If the denaturation step of PCR was omitted, what would happen to the PCR process?
How is PCR used to determine that a person is infected with a specific bacterium?
Now that we have PCR, why would we use culture techniques?
If a DNA polymerase other than the polymerase from Thermus aquaticus was used in the PCR process, what would happen?
List the stages of PCR and describe the function or purpose of each.© BrainMass Inc. brainmass.com October 6, 2022, 1:44 pm ad1c9bdddf
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IF you omit the denaturation step 1) the cDNA or DNA that is being amplified remains double stranded and primer sequences won't bing 2) If at least one denaturation step has occurred and is not repeated than the primers will have bound but do not detach to allow further amplification
Specific primer sequences can be designed only to target the specific DNA of a bacterium and not the DNA of other bacteria or the host. A positive PCR band indicates specific bacterium.
PCR may not always be cost effective. For example there are hundreds of thousands of bacterial strains, therefore you would have to use hundreds of thousands of specific primers and PCR reaction, which could get expensive. Culture allows you to narrow the field prior to picking specific primers, or treatment options. Often you are just trying to establish a diagnosis for selection of the most common bacterial forms and treat with an appropriate antibiotic (ie aerobic or anaerobic, rod or sphere, gram positive or gram negative). However, sometimes there is an outbreak particularly in a hospital setting that you may be specifically worried about ie MRSA in which case a specific PCR to confirm or not can be made within a few hours. In some other cases you may want to use less specific primers for PCR to put you in a ball park faster. For example you could design a series of primers that establish aerobic, anaerobic and antibiotic resistance sequences. This allows a simple direct approach for treatment without specifically identifying the microbe. Another reason not to completely remove culture is that PCR is not 100% and in some cases more than 1 strain of bacteria can present so the combination of the techniques may be required.
The vast majority of proteins are denatured and rendered inoperable by heat. The thermas aquaticus (or Taq) enzyme is essentially heat resistant and can survive the high temperatures need for PCR denaturation (Thank Dr. Mullis, inventor of PCR)
Read: http://en.wikipedia.org/wiki/Polymerase_chain_reaction where you will find the following
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.
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, 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 dNTPs 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 per minute. Under optimum conditions, i.e., if there are no limitations due to limiting substrates or reagents, at each extension step, the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific DNA fragment.
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.