DNA

Highway to Nobel

A chemical reaction changed biotech forever and it was discovered on a California road, E&T reports.

An "elegantly simple" technique, as she describes it, is helping Professor Connie Mulligan of the University of Florida track down the answers to a question that has puzzled anthropologists for decades: how did humans leave the Horn of Africa region more than 40,000 years ago and ultimately colonise the world? The initial emigration from Africa shaped how humans settled into the populations that exist today, but there is little evidence of how it happened.

Mulligan's approach is to use the evidence locked up in our own bodies. She is analysing the DNA in the human genome to track mutations. By comparing mutations from populations in the Horn of Africa to mutations in those just outside Africa, such as the Arabian peninsula, she can make inferences about the age of those mutations, and implications for the migration history of the people.

Using the data acquired, it is possible to determine how long ago those populations split from each other by calculating how many mutations have occurred since their divergence and how many single mutations occur in the sample.

One of the most important tools in Mulligan's work is the polymerase chain reaction (PCR). Invented more than a quarter of a century ago, the PCR has come to permeate biological research, from Mulligan's anthropological detective work through to new technologies such as synthetic biology.

DNA amplification

PCR allows the creation of billions of copies from a single DNA template resulting in "a billion similar molecules in an afternoon. The reaction is easy to execute. It requires no more than a test tube, a few simple reagents, and a source of heat," wrote the inventor of the technique, biochemist and chemistry Nobel Prize winner Kary Mullis in his 1990 article on the technology for Scientific American.

Although scientists working in the early 1980s wanted something better than cloning and were working on alternatives, Mullis stumbled upon the concept that soon transformed genetics.

While driving on California Highway 128 one night, Mullis pondered ways to analyse specific sections of DNA in the human genome. He contemplated how to inspect a specific human DNA sequence using a short stretch of DNA - an oligonucleotide marker - to locate single mutations without having to clone the DNA first. One possible application for this was DNA testing of a foetus to determine if it had a genetic disease with enough time to be able to abort it, if the parents chose.

Before PCR, scientists were required to clone DNA to generate enough genetic material to analyse. This is a laborious process that involves coaxing sections of DNA into bacteria that then helpfully created copies of the DNA as they made more of themselves.

"If you wanted to examine a human DNA sequence closely, you had to clone it. Chop up the DNA into pieces of several thousand base pairs, isolate each of those by growing them in a particular bacterial colony, figure out which colony contained your favourite piece, pick it off a plate and grow it up. That was the magic of cloning," wrote Mullis in a manuscript called 'Night on Highway 128'.

However, this process of cloning took months to complete and was "fuzzy at best", Mullis says. That night, his contemplation of ways to detect these mutations faster and get rid of imposing nucleotides that would interfere with DNA amplification led him to develop the method now known as PCR.

Mullis wrote: "I could design the [oligonucleotides] some distance from each other. After three cycles they would make a double stranded molecule corresponding exactly to the DNA template between them, and that would double in concentration every subsequent cycle... Thirty cycles would be somewhere around a billion [molecules]."

He recalls that, at first, "no one seemed interested because no one believed something that simple could work." However, soon the idea caught the eyes of the company for which he worked, Cetus, and then the eyes of the Chemistry Nobel Prize committee. In 1993, ten years after the journey on Highway 128, he was awarded the prize.

Multiplication

Not only did the DNA strands multiply exponentially and quickly, but so did the applications for the technique.

Researchers are using this type of DNA manipulation to study human evolution faster and more effectively than ever before. One of many projects that are currently ongoing to study human evolution is the Genographic Project, a five-year research, carried out by Spencer Wells, National Geographic's explorer in residence, and his team of field researchers and scientists.

Part of the project is a Genographic Project Public Participation Kit. Participants are able to buy the kit and send off their own DNA sample to be added to the project's database. The website claims that, "with a simple and painless cheek swab", any user can not only "reveal your deep ancestry along a single line of direct descent" but also "place you on a particular branch of the human family tree." The site claims this is anonymous and that all results will be placed in the public domain. The project was funded by the National Geographic Society, computer technology company IBM, and the scientific and historical and funding organisation Waitt Family Foundation.

A key to the project was getting samples not from intermixed populations but specifically from unadmixed, indigenous tribes, which the project geneticists believe are a link between ancient and current migration patterns. On the other hand, the project has encountered opposition from sceptics and certain native groups such as some North American tribes, who refuse to participate in the study. They are of the opinion that the project's findings about their genetic history will disturb and even threaten some natives' land, legal rights, identity, and cultural beliefs.

The International Hapmap project

Another initiative that would not have been possible without PCR is the International HapMap Project, an ongoing effort between ten countries. Its name comes from its purpose, to map out blocks of human genome haplotypes, or segments of associated sequences, in order to quickly assess each individual's unique genetic variation without having to sequence their entire genome.

According to a December 2003 Nature article, the HapMap Project "will allow the discovery of sequence variants that affect common disease, will facilitate development of diagnostic tools, and will enhance our ability to choose targets for therapeutic intervention." The Nature article also claims that the "project is committed to…ensuring that project data remain freely available in the public domain at no cost to users." Since 2003, the official HapMap website has been maintaining an updated list of data downloadable for any interested user, and updates on HapMap collaborations, policies, and research findings.

The Human Genome Project was another worldwide effort to map out the human genome. What sets this project apart from the others, though, is that one of its specific goals was to enhance the genetic and biomedical industries and technologies. The US Department of Energy Genome Program and the US National Institute of Health sponsored the 13-year effort for 18 countries to study roughly 20,000 human DNA genes. Mulligan says that it is the only large-scale science project she knows of to come in under-budget and ahead of schedule.

Even animal conservation efforts are possible with PCR-amplified DNA. Researchers at the National Centre for Biological Sciences and the Wildlife Conservation Society-India Program are using genetic identification approaches and non-invasive DNA sampling to study conservation, identification, and management of rare species such as tigers. Ngo Thi Kim Cuc of the Institute of Animal Breeding and Genetics in Germany uses PCR amplification techniques to develop conservation measures and to study genetic variation in chickens, pigs, and the ruminant mammals, such as goats and giraffes.

PCR applications even extend to smaller members of the animal kingdom. Feed on this: forensic entomology, which uses PCR amplification to study human blood ingested by insects at crime scenes. It is a useful investigation method for forensic scientists.

 Since its invention 25 years ago, PCR has led to numerous innovations in medical, evolutionary, and forensic research fields, among others. Rapid diagnosis of infectious or hereditary diseases, the historical patterns of human migration and the identification of genetic fingerprints are all now being revolutionised thanks to this technique. Today, applications across various disciplines for PCR are being amplified and multiplied almost as fast as the DNA strands that it creates. 

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