Bacteria | Turkish Chemistry
May 17

Plasmids

A plasmid is an independent, circular, self-replicating that carries only a few genes. The number of plasmids in a generally remains constant from generation to generation. Plasmids are autonomous and exist in cells as extrachromosomal genomes, although some plasmids can be inserted into a bacterial chromosome, where they become a permanent part of the bacterial genome. It is here that they provide great functionality in molecular science.

Plasmids are easy to manipulate and isolate using (see also alkaline lysis)

They can be integrated into mammalian genomes, thereby conferring to mammalian cells whatever genetic functionality they carry. Thus, this gives you the ability to introduce genes into a given organism by using to amplify the hybrid genes that are created in vitro. This tiny but mighty plasmid is the basis of recombinant technology. Plasmids

A plasmid is an independent, circular, self-replicating that carries only a few genes. The number of plasmids in a generally remains constant from generation to generation. Plasmids are autonomous and exist in cells as extrachromosomal genomes, although some plasmids can be inserted into a bacterial chromosome, where they become a permanent part of the bacterial genome. It is here that they provide great functionality in molecular science.

Plasmids are easy to manipulate and isolate using (see also alkaline lysis) They can be integrated into mammalian genomes, thereby conferring to mammalian cells whatever genetic functionality they carry. Thus, this gives you the ability to introduce genes into a given organism by using to amplify the hybrid genes that are created in vitro. This tiny but mighty plasmid is the basis of recombinant technology.

There are two categories of plasmids. Stringent plasmids replicate only when the chromosome replicates. This is good if you are working with a that is lethal to the . Relaxed plasmids replicate on their own. This gives you a higher ratio of plasmids to chromosome.

So how do we manipulate these plasmids?

    1. Mutate them using restriction enzymes, ligation enzymes, and PCR. Mutagenesis is easily accomplished by using restriction enzymes to cut out portions of one genome and insert them into a plasmid. PCR can also be used to facilitate mutagenesis. Plasmids are mapped out indicating the locations of their origins of replication and restriction enzyme sites.

    2. Select them using genetic markers. Some are antibiotic resistant. While this is a serious health problem, it is a godsend to molecular scientists. The gene that confers antibiotic resistance can be added (ligated) to the gene you are inserting into the plasmid. So every plasmid that contains your target gene will not be killed by antibiotics. After you transfect your bacterial cells with your engineered plasmid (the one with the target gene and the antibiotic resistant marker), you incubate them in a nutrient broth that also contains antibiotic (usually ampecillin). Any cells that were not transfected (this means they do not have your target gene in them) are killed by the antibiotic. The ones that do have the gene also have the antibiotic resistant gene, and therefore survive the selection process.

    3. Isolate them (such as with alkaline lysis)

    4. Transform them into cells where they become vectors to transport foreign genes into a recipient organism.

There are some minimum requirements for plasmids that are useful for recombination techniques:

    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.

    3. Restriction enzyme sites in non-essential regions. You don’t want to be cutting your plasmid in necessary regions such as the ORI.

In addition to these necessary requirements, there are some factors that make plasmids either more useful or easier to work with.

    1. Small. If they are small, they are easier to isolate (you get more), handle (less shearing), and transform.

    2. Multiple restriction enzyme sites. More sites give you greater flexibility in cloning, perhaps even allowing for directional cloning.

    3. Multiple ORIs. It is important to note that two genes must have different ORIs if they are going to be inserted in the same plasmid.

 

May 17

PCR (polymerase chain reaction)

Let’s say you have a biological sample with trace amounts of DNA in it. You want to work with the DNA, perhaps characterize it by sequencing, but there isn’t much to work with. This is where PCR comes in. PCR is the amplification of a small amount of DNA into a larger amount. It is quick, easy, and automated. Larger amounts of DNA mean more accurate and reliable results for your later techniques.

The techniques was developed by Nobel laureate biochemist Kary Mullis in 1984 and is based on the discovery of the biological activity at high temperatures of DNA polymerases found in thermophiles ( that live in hot springs).Most DNA polymerases (enzymes that make new DNA) work only at low temperatures. But at low temperatures, DNA is tightly coiled, so the polymerases don’t stand much of a chance of getting at most parts of the molecules.

But these thermophile DNA polymerases work at 100C, a temperature at which DNA is denatured (in linear form). This thermophilic DNA polymerase is called Taq polymerase, named after Thermus aquaticus, the it is derived from.

Taq polymerase, however, has no proofreading ability. Other thermally stable polymerases, such as Vent and Pfu, have been discovered to both work for PCR and to proofread.

You’ll need four things to perform PCR on a sample:

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      1. The target sample. This is the biological sample you want to amplify DNA from.

      2. A primer. Short strands of DNA that adhere to the target segment. They identify the portion of DNA to be multiplied and provide a starting place for replication.

      3. Taq polymerase. This is the enzyme that is in charge of replicating DNA. This is the polymerase part of the name polymerase chain reaction.

      4. Nucleotides. You’ll need to add nucleotides (dNTPs) so the DNA polymerase has building blocks to work with.

There are three major steps to PCR and they are repeated over and over again, usually 25 to 75 times. This is where the automation is most appreciated.

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          1. Annealing temperature. Starts at the low end of what you think will work, then move up as necessary. If the temperature is too low, the primers will make more mistakes and you’ll get too many bands when you run your sample on a gel. If the temperature is too high you will get no results and your gel will be blank. You want to be about 3C to 5C below the melting temperature (Tm). A rough formula for determining Tm is Tm=4(G + C) + 2(A + T).

          2. Magnesium concentration. You want your Mg2+ concentration to be about 1.5mM to 3mM. If you go too high, the polymerase will make more mistakes.

          3. Think carefully about primer design. Both primers should have approximately the same Tm so they both anneal at the same temperature. Two out of three bases on the 3′ end should b G or C to get good hybridization (G and C have three H-bonds so you get better polymerization). Lastly, avoid primer dimers, which occur when the primers have ends that will anneal to each other. This will produce NO product.

          4. More is not necessarily better. More polymerase produces more nonspecific product, so don’t just carelessly dump in a bunch of polymerase. Additionally, PCR reactions don’t work if there is too much DNA.

      • 1. Your target sample is heated. This denatures the DNA, unwinding it and breaking the bonds that hold together the two strands of the DNA molecule, leaving you with single stranded DNA (ssDNA).

      2. Temperature is reduced and the primer is added. The primer molecules now have the opportunity to bind (anneal) to the pieces of ssDNA. This labels the portions of DNA to be amplified and provides a starting place for replication.

      3. New pieces of ssDNA are made. Taq polymerase catalyzes the generation of new pieces of ssDNA that are complimentary to the portions marked by the primers. The job of Taq polymerase is to move along the strand of DNA and use it as a template for assembling a new stand that is complimentary to the template. This is the chain reaction in the name polymerase chain reaction.

      PCR is so efficient because it multiplies the DNA exponentially for each of the 25 to 75 cycles. A cycle takes only a minute or so and each new segment of DNA that is made can serve as a template for new ones.

      Perhaps the most important thing to remember is to be very aware of contamination. If, for example, you unknowingly slough off a of skin into your sample, then your DNA may be amplified in the PCR reaction.  Here are some other factors to optimize your results with PCR:

      RT-PCR

      Taq polymerase does not work on RNA samples, so PCR cannot be used to directly amplify RNA molecules. The incorporation of the enzyme reverse transcriptase (RT), however, can be combined with traditional PCR to allow for the amplification of RNA molecules. After you add your RNA sample to the PCR machine, add a DNA primer as usual and allow it to anneal to your target molecule. Then add RT along with dNTPs, which will elongate the DNA primer and make a cDNA copy of the RNA molecules and run the PRC reaction as usual. The product of RT-PCR is a double stranded DNA molecule analogous to the target segment of the RNA molecule.

May 12

Alkaline Lysis

Alkaline lysis is the method of choice for isolating circular plasmid DNA, or even RNA, from bacterial cells. It is probably one of the most generally useful techniques as is a fast, reliable and relatively clean way to obtain DNA from cells. If necessary, DNA from an alkaline lysis prep can be further purified.

Alkaline lysis depends on a unique property of plasmid DNA. It is able to rapidly anneal following denaturation. This is what allows the plasmid DNA to be separated from the bacterial chromosome.

Typically, you will grow up cells that contain the plasmid you want to isolate, then you will lyse the cells with alkali and extract the plasmid DNA. The cell debris is precipitated using and potassium acetate. This is spun down, and the pellet is removed. Isopropanol is then used to precipitate the DNA from the supernatant, the supernatant is removed, and the DNA is resuspended in buffer (often TE). A mini prep usually yields 5-10 ug. This can be scaled up to a midi prep or a maxi prep, which will yield much larger amounts of DNA (or RNA).

Specific protocols for alkaline lysis differ widely from lab to lab, and even from scientist to scientist. The basic principles behind the procedure, however, are fairly uniform. Here they are:

1. Spin down your cells

. Your DNA is still in the cells, so it is in the pellet at this stage.

 

2. Discard the supernatant. Pieces of cell wall are released from the bacteria and are floating around in the supernatant. These cell wall pieces can inhibit on your final DNA, so it is important to get rid of all of the supernatant and to even invert the tube and wipe the lip with a Kim-wipe or Q-tip.

3. Resuspend the cells in buffer (often Tris) and EDTA. EDTA chelates divalent metals (primarily magnesium and calcium). Removal of these cations destabilizes the cell membrane. It also inhibits DNases. should also be added to maintain osmolarity and prevent the buffer from bursting the cells.

4. Lyse the cells with sodium hydroxide (NaOH) and . This highly alkaline solution gave rise to the name of this technique. Mix this by gentle inversion and incubate on ice for five minutes (but no longer, or your DNA will be irreversibly denatured). Three things happen during this stage:

a. pops holes in the cell membranes. (sodium dodecyl (lauryl) sulfate) is a detergent found in many common items such as soap, shampoo and toothpaste.

b. NaOH loosens the cell walls and releases the plasmid DNA and sheared cellular DNA.

. NaOH denatures the DNA. Cellular DNA becomes linearized and the strands are separated. Plasmid DNA is circular and remains topologically constrained.

5. Renature the plasmid DNA and get rid of the garbage. Add potassium acetate (KAc), which does three things:

a. Circular DNA is allowed to renature. Sheared cellular DNA remains denatured as single stranded DNA (ssDNA).

b. The ssDNA is precipitated, since large ssDNA molecules are insoluble in high salt.

. Adding sodium acetate to the forms KDS, which is insoluble. This will allow for the easy removal of the from your plasmid DNA.

Now that you’ve made it easy to separate many of the contaminants, centrifuge to remove cell debris, KDS and cellular ssDNA. Your plasmid DNA is in the supernatant, while all of the garbage is in the pellet.

    6. Precipitate the plasmid DNA by alcohol precipitation (ethanol or isopropanol) and a salt (such as ammonium acetate, lithium chloride, sodium chloride or sodium acetate) and spin this down. DNA is negatively charged, so adding a salt masks the charges and allows DNA to precipitate. This will place your DNA in the pellet.

    7. Rinse the pellet—your plasmid DNA—in ice-cold 70% EtOH and air-dry for about 10 minutes to allow the EtOH to evaporate.

    8. Resuspend your now clean DNA pellet in buffer (often Tris) and EDTA plus RNases to cleave any remaining RNA. Your DNA is now back in solution.

DNA of this purity is good for a number of uses, such as in vitro transcription or translation or cutting with some enzymes. If you are sequencing or transforming this DNA into mammalian cells, you’ll want to use additional purification techniques such as phenol extraction, Qiagen column purification, or silica-based purification. 

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