The Tool Kit of Genetic Engineering: Quick Revision

A brief note on The Tool Kit of Genetic Engineering. Read it before a test. Try each question at the end before you open its answer.

The big picture

• Genetic engineering: change the genetic material and put it into a host.
• Bioprocess engineering: keep the culture sterile and grow only the wanted cells, in large amounts.
• Three basic steps: identify the DNA with the wanted gene, introduce it into a host, maintain it in the host and its offspring.
• Tool kit: restriction enzymes, polymerases, ligases, vectors and a host.

Restriction enzymes

• Endonuclease cuts inside the DNA. Exonuclease removes nucleotides from the ends.
• Recognition sequence: a palindrome, the same on the two strands when both are read from 5 prime to 3 prime.
• EcoRI: GAATTC, cut between G and A, sticky end AATT. The name: Escherichia coli, strain R, first enzyme.
• First one isolated: Hind II. The count in NCERT: over 900 enzymes from over 230 strains of bacteria.
• Sites by chance: a site of n bases appears once in about $4^n$ bases.
• Fragments: straight DNA with k sites gives k + 1. Circular DNA with k sites gives k.

Joining and carrying

• DNA ligase: seals the sugar-phosphate backbone. It makes the joint permanent.
• Vector: origin of replication (sets the copy number), selectable marker (ampR, tetR, chlR, kanR), few cloning sites, best of all one.
• pBR322: p plasmid, B and R for Bolivar and Rodriguez, 322 the number in their laboratory. It has ori, ampR, tetR, rop and cloning sites.
• Host: must be made competent to take up the DNA.

Practice with new numbers

A program to try

The program tests a list of enzymes. It tells which sites are palindromes and how often each should cut.

Mistakes to avoid

• Mixing up ligase and the restriction enzyme. The enzyme cuts. Ligase joins.
• Using k + 1 for a plasmid. A circle with k cuts gives k pieces.
• Reading the number in pBR322 as its size. It is a serial number.
• Choosing a vector with many cloning sites. Few sites, best of all one.

Check yourself

1. Which enzyme removes nucleotides from the ends of DNA?

2. A circular plasmid has three sites for an enzyme. How many fragments does it give?

3. Which part of a vector shows which cells took it up?

4. What does the I in EcoRI stand for?

Sources

• NCERT (2023). Biology, Textbook for Class XII, chapter 9.
• Clark M. A., Douglas M. and Choi J. (2018). Biology 2e, OpenStax, chapter 17.