Question

6. Why can we combine EcoR1 and Pstl in the same reaction? Why? Could we do a double digest with any two enzymes? Why or why not?

Answer 1

Q.6. Answer-

1. For cloning experiments, double digest of DNA is carried out to avoid self ligation of DNA. So that for double digest restriction enzymes that has unique restriction sites are used to create cohesive ends with different sequence.

2. Here EcoRI has 5' -G*ATC -3' recognition sequence and PstI has 5'- CTGCA*G (* = cut site). EcoRI produces 5' overhang and PstI produces 3' overhang. As this two enzymes produce different cohesive ends after cleavage so that these two enzymes can be used in same reaction.

- ECORI 5? — он аяттс GAATTC- -CTTAAG — -GTTAAO overhang HOG Psti (TGCAG- GACGT C- Ф. — Стся он y - 31_ overhang - GO HOAC GTC

3. The restriction enzyme that recognizes different palindrome sequence and cut at its specific site so that we can use such enzymes together in double digest but some enzymes recognizes same sequence and cleave at same site are known as isoschizomers or cut at different positions in that sequence are known as neoschizomers. Such enzymes cannot use in double digest reactions.

SacI GAGCTC Sst I GAGCTC Isochizomers Smal ccc "GGG XmaI e* CC GGG Neoschizomers