Question

You are isolating C. elegant genomic DNA to b used in a subsequent experiment to amplify osm-3 gene by PCR. You add worms to a tube containing lysis buffer and proteinase K, and then use the PCR machine to hold the tube at 65°C for 90 minutes and then at 95°C for 15 minutes. What is the purpose of these two temperature steps?

Answer 1

Polymerase chain reaction (PCR) has a three-step process

These are

• Denaturation: at the 95-degree temperature unwind the double-stranded DNA (ds DNA).
• Annealing: after denaturation DNA become two single strands and at 50-degree temperature primer add at every single strand of the DNA.
• Extension: after annealing, extension occurs at 72-degree temperature and completion of extension each single strand templet converts into dsDNA (one dsDNA converts in two dsDNA in a Single cycle of PCR).

Therefore, in the above experiment, C. elegant DNA at the two temperature 65 degrees and 95 degrees required for annealing and denaturation of the DNA.

• At 65 degree for 90 minutes, osm-3 gene primers added at single-strand DNA. Therefore, osm-3 will amplify.
• After the extension, we hold the DNA at 95 degrees for 15 minutes for denaturation the DNA.

Note: After completion of PCR 30 cycles, conformation test for the gene (osm-3) perform agarose gel electrophoresis to observe the gene (osm-3), band in the agarose gel.