Unit 20: Biotechnology — Long Questions

Components (tools) of recombinant DNA technology:
1. Gene of interest — the gene to be manipulated, obtained by direct synthesis, reverse transcription of mRNA, or cleavage from the chromosome using restriction endonucleases.
2. Restriction endonucleases — molecular scissors that cut DNA at specific palindromic restriction sites, producing sticky ends.
3. Vectors — molecular carriers (plasmids, bacteriophages, cosmids, BACs, YACs, PACs, animal viruses) that carry the gene of interest into host cells.
4. DNA ligase — molecular glue that joins DNA fragments by forming phosphodiester linkages.
5. Expression system — a suitable host (usually bacteria, because of short generation time and simple genetic system) that takes up and expresses the recombinant DNA.

Steps of recombinant DNA technology:
1. Formation of recombinant vector: (i) DNA cleavage — the source DNA and vector DNA are cut with the same restriction endonuclease, producing compatible sticky ends; (ii) production of recombinant DNA — the gene of interest and cleaved vector are incubated with DNA ligase, which joins them into a recombinant vector.
2. Cloning of recombinant vector: the recombinant DNA and the expression system (bacteria) are incubated together; the bacteria take up the recombinant DNA and, as they multiply, form clones each carrying identical copies of the gene of interest.
3. Screening of clone library: (i) selecting for the vector by antibiotic resistance — bacteria are grown on a medium containing an antibiotic (e.g., ampicillin) so only cells with the plasmid survive; (ii) selecting for the gene of interest by blue-white screening — insertion of the gene disrupts the lacZ' gene, so recombinant colonies stay white while non-recombinant ones turn blue; (iii) identifying the specific gene by DNA hybridization — a radioactive probe complementary to the gene of interest hybridizes only with colonies containing it, located by autoradiography.

Restriction endonucleases are enzymes that cut specific nucleotide sequences in DNA; hence they are called molecular scissors. There are many kinds of restriction endonuclease, and each kind recognizes a specific sequence of nucleotides in DNA, called the restriction site or recognition region. The enzyme binds to the DNA at this sequence and cuts the DNA at a particular place within the site.

Restriction sites have palindromic sequences of four to eight nucleotides, meaning the nucleotides at one end of the site are complementary to those at the other end — the two DNA strands carry the same sequence running in opposite directions at that site. The restriction endonuclease binds to and cleaves both strands. After cleavage, both resulting DNA fragments have single-stranded, sticky ends (a few nucleotides long) that are complementary to each other.

These enzymes are especially valuable because any two DNA fragments cut by the same restriction endonuclease have matching sticky ends and so can be joined together with DNA ligase. This property is exploited in recombinant DNA technology: the source DNA (to isolate the gene of interest) and the vector DNA are cut with the same restriction endonuclease, producing compatible sticky ends that allow the gene of interest to be inserted into the vector.

Polymerase chain reaction (PCR) is a technique by which multiple identical copies of a gene of interest are made using the enzyme DNA polymerase, which repeatedly copies a given piece of DNA. PCR was developed by Kary Mullis in 1983, who won the Nobel Prize in Chemistry in 1993.

Steps of PCR 1. Denaturation: an excess of primer (20–30 nucleotide sequence) is mixed with the DNA fragment and heated to about 98°C, which breaks the double-stranded DNA into single strands that act as templates.
2. Annealing of primer: the mixture is cooled to about 60°C, allowing the forward and backward primers to hybridize to complementary regions of the single-stranded DNA.
3. Extension of primer: heat-stable Taq polymerase (isolated from Thermus aquaticus) and all four nucleotides are added; at the optimum temperature of 72°C, Taq polymerase adds nucleotides to the 3' end of the primer, synthesizing a complementary copy of the rest of the fragment, giving two copies of the original DNA.
4. Repeating the cycle: steps 1–3 are repeated without adding more polymerase (it is heat-stable); each cycle (1–2 minutes) doubles the number of DNA molecules, so after 20 cycles a single DNA fragment yields more than one million copies.

Applications of PCR diagnosing infectious agents; detecting genetic mutations responsible for genetic diseases and cancers; preparing DNA fingerprints in criminal investigations from tiny samples of blood or hair; detecting genetic defects in early embryos from a few sloughed-off cells; and examining DNA of extinct species. PCR is also used to build genomic libraries for DNA sequencing.

A gene is transferred into a target cell through its cell membrane or cell wall without destroying the cell, using one of the following common techniques:
(a) Electroporation: pores are created in the cell wall/membrane of the target cell using an electrical current; these pores allow the gene to enter, and close again once the current is discharged.
(b) Microinjection: a fine needle is used to directly inject the gene into the target cell's nucleus.
(c) Biolistic transformation: the gene of interest, coated with heavy metal ions, is fired into the target cell by mechanical force (e.g., a gene gun).
(d) Protoplast-mediated technique: the cell wall and membrane are removed from the target cell to form a protoplast, which is then mixed with a medium containing the gene of interest.
(e) Chemical methods: naked DNA is fused with a chemical agent; the DNA–chemical complex enters the cell cytoplasm by diffusion or endocytosis.
(f) Virus-mediated gene transfer: viruses such as adenovirus, adeno-associated virus, HIV, lentivirus and other retroviruses are used to carry and deliver the gene into the host cell.
In the specific case of producing human insulin, recombinant plasmids carrying the human insulin gene are introduced into Escherichia coli by transformation, after which the bacteria multiply and express the human gene to produce the insulin polypeptide chains.