[mage lang=”en|es|fr|en” source=”flickr”]how does telomerase work[/mage]
How does PCR work? How is the DNA bound to the primers replicated?
When Taq polymerase replicates the DNA strands, how is the area of DNA where the primers are bound replicated? In normal DNA replication the area where primers are bound are not repilcated by the main DNA polymerase.
Like the telomeres are cut short because of primers in normal DNA replication & telomerase is needed, how is this same type of thing overcome in a PCR with Taq polymerase??
I think you don’t have the whole pcr picture in you. Here’s how it wokrs and this should contain the answer to your question too.
In pcr everything is controlled by temperature. By giving specific temperatures .. the pcr reaction can be navigated.
First you heat the pcr to like 95 degrees C. 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. So now you have parted the two DNA strands in doble helix.
Next step is the annealing step. At this point you lower the temperature because you want a peice of DNA to bind to your DNA product from the initial 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. 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.
Next step is the elongation or extension step and holds the answer to your question ……. usually the temperature for this is 72 degrees .. but could vary sometimes. So remember you added dNTP mixture in to your pcr??? Those dNTPs will hold the nucleotides necessary to add nucleotides to extend your DNA sequence.
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.
You will do this for like 30 – 35 cycles and then finally you will have a final elongation time.
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 (once again using the dNTPs you added to the mixture).
Final hold: This step at 4-15°C for an indefinite time may be employed for short-term storage of the reaction.
Hope this helps. Good luck.