Question

When plasmid pD21 is digested with the following enzymes, these bands are produced (all in kb):

HinIII : 3.82, 0.18

SaII : 2.35, 1.65

EcoRV : 3.0, 1.0

HindIII + SaII : 2.35, 1.20, 0.27, 0.18

HindIII + EcoRV : 1.87, 1.00, 0.95, 0.18

SaII + EcoRV : 1.60, 1.40, 0.75, 0.25

Draw a restriction map based on the fragments that were produced.

Answer 1

As plasmid pD21 is circular, number of restriction sites for each enzyme will be 2.

First we compare the digestion of HindIII with HindIII + SalI. We find that, 0.18 kb fragment due to digestion with only HindIII & 2.35 kb fragment due to digestion with only SalI stay intact when digestion with HindIII + SalI. Based on this data initial restriction map is drawn.

Next we compare the data of SalI with SalI + EcoRV. Only possibility is that 2.35 kb fragment due to digestion with only SalI is divided into two fragments of 1.60 kb & 0.75 kb when digested with SalI + EcoRV. Also, 1.65 kb fragment due to digestion with only SalI is divided into two fragments of 1.40 kb & 0.25 kb when digested with SalI + EcoRV. Now, to find the order of this fragments we compare the data with digestion of HindIII + EcoRV. As one 1.60 kb fragment is generated with digestion of SalI + EcoRV & one 1.87 kb fragment is generated with digestion of HindIII+ EcoRV, These two fragments must present side by side. Also, 0.18 kb fragment due to digestion with only HindIII stay intact when digestion with HindIII + EcoRV. Further, we can find a 1 kb fragment when digested with only EcoRV. Considering all of this final restriction map is shown in below.