Biotechnology and Health Care: MDCAT Biology notes

Recombinant DNA technology

Recombinant DNA is DNA containing lengths of nucleotides from two or more different sources, e.g. a human gene inserted into a bacterial plasmid. Its tools are:

Tool	Role
Gene of interest	Isolated from a chromosome, or made from mRNA
Restriction endonuclease ("molecular scissors")	Cuts phosphodiester bonds at a specific base sequence, often leaving sticky ends. Bacteria use these enzymes to cut the DNA of invading viruses. Example: EcoRI from E. coli
DNA ligase ("molecular glue")	Joins DNA fragments by sealing the backbone
Vector	Carries the gene into a host: plasmids, bacteriophages, viruses
Expression system (host)	Usually a bacterium such as E. coli that makes the product

The plasmid pBR322 carries resistance genes for ampicillin and tetracycline, used to select transformed cells. Bacteria are made to take up recombinant plasmids by treatment with calcium chloride ($CaCl_2$).

Complementary DNA

Reverse transcriptase copies mRNA into complementary DNA (cDNA), which lacks introns and can therefore be expressed by bacteria.

Polymerase chain reaction (PCR)

PCR rapidly produces millions of copies of a chosen DNA fragment. Each cycle: denature the DNA at about 95 °C, let short single-stranded DNA primers bind, then extend them with DNA polymerase using a DNA template. The copy number doubles every cycle. The polymerase used is Taq polymerase from Thermus aquaticus, a hot-spring bacterium, because it withstands the high denaturation temperature.

Gel electrophoresis and probes

Gel electrophoresis separates charged molecules (DNA, RNA, proteins) by size; smaller fragments move further through the gel. It is used in DNA fingerprinting. A gene (DNA/RNA) probe is a short single strand of nucleic acid, often made of radioactively labelled nucleotides, that binds (hybridises) to a complementary sequence. Probes are mainly used to diagnose infectious and genetic diseases.

Products for health care

• Insulin (Humulin) made by genetically modified bacteria for diabetics; also growth hormone and interferon.
• Vaccines: the gene for a pathogen's antigen is cloned and the antigen is used as a vaccine. mRNA COVID-19 vaccines instruct our cells to make the viral spike protein. The BCG vaccine against tuberculosis uses a weakened strain of Mycobacterium bovis.
• Transgenic organisms carry a foreign gene, e.g. transgenic mice used to test the safety of polio vaccine. Microinjection inserts a gene directly into an egg.

Monoclonal antibodies

In somatic cell hybridisation, an antibody-producing B lymphocyte (from the spleen of an immunised mouse) is fused with a myeloma (cancer) cell. The hybrid cell (hybridoma) divides indefinitely and its clone makes identical monoclonal antibodies. They are used to detect specific tumour markers and diagnose disease.

Gene therapy

Gene therapy inserts a normal gene into a patient's cells to correct a genetic disorder, using a vector such as a modified virus. In SCID, caused by deficiency of adenosine deaminase (ADA), the enzyme can be given or the gene inserted into blood cells. In cystic fibrosis, liposomes coated with the healthy gene are sprayed into the airways.

Common MDCAT traps

• Hybridomas fuse B lymphocytes with myeloma cells, not with other spleen cells or macrophages.
• Restriction enzymes cut phosphodiester bonds, not hydrogen bonds; ligase joins.
• PCR primers are DNA, not RNA or protein.
• cDNA is made from mRNA by reverse transcriptase.
• Milk is the product most widely pasteurised.

Quick revision

• pBR322: ampicillin and tetracycline resistance.
• Taq polymerase is heat stable.
• Probes detect specific sequences.
• SCID: adenosine deaminase deficiency.
• $CaCl_2$ makes bacteria take up plasmids.