PCR extension is template-directed construction of DNA from an annealed PCR primer by a DNA polymerase. Incoming deoxynucleoside triphosphates supply the new nucleotides. Each addition lengthens the growing strand at its end, so the product grows while the template is read . The direction refers to the nucleic acid backbone, not to left or right on a page.[ref]
Figure 1.
PCR extension follows the paired end on either template orientation
. Original eight-position DNA example with separated complementary templates. Dark rails are existing templates; coloured rails are new strands. The blue PCR primer grows rightward and the green PCR primer grows leftward. Orange marks the material added by DNA polymerase; both arrows point from the original paired end toward the new end. Labels at each end show that both products grow . The templates stop at the displayed boundaries for this teaching example; real starting templates can extend farther. Four-nucleotide PCR primers are deliberately too short to constitute an experimental design. Details.
For a complete worked copying step, start with template and paired PCR primer . The next four template letters are GGAC, encountered in the template's -to- direction. The base pairing rule therefore selects CCTG in order, giving
(1)
The PCR primer supplies four nucleotides, and PCR extension adds four more; this copying event consumes four deoxynucleoside triphosphates. On the oppositely oriented template , the other PCR primer is . It pairs at the right and extends leftward. Reading that template from right to left encounters CTGA after the paired region, so addition of GACT gives . Both finished products are the reverse complements of their respective templates.
The direction argument is short: the PCR primer and its template are antiparallel nucleic acid strands; extension takes place at the PCR primer's end; hence forward progress along the new strand is backward progress along the template. Reflecting a drawing reverses its arrows on the page without changing either chemical direction. This is why a leftward arrow can represent synthesis.
The time needed depends on how much DNA remains to be copied and the chosen DNA polymerase, while temperature must support that enzyme's activity.[ref] Separating PCR annealing and PCR extension into different holds is convenient, but some compatible conditions allow a combined hold. The three chemical jobs therefore need not always require three distinct temperatures.[ref]

Ancestors (6)

  1. PCR thermal cycle
  2. Polymerase chain reaction
  3. Molecular genetics
  4. Genetics
  5. Biology
  6. Home

Incoming links (4)

View article source

Discussion (0) New discussion

There are no discussions about this article yet.

Articles by others on the same topic (0)

There are currently no matching articles.
See all articles in the same topic Create my own version