1 The enzyme PspN4I cleaves the sequence GGNNCC N any of the

1. The enzyme PspN4I cleaves the sequence GG/NNCC (N= any of the four bases). How many times would you expect PspN4I to digest a 30 kb genome? Round off to the nearest whole number.

2. Sequence A, which contains two BstBI sites (TT/CGAA), was digested with BstBI. The resulting fragment was then ligated into the unique TaqI restriction site (T/CGA) within vector B.

Sequence A: CAG TT/CGAA TTC • • • • • • • • • • • • • • • • GGC TT/CGAA AAG Vector B: TGG T/CGA CAC

Which enzyme (s) could be used to release the cloned DNA fragment from the recombinant vector B? BstBI, TaqI , both, or neither.

PLEASE ANSWER BOTH

Solution

1. The enzyme PspN4I cleaves the sequence GG/NNCC (N= any of the four bases). How many times would you expect PspN4I to digest a 30 kb genome? Round off to the nearest whole number.

A : In a completely randomized DNA sequence, with 25 % A\'s, 25 % C\'s, 25 % G\'s, and 25 % T\'s we can predict the avalablity of the restiction site usng the probablity.

In case of PspN4I the restiction site is a recognized by 6 bases(GGNNCC), but you can see the probality is 1/1 for N and 1/4 for other bases. hene we calculate the frequency of cutting to be (1/4)^4 = 0.0039. This means to say that we can find a restiction site for 1/0.0039 = 256.41 bases.

For a 30 kb genome we can find 30000/256.41 = 117 restiction sites of PspN4I.

2. Sequence A, which contains two BstBI sites (TT/CGAA), was digested with BstBI. The resulting fragment was then ligated into the unique TaqI restriction site (T/CGA) within vector B.

Sequence A: CAG TT/CGAA TTC • • • • • • • • • • • • • • • • GGC TT/CGAA AAG Vector B: TGG T/CGA CAC

Which enzyme (s) could be used to release the cloned DNA fragment from the recombinant vector B? BstBI, TaqI , both, or neither.

A: After the ligation of the cut fragment we can have a construct with following sequence

TGG T*CGAA TTC • • • • • • • • • • • • • • • • GGC TT*CGA CAC

The * in sequence is the point of ligation. You can see from the ligated product the site off TaqI is conserved and hence it can be released with TaqI.

However, the vector does not contain the match for 1st and 6th base of BstBI and cannot be used to release the insert.