Dna | 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 bacteria (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 bacteria 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 bacteria (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 bacteria 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 bacteria 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 in it. You want to work with the , 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 into a larger amount. It is quick, easy, and automated. Larger amounts of 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 polymerases found in thermophiles (bacteria that live in hot springs).Most polymerases (enzymes that make new ) work only at low temperatures. But at low temperatures, is tightly coiled, so the polymerases don’t stand much of a chance of getting at most parts of the molecules.

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

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

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

      4. Nucleotides. You’ll need to add nucleotides (dNTPs) so the 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 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 .

      • 1. Your target sample is heated. This denatures the , unwinding it and breaking the bonds that hold together the two strands of the molecule, leaving you with single stranded (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 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 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 exponentially for each of the 25 to 75 cycles. A cycle takes only a minute or so and each new segment of 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 piece of skin into your sample, then your 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 primer as usual and allow it to anneal to your target molecule. Then add RT along with dNTPs, which will elongate the 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 molecule analogous to the target segment of the RNA molecule.

May 14

Southern blotting

Southern blotting

Southern blotting was named after Edward M. Southern who developed this procedure at Edinburgh University in the 1970s. To oversimplify, DNA molecules are transferred from an agarose gel onto a membrane. Southern blotting is designed to locate a particular sequence of DNA within a complex mixture. For example, Southern Blotting could be used to locate a particular gene within an entire genome.

The amount of DNA needed for this technique is dependent on the size and specific activity of the probe. Short probes tend to be more specific. Under optimal conditions, you can expect to detect 0.1 pg of the DNA for which you are probing.

This diagram shows the basic steps involved in a Southern blot.

dSouthern blot

Let’s look at this technique in greater detail.  

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    • 1. Digest the DNA with an appropriate restriction enzyme.

    2. Run the digest on an agarose gel.

    3. Denature the DNA (usually while it is still on the gel).
    For example, soak it in about 0.5M NaOH, which would separate  double-stranded DNA into single-stranded DNA. Only ssDNA can transfer.

    A depurination step is optional. Fragments greater than 15 kb are hard to transfer to the blotting membrane. Depurination with HCl (about 0.2M HCl for 15 minutes) takes the purines out, cutting the DNA into smaller fragments. Be aware, however, that the procedure may also be hampered by fragments that are too small.

    Be sure to neutralize the acid after this step, or the base after the prior step if you don’t depurinate.

    Transfer DNA to membrane4. Transfer the denatured DNA to the membrane. Traditionally, a nitrocellulose membrane is used, although nylon or a positively charged nylon membrane may be used. Nitrocellulose typically has a binding capacity of about 100µg/cm, while nylon has a binding capacity of about 500 µg/cm. Many scientists feel nylon is better since it binds more and is less fragile. Transfer is usually done by capillary action, which takes several hours. Capillary action transfer draws the buffer up by capillary action through the gel an into the membrane, which will bind ssDNA.

    You may use a vacuum blot apparatus instead of capillary action. In this procedure, a vacuum sucks SSC through the membrane. This works similarly to capillary action, excepts more SSC goes through the gel and membrane, so it is faster (about an hour). (SSC provides the high salt level that you need to transfer DNA.)

    After you transfer your DNA to the membrane, treat it with UV light. This cross links (via covalent bonds) the DNA to the membrane. (You can also bake nitrocellulose at about 80C for a couple of hours, but be aware that it is very combustible.)

    5. Probe the membrane with labeled ssDNA. This is also known as hybridization.
    Whatever you call it, this process relies on the ssDNA hybridizing (annealing) to the DNA on the membrane due to the binding of complementary strands.
    Probing is often done with 32P labeled ATP, biotin/streptavidin or a bioluminescent probe.

    A prehybridization step is required before hybridization to block non-specific sites, since you don’t want your single-stranded probe binding just anywhere on the membrane.

    To hybridize, use the same buffer as for prehybridization, but add your specific probe.

    6. Visualize your radioactively labeled target sequence. If you used a radiolabeled 32P probe, then you would visualize by autoradiograph. Biotin/streptavidin detection is done by colorimetric methods, and bioluminescent uses luminesence.

32P labeled ATP
Treat the dsDNA fragment that you are using as a probe with a limiting amount of Dnase, which causes double-stranded nicks in DNA. Add 32P, dATP, and other dNTPs to DNA polymerase I, which has 5′ to 3′ polymerase activity and 5′ to 3′ exonuclease activity.

Nick translation occurs and as the nick is translated down the DNA strand, the polymerase activity continues to nick while the exonuclease activity continues to fill in the nick. As this happens, 32P becomes incorporated into, and thus labels, the DNA. Heat the DNA to make it single stranded, then immediately place it on ice to keep the two strands from reannealing to each other. (If the DNA is on ice, the DNA passes through the annealing temperature too quickly for the DNA to rehybridize into double-stranded DNA.)

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