Genetic Technology

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Cambridge International AS & A Level Biology (9700)

Genetic Technology

The big idea: Enzymes that cut, copy and join DNA let us move a gene into another organism and have it expressed. The same tools copy DNA by PCR, sort it by size on a gel and read whole genomes, which puts genetic technology to work in medicine and farming.

Summary — What This Chapter Covers

Recombinant DNA, genetic engineering and three sources of a gene.
Restriction endonucleases, DNA ligase, plasmids, DNA polymerase and reverse transcriptase; promoters; fluorescent marker genes; gene editing.
PCR with Taq polymerase; gel electrophoresis; microarrays; sequence databases.
Recombinant human insulin, factor VIII and adenosine deaminase; genetic screening; gene therapy for SCID and inherited eye diseases; ethics.
GM salmon, herbicide-resistant soybean, insect-resistant cotton; ethics of GMOs in food production.

1. Principles of Genetic Technology

19.1

Recombinant DNA and genetic engineering

Recombinant DNA is DNA made by joining together pieces of DNA from two or more different sources, usually from different species.

Genetic engineering is the deliberate manipulation of genetic material to modify specific characteristics of an organism. This may involve transferring a gene into an organism so that the gene is expressed (transcribed and translated into its protein). The organism that receives the gene is a genetically modified organism (GMO) or transgenic organism.

Where the gene comes from

Extracted from the DNA of a donor organism: cut out with restriction enzymes. Eukaryotic genes contain introns, which bacteria cannot remove.
Synthesised from the mRNA of a donor organism: mRNA is extracted from cells that are making the protein (so it is plentiful), and reverse transcriptase makes complementary DNA (cDNA). The cDNA has no introns, so bacteria can express it.
Synthesised chemically from nucleotides: using a known base sequence (e.g. from a database, or worked out from the amino acid sequence); a machine joins nucleotides in the right order.

The tools for transferring a gene

Tool	Role
Restriction endonucleases	Enzymes (from bacteria) that cut DNA at specific base sequences called restriction sites, often palindromic, e.g. EcoRI cuts G↓AATTC. Many make staggered cuts, leaving short single-stranded sticky ends. Cutting the gene and the plasmid with the same enzyme gives complementary sticky ends that can pair by hydrogen bonding
DNA ligase	Joins the sugar-phosphate backbones of the gene and plasmid by forming phosphodiester bonds, sealing the recombinant DNA
Plasmids	Small circular DNA molecules in bacteria, used as vectors to carry the gene into a host cell. They replicate independently in the host, so the gene is copied when bacteria divide. Bacteria take them up when treated with calcium ions and a heat shock (or electroporation)
DNA polymerase	Builds a new DNA strand complementary to a template: used to make cDNA double-stranded and to copy DNA in PCR
Reverse transcriptase	Enzyme (from retroviruses) that makes single-stranded cDNA using mRNA as a template

Why a promoter may also be needed

promoter

Marker genes coding for fluorescent products

marker gene GFP expressed

Gene editing

Gene editing insertion, deletion or replacement specific sites

Microarrays

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Also in the full note

The polymerase chain reaction (PCR)
Gel electrophoresis
Sequence databases
Recombinant human proteins
Genetic screening
Gene therapy
Social and ethical considerations
Meeting the global demand for food

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Genetic Technology