Biotechnology: Principles and Processes flash cards

1. 1.Define biotechnology (as per EFB).

   The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services. It uses live organisms or their enzymes to make useful products.

   Hint: European Federation of Biotechnology definition.

2. 2.What are the two core techniques that built modern biotechnology?

   (1) Genetic engineering — techniques to alter genetic material (DNA/RNA) and introduce it into host organisms to change the phenotype. (2) Maintenance of sterile (contamination-free) conditions in chemical engineering to grow only the desired microbe/eukaryotic cell for products.

   Hint: One is about DNA, the other about a clean environment.

3. 3.What is genetic engineering?

   Techniques to alter the chemistry of genetic material (DNA and RNA), introduce these into host organisms and thus change the phenotype of the host organism.

   Hint: Altering DNA/RNA and putting it into a host.

4. 4.Why is recombinant DNA (rDNA) technology needed instead of traditional sexual/asexual reproduction?

   In sexual reproduction the whole genome is mixed unpredictably (offspring vary), and asexual reproduction preserves the genome but does not allow variation. rDNA technology lets us introduce ONE specific desired gene into an organism without altering the rest of the genome.

   Hint: Precision — only the desired gene.

5. 5.What is recombinant DNA?

   DNA formed by joining (combining) DNA segments from two different sources — e.g., a foreign gene inserted into a vector/plasmid.

   Hint: 'Recombined' from two sources.

6. 6.Name the three basic steps common to all recombinant DNA technology.

   (1) Identification of DNA with desirable genes. (2) Introduction of the identified DNA into the host. (3) Maintenance of the introduced DNA in the host and transfer of the DNA to its progeny.

   Hint: Identify, introduce, maintain.

7. 7.Who first constructed recombinant DNA, and how?

   Stanley Cohen and Herbert Boyer (1972). They isolated an antibiotic-resistance gene by cutting a piece of DNA from a plasmid (of Salmonella typhimurium) using restriction enzymes, and linked it to a native plasmid of E. coli using DNA ligase.

   Hint: Cohen and Boyer, plasmid + ligase.

8. 8.Name the three key groups of tools required for recombinant DNA technology.

   (1) Restriction enzymes (and other enzymes like polymerases, ligases). (2) Cloning vectors (plasmids, bacteriophages). (3) Host organisms (competent host into which rDNA is introduced).

   Hint: Enzymes, vectors, hosts.

9. 9.What are restriction enzymes and to which broader class do they belong?

   Restriction enzymes are molecular scissors that cut DNA at specific recognition sequences. They belong to a larger class called nucleases.

   Hint: 'Molecular scissors' = nucleases.

10. 10.Distinguish exonucleases from endonucleases.

    Exonucleases remove nucleotides from the ends of the DNA. Endonucleases cut the DNA at specific positions WITHIN the DNA (internal sites).

    Hint: 'Exo' = ends; 'endo' = within.

11. 11.Which type of nuclease is a restriction enzyme, and why is that important?

    A restriction enzyme is an endonuclease — it makes cuts at specific internal positions, so it can cut precisely at a desired recognition sequence rather than nibbling the ends.

    Hint: Cuts within, at specific sites.

12. 12.QUESTION: Which of these is NOT true of restriction endonucleases? (a) They cut within DNA (b) They recognise palindromic sequences (c) They remove nucleotides from ends (d) They produce sticky ends when cutting away from the centre of palindrome.

    (c) They remove nucleotides from ends. That describes EXONUCLEASES. Restriction endonucleases cut at internal specific sites; the other statements are correct.

    Hint: Endo vs exo.

13. 13.How does a restriction endonuclease 'recognise' its cut site?

    It inspects the length of the DNA sequence, and after finding its specific recognition sequence, it binds the DNA and cuts each of the two strands of the double helix at specific points in their sugar-phosphate backbones.

    Hint: Recognises a specific base sequence, then cuts.

14. 14.What was the first restriction endonuclease isolated, and the second widely used one?

    Hind II was the first restriction endonuclease isolated and characterised. EcoRI was another well-known enzyme used widely.

    Hint: 'First' = Hind II.

15. 15.On what basis does Hind II always cut DNA?

    Hind II always cuts DNA molecules at a particular point by recognising a specific sequence of six base pairs (a specific recognition sequence).

    Hint: 6 bp recognition sequence.

16. 16.How many restriction enzymes are known today (approximately), and from where are they isolated?

    More than 900 restriction enzymes have been isolated from over 230 strains of bacteria, each recognising different recognition sequences.

    Hint: >900 enzymes, >230 bacterial strains.

17. 17.Explain the naming convention of restriction enzymes using EcoRI.

    EcoRI: 'E' = genus Escherichia; 'co' = species coli; 'R' = strain (RY13); 'I' (Roman numeral) = the order in which the enzyme was isolated from that strain. The first letter is the genus, next two letters the species.

    Hint: Genus + species + strain + order isolated.

18. 18.What is a palindromic sequence in DNA?

    A sequence of base pairs that reads the same on the two strands when orientation of reading is kept the same (both read 5'→3'). Restriction enzymes recognise palindromic recognition sites.

    Hint: Reads same 5'→3' on both strands.

19. 19.QUESTION: Given the EcoRI site 5'-GAATTC-3' / 3'-CTTAAG-5', explain why this is palindromic.

    Reading the top strand 5'→3' gives GAATTC; reading the bottom strand 5'→3' also gives GAATTC. Because both strands read identically in the 5'→3' direction, it is a palindrome.

    Hint: Read both strands 5'→3'.

20. 20.What are 'sticky ends' (cohesive ends) and how are they produced?

    Restriction enzymes cut the strands of DNA a little away from the centre of the palindrome, but between the same two bases on opposite strands. This leaves single-stranded overhanging stretches called sticky ends. They are 'sticky' because they form hydrogen bonds with complementary cut ends.

    Hint: Overhanging single-stranded stretches.

21. 21.Why are sticky ends so useful in rDNA technology?

    Sticky ends from the same restriction enzyme are complementary, so they base-pair (hydrogen bond) with each other. This makes it easy for DNA ligase to join the two fragments — facilitating the action of the enzyme ligase.

    Hint: They facilitate ligase joining.

22. 22.Why must the SAME restriction enzyme cut both the vector and the source DNA (insert)?

    Using the same enzyme produces the same kind of sticky ends on both the vector and the insert, so their complementary overhangs can base-pair, allowing them to be joined into a recombinant DNA molecule.

    Hint: Matching complementary sticky ends.

23. 23.What is the role of DNA ligase in rDNA technology?

    DNA ligase joins (seals) the cut DNA fragments by forming phosphodiester bonds — it 'glues' the foreign DNA fragment into the vector after their sticky ends have base-paired.

    Hint: Molecular glue; seals the nicks.

24. 24.When source DNA and vector are cut by the same restriction enzyme and mixed with ligase, what products can form?

    The insert can join to the vector to form recombinant DNA, but the vector ends and insert ends can also re-join to themselves. Ligase seals whatever complementary sticky ends come together.

    Hint: Insert + vector, but also self-ligation.

25. 25.What is gel electrophoresis used for?

    It is a technique to separate DNA fragments (which are negatively charged) according to their size by forcing them to move through a gel matrix under an electric field.

    Hint: Separates DNA by size.

26. 26.Toward which electrode does DNA move during electrophoresis, and why?

    DNA moves toward the anode (positive electrode) because DNA is negatively charged (due to phosphate groups).

    Hint: Negative DNA → positive pole.

27. 27.Which matrix is commonly used in gel electrophoresis and what is its source?

    Agarose — a natural polymer extracted from sea weeds. It acts as a molecular sieve.

    Hint: Agarose, from seaweed.

28. 28.How does the size of a DNA fragment affect its migration in agarose gel?

    Smaller fragments move farther (faster) through the sieving agarose matrix; larger fragments move less (slower). So separation is by size.

    Hint: Smaller = farther.

29. 29.How are separated DNA fragments visualised in a gel, and what is the result called?

    The gel is stained with ethidium bromide and exposed to UV radiation; DNA appears as bright orange bands. The separated bands are cut out and extracted — a process called elution.

    Hint: Ethidium bromide + UV → orange bands; elution.

30. 30.What is 'elution' in the context of gel electrophoresis?

    Elution is cutting out the separated DNA bands from the agarose gel and extracting (purifying) the DNA fragments from the gel piece. These purified fragments are then used to construct recombinant DNA.

    Hint: Extracting DNA from the gel band.

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