DNA? - Methylation, which also can occur in eukaryotic DNA - Cellular DNA is methylated, enzyme cannot recognize cut site They cut at very specific sequences called DNA restriction sites - Many DNA restriction sites have axis of symmetry through midpoint - DNA restriction sites usually 4 to 8 bp long

What are the limitations of RT-PCR? What is more accurate? Why don't we use that then? What is a better choice to use than RT-PCR?

Limitations: - EtBr not a very sensitive stain - cannot directly quantitate amount of mRNA Northern blot analysis of RNA levels more accurate (a direct way to measure mRNA levels and can be qualitative or quantitative) - no amplification required (no worry of primer binding differences between samples) - It's HARD! (More difficult because working with RNA and can't work with extremely small genes) A better way: real-time RT-PCR

Describe the process of electroporation

Metal plates and small "tube" or hole you put bacteria in with small pipet, make sure the liquid is touching both sides of the tube so that the electrical current runs completely across the solution the bacterial cells are in DNA and bacterial cells are in solution and current opens up the membrane of the bacterial cells temporarily so the DNA can get in the cell Then the solution is put on selective plates to see which cells were transformed

What are the advantages of using PCR? Limitations?

PCR Advantages: - Faster gene cloning (amplify a sequence out of a genome and put into vectors to create transgenic lines, etc.) - Fast detection of genetic variation (take any gemonic sample, amplify one region, and see what size it is to compare to the norm and see if any mutations are present PCR Limitations: -Requires use of specific primers: DNA sequence of desired product must be known to design primers (can't amplify if you don't already know some about the sequence) - Taq polymerase does not proofread: Mismatches uncorrected & replicated (will have mutations/errors) - Sensitivity of PCR can amplify from wrong template (no contamination from other DNA sources!) *even one molecule of DNA can contaminate - Product length limited: by enzyme & conditions 20 kb (this estimate is on the high end - can't indefinitely amplify so need smaller products)

What is the purpose of the Pre-hybridization step? What is used, how, and why?

Pre-hybridization step - Blocks non-specific sites on the membrane - Do not want probe binding anywhere - Typical blocker used: salmon sperm ssDNA Why? (Create membrane with DNA on it, and have all the white area open area on membrane without nucleic acid bound. Probe is nucleic acid - you want your probe to prefer the single stranded DNA you are trying to identify. If you have open positively charged membrane, probe will bind to this and you will have a black x-ray) - Squeeze a salmon, they give up their sperm - DNA rich - Most people are not probing for salmon sperm sequences - good source of nonspecific DNA *Block single stranded DNA all over the membrane - southern blots normally use salmon sperm ssDNA - likelihood that your probe will recognize the salmon sperm single stranded DNA is low. Add single stranded DNA solution and put membrane in Tupperware so the single stranded DNA of the salmon sperm will soak in (nonspecific single stranded DNA so no more exposed membrane before adding probe)

How do you prepare the probe for the southern blot? What process is used to label a DNA fragments?

Preparing the probe: -Small piece of DNA used to find another piece of DNA -Must be labeled to be visualized (Not necessarily radioactive or fluorescent, but it's a nucleotide sequence (one way is taking a PCR product - a known sequence - and "label" it by adding radioactivity to it) -Usually prepared by making a radioactive copy of a DNA fragment DNA fragment is labeled by the Random Hexamer Labeling Proces

Why is gene cloning so important? What is it used to study?

Provides a pure sample of an individual gene - and LOTS of it Used to study characteristics, function, importance of gene: - gene sequence - gene expression - gene overexpression (in different organisms, tissues, etc) - manipulate DNA sequence, test mutant gene function - protein function - protein dimerization with other proteins (ex. Yeast 2 hybrid assay - Gal-4 functions as a dimer and separate to see their diff functions and put back together) - protein's DNA-binding capacity - on and on and on

What is Q-PCR? What are the 2 steps? What is the same and what is different than RT-PCR? What are the 2 protocol examples?

Quantitative PCR (also called real time PCR) Still two steps like RT-PCR; step one is the same as RT-PCR 1. cDNAs produced from RNA - Using 1. dT primer (binds poly A tail and allows mRNA to be transcribed) or 2. specific gene primer 2. How you detect your mRNA levels - still indirect, 2 protocals for detection 2 protocol examples: 1. Use SYBR-green to stain dsDNA PCR products (not specific to your gene and may have multiple products from primers so must test primers ahead of time) 2. Taqman with primers + fluorescently-labeled probe (fluorescent labeled reporter gene and quencher)

What is RT-PCR and what does it do ?

Reverse transcriptase (RT)-PCR Detects and quantifies RNA in sample Two steps: 1.cDNAs produced from RNA - Using 1. dT primer (binds poly A tail and allows mRNA to be transcribed) or 2. specific gene primer 2. PCR amplification of cDNAs - viruses with RNA genomes can be detected (HIV, measles, mumps) *An indirect way to measure mRNA levels - RNA is not stable, so using it in the lab can be difficult because it easily degrades and is easily contaminated so it is easier to take RNA and make cDNA from it for indirect determination of it

What is in an SDS-PAGE gel and what are the purposes of these components? What do the primary and secondary proteins that are added to the membrane do?

SDS-PAGE stands for sodium dodecyl (lauryl) sulfate-polyacrylamide gel electrophoresis SDS (sodium dodecyl) coats the molecules of proteins so they're all one charge (negative charge so the proteins go toward the positive charge) The PA in PAGE stands for polyacrylamide which is similar to agarose that it forms a matrix (is a neurotoxin though) Primary antibody = the one that has to recognize your specific protein; they bind to the protein on the membrane Secondary protein had a molecule linked to it for detection - so it binds to the primary antibodies where they bind on the proteins and emit some sort of signal so we know where they are bound

What are the different types of blots, their macromolecules, and functions.

Southern Blot: DNA (E.M. Southern, 1970's) - To identify a DNA sequence of interst - mutation, repeats that vary within individuals *Southern blot named after man who discovered it (so capitalized) northern blot: RNA - works like a Southern - Determine if different mRNAs for 1 gene are produced - Is gene expressed? western blot: Protein - same concept, different gel, etc - Is protein present? What tissues? - "probes" - flourescent or enzyme-linked antibodies

What are the 2 types of plasmids?

Stringent plasmids replicate only when the chromosome replicates. - This is good if you are working with a protein that is lethal/sickly to the cell - Not many copies because depends on chromosome replication Relaxed plasmids replicate on their own. This gives you a higher ratio of plasmids to chromosome. - Independent of cell division or chromosome replication so many copies made this way

What is Taqman? How does it work?

Taqman: a type of Q-PCR A fluorescent reporter dye and a quencher dye are attached to the 5' and 3' ends respectively to a Taqman DNA probe used in Q-PCR. The Taqman probe (which is between the reporter and quencher) is an oligonucleotide that is complementary to one stand of the PCR product *The probe remains together When you use a TaqMan detector, during the primer annealing step, both a primer and TaqMan detector bind to the template DNA When you use a Taqman detector in real-time PCR, during the primer elongation step, the detector is digested by Taq polymerase which separates the reporter from the quencher - since the reporter is not quenched, it releases a color *Taq binds to the forward primer and starts polymerizing, when it hits the probe it knocks the quencher off so that the reporter lets off a signal - every cycle that happens, you get more signal produced (and this is how you quantify it)

What are you hybridizing your probe to? What happens during the hybridization step? Why must you wash off the excess probe?

That probe is what you use in your hybridization step - you are hybridizing your probe to match your single stranded DNA sequence Hybridization step - Membrane incubated with radioactive ssDNA probe - Binds complementary sites on membrane Wash off excess probe...wash....wash...wash - if any unwanted probe around for next step = all black film

Describe the process of Random Hexamer Labeling Process

The DNA fragment is labeled by the Random Hexamer Labeling Process: 1. The template DNA is denatured by boiling. 2. A mixture of hexamers (6 nucleotides) containing all possible sequences is added and allow to base pair. 3. DNA polymerase is added with radioactive nucleotides. 4. The mixture is boiled to separate the strands and is ready for hybridization. *Can take any PCR product we want and do this technique to be able to identify that particular sequence - Take template DNA (PCR product), denature in boiling water, and basically do one stage of PCR/1 cycle, add hexomers or six nucleotides of random sequences (don't use a particular primer but it will bind sometimes and give you some product), DNA polymerase is added and have one of 4 nucleotides that is radioactive

What does the Random Hexamer Labeling process produce? How does it do this?

The Random Hexamer Labeling Process produces a radioactive single-stranded DNA copy of both strands of the template for use as a probe. The hexamers bind at random places and anywhere they bind synthesis will start. Only one nucleotide will be radioactive (say like every C or something) Then your mixture for this probe will have everything still at the bottom (template sequence and radioactive copies - all is added to the membrane, doesn't have to be just the radioactive nucleotides)

During Blue-White selection, what components are included on the plate? What happens to plasmids containing the insertion? Not containing to insertion? What does this technique NOT tell you?

The plate contains - 1. Agar 2. Nutrients 3. X-gal (substrate) 4. Ampicillin (amp) Plasmid containing gene (insert) in MCS: - no functional Beta-galactosidase - "White" colonies Plasmid with no insert: - Functional beta-galactosidase - "Blue" colonies This does not tell you what was inserted in the MCS, only that something is there

What can a southern blot be used for? What can you not do this with and why? What do you need to be able to do to do a blot? What is a blot, what do you generally do, and what results from it and why?

This is a way if you have DNA that you put on a gel, how can I search for a certain molecule that I put on a gel You can't really do this with polymers because if you try to incubate with a probe or anything your sample will wash out and the banding pattern and stuff will be messed up. Need a more permanent way to transfer the DNA once you separate it into a permanent position. A blot is taking a piece of paper and sticking with the gel and forcing the DNA to move up into the paper, that can be fixed with UV light (crosslink of the DNA) and put a DNA probe of your choice to protect it. A "smear" because it's a whole chromosome - the point of the gel is to pull apart them as far as you can get them so you can separate them by size optimally Start with the DNA gel, then end up with an x-ray film exposed to the membrane so only certain sequences on the gel light up where the probe (radioactive probe will bind to these particular sequences)

What is one application for using an agarose gel electrophoresis? What is an example?

To compare a gene to a known wild-type to view mutations. Ex. DNA electrophoresis gel depicting genotype assay for mice carrying clock gene "knockouts" Comparing mPer2 homozygous mouse and mPer2 heterozygous mutant mouse and a wild-type mouse. They used a DNA ladder for known sizes. The mutant mPer2 genes were larger than the mPer2 wild-type allele, so they didn't travel as far on the gel as the wild-type.

What types of DNA samples are used with an agarose gel electrophoresis? What is the DNA loading buffer used for? What are DNA ladders and what are they used for?

Types: - Restriction digestions of DNA - PCR products DNA loading buffer: - Sinks DNA into wells as it is loaded into gel - Visualize how far samples have run on gel DNA ladders: - Several DNA fragments of known amounts, sizes - Allows comparison of sample's sizes - Allows quantitation of samples

How does the Western Blot differ from the others? What are the different detection methods for a western blot?

Uses a different type of gel electrodes instead of capillary action - pushes proteins with voltage instead Detection mechanisms also differ 1. Radioactive isotopes that have been engineered within the antibody 2. Enzyme-linked antibodies - adds substrate to membrane and if antibody is bound to protein, enzyme reacts with substrate (light or color change) 3. Chlorophyll on antibody (reporter gene stuff?)

With a southern blot (or any blot really) why must you use restriction enzymes to break up your DNA (or whatever the blot uses)? How does the gel usually turn out with a southern blot and why? Why is NaOH used in a southern blot? What is used during transfer of the ssDNA onto the membrane and how does it work? In what way does the DNA end up on the membrane?

Whole chromosomes cannot run through a gel so the restriction enzymes cut at specific locations to leave you with restriction fragments Usually no distinct bands because so much DNA is present (more of a smear) Only ssDNA can transfer to a membrane - low enough concentration not to degrade it and just make it ssDNA Tray containing buffer solution, blotting paper which is essentially fancy cardboard to put under the gel, membrane filter, paper towels, and then something heavy on top to weigh down - liquid is forced in the bottom of the tray up through the paper towel so the DNA travels to the membrane - nylon membrane is positively charged and DNA negative so should attract it (capillary action moves DNA onto membrane) DNA should be on the membrane in the same pattern it was on the gel. Some areas are colored because DNA is there and white area of the membrane is naked - needs to be fixed before can do the hybridization step

How do you use a BAC to sequence an entire genome? How does the YAC process compare? What else can BACs/YACs be used for?

You have a massive BAC and about half of the vector is unknown DNA 1. Lay primer on known sequence and start sequencing through (can't go all the way through) 2. Bridge, make another primer, and sequence *Repeat multiple times until you get to the other end of the known sequences 3. Take all the sequenced pieces and stack them up until you have a continuous sequence all along - this is just one piece of the genome 4. Each and every BAC in the tube is one piece of the genome, and must sequence all of them to get the entire gemone (takes a long time) YAC works the same just linear Can be used to sequence or to analyze a big piece of the genome such as the promoter without error (can use this as well just must find the specific BAC with it)

What are restriction enzymes? How many of these are in prokaryotes? Why do bacteria produce these enzymes? How are they used for vectors?

a.k.a. restriction endonucleases - all organisms contain endonucleases - only prokaryotes contain restriction endonuc. Over 1200 different enzymes have been characterized from prokaryotes (400 isolated) Why do bacteria produce these enzymes? - Protection from foreign DNA → phages (bacterial viruses) - Restricts from cutting self DNA Once you have your piece of DNA, to put into your vector and to test what you have in it requires restriction enzymes (restriction endonucleases)

What is an example of a plasmid used as a cloning vector? What is included in it?

pUC19 plasmid Large circular DNA which has an ori site where replication would begin, an amp resistance site (amp), and a lacZ+ coding sequence that is used as part of a reporter to tell you if something is in the cloning site You must choose/design an enzyme which is compatible with the ends of the piece you are wanting to put in the vector so the cDNA can be put in - can't choose an enzyme to be put into the vector that cuts all sites, needs an enzyme that will only open up to vector once