Thursday, February 15, 2007

 

DNA method gives new perspective on the Mysteries of Nature

What caused the extinction of the mammoth [2] while other ice age mammals like the musk ox survived to present day? A new scientific methodological approach to detect genetic material will help researchers to solve the many mysteries of the past.

"I’m confident that the new methodological approach will be of great importance to molecular biology", says Professor Eske Willerslev at the Centre for Ancient Genetics, University of Copenhagen. One of his PhD students recently came up with a brilliant idea enabling researchers to get a full view of total ecosystems or populations dating thousands of years back in time. What usually has taken the DNA-researchers several years of laboratory work can now be done in just a few hours.

The automation of a long research process

Professor Eske Willerslev and his team find DNA traces of ancient life in areas where the ground is permanently frozen like in Siberia or Alaska. Here, inside the frozen ground, the team is able to find ancient DNA material from animals and plants that used to live in the area thousands of years ago. In order to detect the types of DNA material in a sample, the researchers normally use a DNA primer - a kind of 'fishing hook' attached to a specific piece of DNA. That particular piece of DNA is then being multiplied, cloned and sequenced (see "DNA Sequencing") which makes it possible for the researchers to identify it. However, this procedure is slow, and it takes years just to identify a fraction of the most common animals and plants available from the many DNA samples.

The technology

A new sequencing machine capable of interpreting millions of pieces of DNA in just a few hours was recently introduced. The machine alone brought in a revolution to the field, but has certain disadvantages and shortcomings. Firstly, an analysis made by the machine is quite expensive. Each analysis costs approximately DKK 45,000 and although the machine reads extensive amounts of DNA material, the cost is still considerable to a research project. Secondly, a vital problem arises when researchers try to benefit from the machine's enormous capacity by analysing samples from multiple locations or specimens in a single run in order to reduce costs. The machine simply cannot separate more than 16 samples from each other.

Eske Willerslev went to check out the machine for himself at the Danish Cattle Research Centre in Foulum - the only place in Denmark, which operates the new sequencing machine. He realised to his great disappointment that the researchers at the University of Copenhagen could not make use of the machine for their respective projects due to the disadvantages mentioned above.

A simple but brilliant idea!

Then Jonas Binladen, a PhD student from his team, came up with a simple but brilliant idea: By attaching a 'finger-print' to the tagged primers ('fishing hooks' used to amplify DNA from each sample), one should - in theory - be able to localise each of the million sequences produced in each run, to its original sample or specimen. By making it possible to process amplification products from multiple samples or specimens in the same run, the team could make use of the machine's great capacity.

The research team now wanted to test the idea. And it really did work! The results are now being published in the scientific web magazine PLoS ONE Publication [1].

According to Eske Willerslev, the new approach have great scientific potentials:

"Today, when using conventional methods to detect ancient DNA, we are only able to test a limited number of samples providing us with a somewhat random image of life in the past. Due to this new method, our knowledge will be put into a whole new perspective. For instance, finding out if species became endangered due to a dramatic change in the climate or if the decline in numbers started many years earlier than we originally thought or estimated".

Source: University of Copenhagen PR 15 February 2007

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[1] Based on:

Binladen J, Gilbert MTP, Bollback JP, Panitz F,
Bendixen C, et al. (2007) The Use of Coded PCR Primers Enables
High-Throughput Sequencing of Multiple Homolog Amplification Products
by 454 Parallel Sequencing. PLoS ONE 2(2): e197.
doi:10.1371/journal.pone.0000197

Abstract

Background

The invention of the Genome Sequence 20TM DNA Sequencing System (454 parallel sequencing platform) has enabled the rapid and high-volume production of sequence data. Until now, however, individual emulsion PCR (emPCR) reactions and subsequent sequencing runs have been unable to combine template DNA from multiple individuals, as homologous sequences cannot be subsequently assigned to their original sources.

Methodology

We use conventional PCR with 5'-nucleotide tagged primers to generate homologous DNA amplification products from multiple specimens, followed by sequencing through the high-throughput Genome Sequence 20TM DNA Sequencing System (GS20, Roche/454 Life Sciences). Each DNA sequence is subsequently traced back to its individual source through 5' tag-analysis.

Conclusions

We demonstrate that this new approach enables the assignment of virtually all the generated DNA sequences to the correct source once sequencing anomalies are accounted for (miss-assignment rate less than 0.4%). Therefore, the method enables accurate sequencing and assignment of homologous DNA sequences from multiple sources in single high-throughput GS20 run. We observe a bias in the distribution of the differently tagged primers that is dependent on the 5' nucleotide of the tag. In particular, primers 5' labelled with a cytosine are heavily overrepresented among the final sequences, while those 5' labelled with a thymine are strongly underrepresented. A weaker bias also exists with regards to the distribution of the sequences as sorted by the second nucleotide of the dinucleotide tags. As the results are based on a single GS20 run, the general applicability of the approach requires confirmation. However, our experiments demonstrate that 5' primer tagging is a useful method in which the sequencing power of the GS20 can be applied to PCR-based assays of multiple homologous PCR products. The new approach will be of value to a broad range of research areas, such as those of comparative genomics, complete mitochondrial analyses, population genetics, and phylogenetics.

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[2] Excerpt from "Supernova Explosion May Have Caused Mammoth Extinction"

A distant supernova that exploded 41,000 years ago may have led to the extinction of the mammoth, according to research conducted by nuclear scientist Richard Firestone of the U.S. Department of Energy's Lawrence Berkeley National Laboratory (Berkeley Lab).

Firestone, who collaborated with Arizona geologist Allen West on this study, unveiled this theory September 24, 2005 at the 2nd International Conference "The World of Elephants" in Hot Springs, South Dakota (see "Evidence of a Catastrophic Impact Event at the End of the Clovis Era"). Their theory joins the list of possible culprits responsible for the demise of mammoths, which last roamed North America roughly 13,000 years ago. Scientists have long eyed climate change, disease, or intensive hunting by humans as likely suspects.

Now, a supernova may join the lineup. Firestone and West believe that debris from a supernova explosion coalesced into low-density, comet-like objects that wreaked havoc on the solar system long ago. One such comet may have hit North America 13,000 years ago, unleashing a cataclysmic event that killed off the vast majority of mammoths and many other large North American mammals. They found evidence of this impact layer at several archaeological sites throughout North America where Clovis hunting artifacts and human-butchered mammoths have been unearthed. It has long been established that human activity ceased at these sites about 13,000 years ago, which is roughly the same time that mammoths disappeared.

They also found evidence of the supernova explosion's initial shockwave: 34,000-year-old mammoth tusks that are peppered with tiny impact craters apparently produced by iron-rich grains traveling at an estimated 10,000 kilometers per second. These grains may have been emitted from a supernova that exploded roughly 7,000 years earlier and about 250 light years from Earth.

"Our research indicates that a 10-kilometer-wide comet, which may have been composed from the remnants of a supernova explosion, could have hit North America 13,000 years ago," says Firestone. "This event was preceded by an intense blast of iron-rich grains that impacted the planet roughly 34,000 years ago."

Source: Berkeley Lab PR September 23, 2005

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Recent posts include:

"Metagenomics: Investigating the invisible life in our environment"

"Reductive Genome Evolution from the Mother of Rickettsia"

"Evolution Of Influenza A Virus (PR + Paper)"

"Shotgun sequencing finds nanoorganisms"

"Research News: Neanderthal Genome Sequencing Yields Surprising Results"

"Latest Sequencing Targets: Gibbon Genome Sequence To Be Added To Primate Tree"

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