[edit] PCR principle and procedure

Figure 1a: An old thermal cycler for PCR

Figure 1b: A very old three-temperature thermal cycler for PCR

PCR is used to amplify specific regions of a DNA strand (the DNA target). This can be a single gene, a part of a gene, or a non-coding sequence. Most PCR methods typically amplify DNA fragments of up to 10 kilo base pairs (kb), although some techniques allow for amplification of fragments up to 40 kb in size.^[3]

A basic PCR set up requires several components and reagents.^[4] These components include:

• DNA template that contains the DNA region (target) to be amplified.
• One or more primers, which are complementary to the DNA regions at the 5' (five prime) and 3' (three prime) ends of the DNA region.
• a DNA polymerase such as Taq polymerase or another DNA polymerase with a temperature optimum at around 70°C.
• Deoxynucleotide triphosphates (dNTPs), the building blocks from which the DNA polymerases synthesizes a new DNA strand.
• Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase.
• Divalent cations, magnesium or manganese ions; generally Mg²⁺ is used, but Mn²⁺ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn²⁺ concentration increases the error rate during DNA synthesis^[5]
• Monovalent cation potassium ions.

The PCR is commonly carried out in a reaction volume of 15-100 μl in small reaction tubes (0.2-0.5 ml volumes) in a thermal cycler. The thermal cycler allows heating and cooling of the reaction tubes to control the temperature required at each reaction step (see below). Thin-walled reaction tubes permit favorable thermal conductivity to allow for rapid thermal equilibration. Most thermal cyclers have heated lids to prevent condensation at the top of the reaction tube. Older thermocyclers lacking a heated lid require a layer of oil on top of the reaction mixture or a ball of wax inside the tube.

[edit] Procedure

The PCR usually consists of a series of 20 to 35 repeated temperature changes called cycles; each cycle typically consists of 2-3 discrete temperature steps. Most commonly PCR is carried out with cycles that have three temperature steps (Fig. 2). The cycling is often preceded by a single temperature step (called hold) at a high temperature (>90°C), and followed by one hold at the end for final product extension or brief storage. The temperatures used and the length of time they are applied in each cycle depend on a variety of parameters. These include the enzyme used for DNA synthesis, the concentration of divalent ions and dNTPs in the reaction, and the melting temperature (Tm) of the primers.^[6]

• Initialization step: This step consists of heating the reaction to a temperature of 94-96°C (or 98°C if extremely thermostable polymerases are used), which is held for 1-9 minutes. It is only required for DNA polymerases that require heat activation by hot-start PCR.^[7]

• Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 94-98°C for 20-30 seconds. It causes melting of DNA template and primers by disrupting the hydrogen bonds between complementary bases of the DNA strands, yielding single strands of DNA.

• Annealing step: The reaction temperature is lowered to 50-65°C for 20-40 seconds allowing annealing of the primers to the single-stranded DNA template. Typically the annealing temperature is about 3-5 degrees Celsius below the Tm of the primers used. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA synthesis.

• Extension/elongation step: The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75-80°C,^[8][9] and commonly a temperature of 72°C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTP's that are complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified. As a rule-of-thumb, at its optimum temperature, the DNA polymerase will polymerize a thousand bases in one minute.

• Final elongation: This single step is occasionally performed at a temperature of 70-74°C for 5-15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.

• Final hold: This step at 4-15°C for an indefinite time may be employed for short-term storage of the reaction.

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لینک: http://www.webmd.com/a-to-z-guides/urine-test

بخشی از متن:

Urine Test

A urine test checks different components of urine, a waste product made by the kidneys. A regular urine test may be done to help find the cause of symptoms. The test can give information about your health and problems you may have.

The kidneys take out waste material, minerals, fluids, and other substances from the blood to be passed in the urine. Urine has hundreds of different body wastes. What you eat, drink, how much you exercise, and how well your kidneys work can affect what is in your urine.

More than 100 different tests can be done on urine. A regular urinalysis often includes the following tests.

• Color. Many things affect urine color, including fluid balance, diet, medicines, and diseases. How dark or light the color is tells you how much water is in it. Vitamin B supplements can turn urine bright yellow. Some medicines, blackberries, beets, rhubarb, or blood in the urine can turn urine red-brown.
• Clarity. Urine is normally clear. Bacteria, blood, sperm, crystals, or mucus can make urine look cloudy.
• Odor. Urine does not smell very strong, but has a slightly "nutty" odor. Some diseases cause a change in the odor of urine. For example, an infection with E. coli bacteria can cause a bad odor, while diabetes or starvation can cause a sweet, fruity odor.
• Specific gravity. This checks the amount of substances in the urine. It also shows how well the kidneys balance the amount of water in urine. The higher the specific gravity, the more solid material is in the urine. When you drink a lot of fluid, your kidneys make urine with a high amount of water in it which has a low specific gravity. When you do not drink fluids, your kidneys make urine with a small amount of water in it which has a high specific gravity.
• pH. The pH is a measure of how acidic or alkaline (basic) the urine is. A urine pH of 4 is strongly acidic, 7 is neutral (neither acidic nor alkaline), and 9 is strongly alkaline. Sometimes the pH of urine is affected by certain treatments. For example, your doctor may instruct you how to keep your urine either acidic or alkaline to prevent some types of kidney stones from forming