Basic Molecular Biology: PCR and Real-Time PCR – Principle of PCR — Transcript

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1. 0:01[Music]
2. 0:10polymerase chain reaction or pcr
3. 0:14is a technique for amplifying specific
4. 0:17dna fragments from a dna template
5. 0:20pcr happens in three basic steps
6. 0:24denaturation annealing and extension
7. 0:28denaturation is the first step of pcr in
8. 0:31this step heat is applied to separate
9. 0:34double-stranded dna into two single
10. 0:37strands following the denaturation step
11. 0:40is the annealing step the temperature is
12. 0:43decreased so that the primers can anneal
13. 0:45to the complementary sequences on the
14. 0:48dna templates
15. 0:50the temperature of the annealing step
16. 0:52depends on the melting temperature of
17. 0:54the primer selection of the annealing
18. 0:57temperature is critical typically the
19. 1:00optimum annealing temperature is 3 to 5
20. 1:03degrees celsius below the melting
21. 1:05temperature too high of an annealing
22. 1:08temperature prevents optimal binding of
23. 1:10the primers to the templates while a
24. 1:13temperature that is too low can lead to
25. 1:15non-specific binding and subsequently
26. 1:18non-specific pcr products extension
27. 1:21occurs after the primers hybridize to
28. 1:24the templates the temperature of this
29. 1:26step is adjusted to the optimum
30. 1:29temperature where the dna polymerase
31. 1:31functions best in extension
32. 1:34dna polymerase adds nucleotides to the
33. 1:37three prime ends of the primers that sit
34. 1:40on templates following the base pair
35. 1:42complementarity rule the length of the
36. 1:44extension steps depends on the extension
37. 1:47rate of the polymerase used and the
38. 1:50length of the desired pcr product these
39. 1:53three steps comprise one pcr cycle the
40. 1:57cycle is repeated multiple times
41. 1:59allowing the generation of a large
42. 2:02number of copies of a specific dna
43. 2:04fragment
44. 2:08[Music]
45. 2:22you