Reality → Life → Genetics → Biotech
Mendel derived three principles from his experiments:
The fruit fly is a famed early model organism for genetic research: easy to breed, with a short life cycle, and salivary gland chromosomes about 100 times larger than normal chromosomes. In thousands of lab experiments with millions of larvae bred, Morgan and his students studied the linkage of genes (i.e., their occurrence on the same chromosome). By exploiting naturally occurring crossovers and the rule that the frequency of segregation of linked genes increases with the distance between them, they could map gene locations on a chromosome .
The Avery-MacLeod-McCarty experiment of 1944 showed that lab-purified DNA causes genetic changes; 8 years later, the Hershey-Chase experiment confirmed DNA's genetic role by showing that viral DNA replicates when injected into bacteria.
Watson and Crick's groundbreaking brief paper presented an elegant solution to the structural problem: the double helix. They found the solution based on study of previous work by other researchers, in particular Franklin's X-ray work, and experimentation with a physical 3D-model.
Recombinant DNA is produced by 'cutting' a DNA sequence with restriction enzymes and 'pasting' a cut-out fragment into another DNA strand with 'gluing' enzymes (ligases). The enzymes act at specific locations of nucleotide sequences across all species and synthetic DNA. In molecular cloning, recombinant DNA in the form of a plasmid is introduced into bacterial cells for multiplication ('copying').
Genetic engineering usually comprises the insertion of lab-produced recombinant DNA into a host's genome. The work involves DNA sequencing and the use of bioinformatics. Commercial applications include the production of drugs and hormones (e.g., insulin) with genetically modified bacteria. New tools and applications may materialize from synthetic biology (2016 Nobel Prize in Chemistry). New and more efficient enzymes for various applications are possible (2018 Nobel Prize in Chemistry). Greatly increased precision and efficiency in gene editing became possible with CRISPR sequences, Cas9 enzymes, and customized guide RNA (2020 Nobel Prize in Chemistry, see also videos from MIT and Zeit).
There is continuing debate about the long-term health impact of genetically modified foods and about the environmental impacts of growing their crops. Public resistance to genetically modified animals and their products is particularly strong and has prevented their entering into the market. Ethical concerns led to restrictions on cloning of animals and humans.
In the Sanger sequencing method, a DNA sample consisting of many copies of a single-stranded DNA segment is in vitro combined with a mixture of the four standard nucleotides (A, C, G, T) and a dideoxynucleotide (a nucleotide modified by removal of a second OH group). If, for instance, the dideoxynucleotide's base is A, then, with the addition of a catalytic enzyme (DNA polymerase) and a primer, replications take place that terminate whenever the dideoxynucleotide encounters the complementary nucleobase T, because the missing OH group prevents bonding. In this way, many short strands of different lengths are created through replication. They can be sorted in a gel exposed to an electric field (electrophoresis): the shortest pieces move fastest and travel the longest distance, whereas the longest pieces move slowest and travel the shortest distance, thus indicating the sequence of all T nucleobases in the DNA sample. Repetition of the process with dideoxynucleotide bases C, G, and T produces a picture of the sample's sequence of all nucleobases. The method has been automated by adding all four types of didenucleotides at once, together with four different fluorescent dyes, each reacting with a specific nucleotide type and emitting light of a different wavelength.
The polymerase chain reaction (PCR) produces thousands to millions of copies of a DNA segment through thermal cycling of a reaction mixture. Primers (short sequences, typically 20 base pairs long) that are complementary to starting and ending points bracket the DNA sequence targeted for amplification. At a temperature of 90°C, double-stranded DNA unwinds and the selected segment replicates under the influence of a heat-tolerant DNA polymerase. When cooled down, complementary single strands join together to form pieces of a double helix. With each repetition of the thermal cycle, the number of double helix pieces is doubled. PCR machines with automated thermal cycling complete billion-fold DNA amplification in a few hours. A key to the PCR technology was the isolation of a heat-tolerant DNA polymerase from thermophile bacteria living in the hot springs of the Yellowstone National Park.
While it took 13 years and an expense of $3 billion for completion of the Human Genome Project in 2001, only 6 years later sequencer machines could complete a human genome in days at a cost of $1 million, and in 2014 the cost allegedly approached $1,000.
Computer models simulate potential chemical reactions and pathways involving biomacromolecules, based on inputs from experiments and the application of classical and quantum physics (see 2013 Nobel Prize in Chemistry).
DNA microarrays allow the identification of thousands of DNA sequences simultaneously. Microscopic probes of synthesized oligonucleotides (assembled according to the DNA sections to be examined) are bonded to small glass plates in an array pattern (the technology was inspired by computer chip manufacturing). When a lab sample of isolated and prepared targets of DNA or RNA sequences is dropped onto the plate, their base pairs will bond to the complementary base pairs of the probes. A laser scanner activates fluorescence of various chemical markers for specific sequences of the sample. Capture of the light signals and computerized analysis render a picture of the relative abundance of the fluorescing sequences.