Biotechnology: Principles and Processes: common doubts

Question 1

What are the two core techniques of modern biotechnology?

Accepted Answer

They are genetic engineering and bioprocess engineering. Genetic engineering alters the genetic material of organisms to change their phenotype, while bioprocess engineering maintains sterile conditions so that only the desired microbe or cell grows in large quantities, as in the manufacture of antibiotics, vaccines and enzymes.

Question 2

Who made the first recombinant DNA and how?

Accepted Answer

Stanley Cohen and Herbert Boyer made the first recombinant DNA in 1972. They isolated an antibiotic resistance gene and linked it to the plasmid of the bacterium Salmonella typhimurium, using restriction enzymes and DNA ligase. When this was transferred into E. coli, the gene was copied in the host.

Question 3

What are the three basic steps in genetically modifying an organism?

Accepted Answer

First, identify the DNA with the desired genes. Second, introduce the identified DNA into the host. Third, maintain the introduced DNA in the host and transfer it to the progeny. Each step needs its own tools, such as restriction enzymes, vectors and competent host cells.

Question 4

What is the difference between exonuclease and endonuclease?

Accepted Answer

Exonucleases remove nucleotides from the ends of DNA, whereas endonucleases cut the DNA at specific positions within it. Restriction enzymes are endonucleases that recognise a particular palindromic sequence. So an exonuclease nibbles from the ends, but an endonuclease cuts in the middle.

Question 5

How is the name EcoRI derived?

Accepted Answer

The first letter E comes from the genus Escherichia, and co from the species coli. The letter R comes from the strain, RY 13, and the Roman numeral I shows that it was the first enzyme isolated from that strain. So R does not stand for restriction.

Question 6

What is a palindromic sequence and what are sticky ends?

Accepted Answer

A palindromic sequence of DNA reads the same in the 5′ to 3′ direction on both strands, such as 5′-GAATTC-3′ with 3′-CTTAAG-5′. A restriction enzyme cuts the strands a little away from the centre, which leaves single-stranded overhangs called sticky ends. They pair easily with matching ends, and DNA ligase joins them.

Question 7

Why does DNA move to the anode in gel electrophoresis?

Accepted Answer

DNA is negatively charged because of its phosphate groups, so it migrates toward the positive electrode (anode) under an electric field. The gel matrix, agarose from seaweeds, sieves the fragments so that smaller ones move farther. DNA is stained with ethidium bromide and seen as orange bands under UV light.

Question 8

What is elution in gel electrophoresis?

Accepted Answer

Elution is the cutting of a stained DNA band out of the agarose gel and extracting the DNA from it. The purified fragments can then be used to construct recombinant DNA by joining with a vector. Only after the bands are visible under UV light can the fragment of interest be selected.

Question 9

What are the features required in a cloning vector?

Accepted Answer

A vector needs an origin of replication (ori), which controls the copy number, and a selectable marker such as an antibiotic resistance gene, which identifies transformants. It also needs a cloning site, ideally just one, where a restriction enzyme cuts. Plasmids and bacteriophages are commonly used vectors.

Question 10

How does insertional inactivation select recombinants in pBR322?

Accepted Answer

Cloning a gene at the BamH I site inactivates the tetracycline resistance gene (tetR). Recombinants therefore still grow on ampicillin but fail to grow on tetracycline, whereas non-recombinants grow on both. This makes the presence of the insert detectable by the loss of one marker.

Question 11

In blue-white selection, which colonies are recombinants?

Accepted Answer

The colourless (white) colonies are recombinants. An insert in the gene for β-galactosidase inactivates the enzyme, so the chromogenic substrate is not converted to a blue product and the colony stays colourless. Blue colonies have an intact enzyme gene and so contain no insert.

Question 12

How is DNA introduced into bacteria and into plant or animal cells?

Accepted Answer

For bacteria, cells are made competent with a divalent cation such as calcium, then incubated on ice with DNA, given a heat shock at 42 °C and returned to ice. In animal cells DNA is micro-injected into the nucleus. In plants, gold or tungsten particles carrying DNA are fired into cells by biolistics (gene gun).

Question 13

Which natural vectors are used for plants and animals?

Accepted Answer

The Ti plasmid of Agrobacterium tumefaciens is used for plants, since the bacterium naturally transfers its T-DNA into plant cells; it is first disarmed so that it does not cause tumours. For animals, disarmed retroviruses are used to deliver the desired genes into cells.

Question 14

Which enzymes are used to break open different cells for DNA isolation?

Accepted Answer

Lysozyme is used for bacterial cells, cellulase for plant cells and chitinase for fungal cells, because each cell wall has a different chemical makeup. Ribonuclease is added to remove RNA, and protease to remove proteins. Finally chilled ethanol precipitates the DNA as fine threads, which are spooled out.

Question 15

What are the three steps of PCR?

Accepted Answer

Denaturation separates the DNA strands by heating, annealing lets primers bind to the complementary regions on each strand, and extension lets a DNA polymerase extend the primers using the nucleotides. Repeating the cycle amplifies the target region many times, up to about a billion copies.

Question 16

Why is Taq polymerase used in PCR?

Accepted Answer

Taq polymerase is stable at the high temperature of denaturation, so it is not destroyed in every cycle. It is isolated from the thermophilic bacterium Thermus aquaticus. An ordinary DNA polymerase would be inactivated by the heating step and would have to be added again each time.

Question 17

Is it enough to put a foreign gene into a host to get the protein?

Accepted Answer

No. The host must also keep the gene and read it, so the gene needs to sit under a suitable promoter and the cells need the right conditions. Small-scale cultures in flasks come first, then the process is scaled up in a bioreactor. Only when the protein is actually expressed is the work useful, and it must still be purified.

Question 18

What is a bioreactor and why is it used?

Accepted Answer

A bioreactor is a vessel in which raw materials are converted into specific products by microbes, plant or animal cells, or their enzymes. Large vessels of 100 to 1,000 litres make production on a big scale possible, while controls keep temperature, pH, oxygen, substrate and stirring at their best values. Stirred-tank designs, including sparged ones that bubble air through the culture, are the commonest.

Question 19

What is downstream processing?

Accepted Answer

Downstream processing consists of the separation and purification steps that follow the biosynthetic stage, carried out before a product is marketed. The product is then formulated with suitable preservatives, and drugs undergo clinical trials as well as strict quality control. It comes after the growth stage and not before.

Question 20

Why are restriction enzymes called molecular scissors and DNA ligase molecular glue?

Accepted Answer

Restriction enzymes cut DNA at specific recognition sites and ligase joins the cut pieces, so one cuts and the other pastes. Together they allow a gene to be cut from one DNA and joined to a vector. Because of these actions, they are the two most important enzymes in gene cloning.

Biotechnology: Principles and Processes: common doubts, answered

About the chapter

Why are restriction enzymes called molecular scissors and DNA ligase molecular glue?

What biotechnology is