Lessons 30–31 · 2 lessons · A. G‘afurov, A. Abdukarimov, J. Tolipova, O. Ishankulov, M. Umaraliyeva, I. Abdurahmonova. Biology, Grade 10, 1st edition. “Sharq” publishing and printing joint-stock company, Tashkent, 2017
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Enzymes used in genetic engineering
Textbook: pp. 131–133
GoalExplain the functions of the enzymes of genetic engineering – polymerase, reverse transcriptase (revertase), ligase and restriction enzymes.
Genetic engineering uses enzymes that cut, join and copy DNA. DNA polymerase (isolated from E. coli by A. Kornberg in the 1950s) builds a new DNA strand by adding complementary nucleotides. Revertase (reverse transcriptase) – an enzyme of some RNA viruses – makes complementary DNA (cDNA) on an RNA template; this is how a gene copy is made from mRNA. DNA ligase sews DNA pieces together by forming phosphodiester bonds between neighbouring nucleotides. Restriction enzymes (restriction endonucleases) find a specific sequence of 4–6 nucleotides they recognise – the restriction site – and cut DNA there. In bacteria they protect against foreign DNA (for example viral DNA). Restriction enzymes make sticky-ended (EcoRI) or blunt-ended (HpaI) fragments; sticky ends are complementary, so different DNA pieces join easily. Enzyme names come from the bacterium they were isolated from: EcoRI – Escherichia coli. Cut fragments are separated by length with electrophoresis.
Worked examples
EcoRI recognises the site G↓AATTC. If this sequence occurs in 3 places, a linear DNA is cut 3 times into 4 pieces; a circular plasmid with 1 site is cut once and opens into one linear piece.
In electrophoresis DNA, being negatively charged, moves towards the positive pole; small fragments move faster and large ones slower, so fragments separate by length in the gel.
Class activity
“Enzymes as tools”: cards with enzyme names and functions (scissors, glue, copier) are given mixed up and matched.
Practice
1
Distinguish the functions of restriction enzyme and ligase.
A restriction enzyme cuts DNA at a specific site; ligase joins DNA fragments.
2
A linear DNA has 5 restriction sites. How many fragments result after cutting? (Number only.)
6
3
What does reverse transcriptase do and why is it needed?
It synthesises DNA on an RNA template; used to obtain a gene copy (cDNA) from mRNA, e.g. in making the insulin gene.
4
Why do sticky ends make joining DNA fragments easier?
They are complementary, so the nitrogen bases pair by hydrogen bonds and ligase seals the rest.