Base | Turkish Chemistry
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 .

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 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 .

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

      2. Temperature is reduced and the primer is added. The primer 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 . The incorporation of the enzyme reverse transcriptase (RT), however, can be combined with traditional PCR to allow for the amplification of RNA . After you add your RNA sample to the PCR machine, add a primer as usual and allow it to anneal to your target . Then add RT along with dNTPs, which will elongate the primer and make a cDNA copy of the RNA and run the PRC reaction as usual. The product of RT-PCR is a double stranded analogous to the target segment of the RNA .

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 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 .

    3. Denature the DNA (usually while it is still on the ).
    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 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 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.)

May 11

Vitamin C Determination by Iodine Titration
Vitamin (ascorbic acid) is an antioxidant that is essential for human nutrition. Vitamin deficiency can lead to a disease called scurvy, which is characterized by abnormalities in the bones and teeth. Many fruits and vegetables contain vitamin , but cooking destroys the vitamin, so raw citrus fruits and their juices are the main source of ascorbic acid for most people.

One way to determine the amount of vitamin in food is to use a redox titration. The redox reaction is better than an acid-base titration since there are additional acids in a juice, but few of them interfere with the oxidation of ascorbic acid by iodine.

Iodine is relatively insoluble, but this can be improved by complexing the iodine with iodide to form triiodide:

I2 + I- <–> I3-

Triiodide oxidizes vitamin to form dehydroascorbic acid:

6H8O6 + I3- + H2O –> 6H6O6 + 3I- + 2H+

As long as vitamin is present in the solution, the triiodide is converted to the iodide ion very quickly. Howevever, when the all the vitamin is oxidized, iodine and triiodide will be present, which react with starch to form a blue-black complex. The blue-black color is the of the titration.

This titration procedure is appropriate for testing the amount of vitamin in vitamin tablets, juices, and fresh, frozen, or packaged fruits and vegetables. The titration can be performed using just iodine solution and not iodate, but the iodate solution is more stable and gives a more accurate result.

Purpose

The goal of this laboratory exercise is to determine the amount of vitamin in samples, such as fruit juice.

Procedure

The first step is to prepare the solutions. I’ve listed examples of quantities, but they aren’t important. What matters is that you know the concentration of the solutions and the volumes that you use.

Preparing Solutions

1% Starch Indicator Solution

 

  1. Add 0.50 g soluble starch to 50 near-boiling distilled .
  2. Mix well and allow to cool before use. (doesn’t have to be 1%; 0.5% is fine)

Iodine Solution

 

  1. Dissolve 5.00 g potassium iodide (KI) and 0.268 g potassium iodate (KIO3) in 200 ml of distilled .
  2. Add 30 ml of 3 M sulfuric acid.
  3. Pour this solution into a 500 ml graduted cylinder and dilute it to a final volume of 500 ml with distilled .
  4. Mix the solution.
  5. Transfer the solution to a 600 ml beaker. Label the beaker as your iodine solution.

Vitamin Standard Solution

 

  1. Dissolve 0.250 g vitamin (ascorbic acid) in 100 ml distilled .
  2. Dilute to 250 ml with distilled in a volumetric flask. Label the flask as your vitamin standard solution.

Standardizing Solutions

 

  1. Add 25.00 ml of vitamin standard solution to a 125 ml Erlenmeyer flask.
  2. Add 10 drops of 1% starch solution.
  3. Rinse your buret with a small volume of the iodine solution and then fill it. Record the initial volume.
  4. Titrate the solution until the is reached. This will be when you see the first sign of blue color that persists after 20 seconds of swirling the solution.
  5. Record the final volume of iodine solution. The volume that was required is the starting volume minus the final volume.
  6. Repeat the titration at least twice more. The results should agree within 0.1 ml.
You titrate samples exactly the same as you did your standard. Record the initial and final volume of iodine solution required to produce the color change at the .

Titrating Juice Samples

 

  1. Add 25.00 ml of juice sample to a 125 ml Erlenmeyer flask.
  2. Titrate until the is reached. (Add iodine solution until you get a color that persists longer than 20 seconds.)
  3. Repeat the titration until you have at least three measurement that agree to within 0.1 ml.

Titrating Real Lemon

Real Lemon is nice to use because the maker lists vitamin , so you can compare your value with the packaged value.

  1. Add 10.00 ml of Real Lemon into a 125 ml Erlenmeyer flask.
  2. Titrate until you have at least three measurements that agree within 0.1 ml of iodine solution.

Other Samples

 

  • Vitamin Tablet – Dissolve the tablet in ~100 ml distilled . Add distilled to make 200 ml of solution in a volumetric flask. 
  • Fresh Fruit Juice – Strain the juice through a coffee filter or cheese cloth to remove pulp and seeds, since they could get stuck in the glassware. 
  • Packaged Fruit Juice – This also may require straining. 
  • Fruits & Vegetables – Blend a 100 g sample with ~50 ml of distilled . Strain the mixture. Wash the filter with a few milliliters of distilled . Add distilled to make a final solution of 100 ml in a volumetric flask.

Titrate these samples in the same way as the juice sample described above.

Titration Calculations

 

  1. Calculate the ml of titrant used for each flask. Take the measurements you obtained and average them.average volume = total volume / number of trials

     

  2. Determine how much titrant was required for your standard.If you needed an average of 10.00 ml of iodine solution to react 0.250 grams of vitamin , then you can determine how much vitamin was in a sample. For example, if you needed 6.00 ml to react your juice (a made-up value – don’t worry if you get something totally different):

    10.00 ml iodine solution / 0.250 g Vit = 6.00 ml iodine solution / X ml Vit

    40.00 X = 6.00

    X = 0.15 g Vit in that sample

     

  3. Keep in mind the volume of your sample, so you can make other calculations, such as grams per liter. For a 25 ml juice sample, for example:0.15 g / 25 ml = 0.15 g / 0.025 L = 6.00 g/L of vitamin in that sample

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