Question

For plant physiology..

The shy2-2 mutation changes the “CCA" which encodes for a proline to a "TCA" which encodes for a serine, this is what causes a change in the conformation of the IAA3/SHY2 protein that leads to the changes in the phenotype that you observed. WT tggccacca accggtggt shy2-2 tagtcassa accagtaat • We are also using control primers that amplify a portion of the ACTIN gene, which remains unaltered in the three genotypes. Tube # DNA Template WT (Ler) WT (Ler) Shy2 Shy2 Primers 3CF/3CDR ACT1F/ACTIR 3CF/3CDR ACT1F/ACTIR 3CF/3CDR ACT1F/ACTIR F2 Tube #5 Tube # 1 12.5ul lul 3CF/3CDR Tube #3 12.5ul lul 3CF/3CDR 12.5ul lul 3CF/3CDR Component Master Mix (MM) Primer Mix Forward/Reverse) DNA Template H20 Total Volume Tube #2 12.5ul lul ACTIF/ACTIR lul (Ler) 10.5 25ul Tube #4 12.5ul lul ACT1F/ACTIR lul (Shy2) 10.5 12.5ul lul ACTIF/ACTIR lul (F2) 10.5 25ul lul (Ler) 10.5 25ul lul (Shy2) 10.5 25ul lul (F2) 10.5 25ul • In the above, why do we add primer DNA and template DNA?

Answer 1

Ans:- The primers DNA and template DNA was added due to reasons mentioned below

Reason for adding Primer DNA

Primers are the small stretch of nuclotides that binds to the complementary sequence present in the DNA template. The primers are required in above reaction because DNA polymerase enzymes cannot amplify the DNA or make copy of DNA untill the free 3' OH group is provided by the primers. So, DNA polymerase start synthesizing the DNA by adding first nucleotides at 3' OH group and strat polymerization steps. That's why it's is extremely essential that primers are always required where you want to make copies of DNA.

Reason for adding template DNA

DNA polymerase need the template from where it can copy the sequence and makes a lot of copies of DNA. So, if DNA template is absent the DNA polymerase won't work. So, for every PCR reaction it is important to add DNA template, where DNA primers can binds and subsequently the DNA polymerase can make copy of DNA.

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