The color genome is cheap and fast

Pyotr Smirnov, Artem Tuntsov, "Gazeta.Ru»From the discovery by Gregor Medel of the laws of inheritance of external signs to the description of the material base of this very heredity – DNA – 90 years have passed.

Over the next half century, scientists not only managed to understand the peculiarities of the genetic apparatus, but also learned to some extent to control these processes. However, most of us are more familiar not with fundamental discoveries explaining the principles of biological systems, but with technological breakthroughs demonstrating the power and splendor of science.

A striking example is the Human Genome project, which ended in 2004, which did not seem to bring new fundamental knowledge about the work, but at the same time added tens of billions of kilobytes of information to continuously replenished databases. For the first time, not a patchwork quilt of deciphered DNA fragments of many people was presented, but the genome of one particular person – one of the discoverers of the structure of James Watson's DNA itself. About a year ago, the decoding of another complete genome was completed – one of the pioneers of decoding Craig Venter.

On Thursday, to the delight of Watson, who lost his job due to "politically incorrect" statements, this short list was replenished with an Asian and a Negro.

In the latest issue of Nature, two research teams immediately announced the completion of projects to determine the complete sequences of two human genomes using the relatively cheap and very fast method of Illumina. It took less than two weeks to decode each genome with all the preparatory work.

The heroes of the narrative in these two stories remained anonymous, it is only known that the object of research by David Bentley and his colleagues is a Yoruba man living in Nigeria, and scientists led by Zhun Wang deciphered the genome of a Beijing resident, also a man. A detailed analysis of all these differences is still ahead. However, it is no less interesting how the genomes were read.

The Negro and the Chinese vs. Watson and VenterIn the decoded Nigerian genome, scientists found 4 million single nucleotide substitutions that distinguish him from Watson and Venter – the so-called "snips" (SNP, single nucleotide polymorphisms.

In addition, scientists have identified about 400 thousand differences of another kind – the so-called CNV substitutions, in which whole DNA fragments from many nucleotides are rearranged, appear in several copies or are completely removed from the genome.

A Chinese differs less from two Caucasians with known genomes – 3 million "snips" were found in his DNA. Perhaps soon we will hear a comment on this from James Watson, who until now has not been particularly shy about claiming that representatives of different races have different mental abilities inherent in their genes.

The patchwork genomeAll modern methods of genome decoding are based on the same principle.

The isolated and purified DNA is first amplified – turned into a large number of copies, then cut into numerous pieces, divided into separate strands, and then the nucleotide sequences are "read". They are put together again already in the memory of supercomputers with the help of complex mathematical algorithms.

To understand what such a procedure looks like, imagine a good rural or even a small district library. In the tens of thousands of volumes that make up it, there will just be about 3 billion letters – so many pairs of nucleotides are in the DNA contained in every cell of our body. So, all these tens of thousands of books must first be multiplied, turning into millions of volumes, then cut into pages, torn into scraps and thoroughly mixed. After that, you are asked to restore the books of the entire library.

It is the first step that helps in this obviously difficult task – the "reproduction" of the source library. Although individual pages were torn to shreds randomly, in the final mash, every phrase is present multiple times, while all these repeating fragments overlap. This allows you to restore integrity.

The 4 methods currently used differ: Applied Biosystems, Illumina, Roche and Helicos – only by the size of the shreds and the methods used to read the letters. Almost 20 years ago, Watson and his colleagues who participated in the "Human Genome" decided to split DNA into chains of 400-800 base pairs – a few lines of text in our analogy. The authors of the latest works were able to limit themselves to pieces of lines with only 30-35 letters in each.

However, the comparison is not entirely appropriate. It is much easier for our contemporaries – firstly, they already have two reference genomes with which pieces of the read text can be compared - after all, they are not so different in humans. And secondly, the possibilities of computer technology have expanded many times over the past decade, especially if they are adjusted for availability and cost.

Bridges, paints and chemicalsIncreasing the speed of genome reading is a truly great achievement.

And it's not just that millions of patches with the text of the genetic code are now being read in parallel. The invention that allowed to deal with the whole genome in less than a month is the so–called "bridge" method.

Known oligonucleotides (short chains consisting of only a few nucleotides) are attached to each DNA fragment from both ends. Then, thanks to these oligonucleotides, these threads are stretched over thin horizontal dies – it really turns out something like bridges. Naturally, all these described addition reactions are explained by chemical interactions and do not require any control from scientists. The only thing that is required of them is to add the right enzymes that control the reactions at the right time and in the right concentration.

Thanks to this independence, all bridges are built on a die in a few seconds, and about 80 million chains are placed on one standard plate in the Illumina installation.

After that, a solution of individual nucleotides is added to the die, tightly cross–linked (again by chemical bond) with multicolored fluorophores - luminous molecules, moreover, each nucleotide corresponds to its own color. Scientists wait for the time necessary to attach the next nucleotide complementarily to the second DNA chain, after which they remove the solution of free "fireflies" and look at which nucleotide has joined at one or another point of the die.

Then the procedure "adding paints – flushing – photo registration" is repeated until all the sequences located on the die are deciphered. In the Illumina installation, this happens 30-35 times – depending on the length of the sections the DNA under study was cut into.

Fast, cheap, reliableDavid Bentley's work published in Nature was devoted not only to the description of the genetic differences between the Negro and Watson, but also to the study of the reliability of the method used.

To evaluate it, a team of almost two hundred scientists tested the equipment on a well-studied DNA section with a length of 162,572 base pairs encoding genes of the so-called main histocompatibility complex.

The accuracy of the method was 99.96%, which fully meets modern requirements. Moreover, the "doubtful" 0.04% were located in the same place where other decryption methods also fail.

According to the authors of the papers published in Nature, the era of individual genomics is just around the corner. And very soon it will become quite accessible to ordinary people.

The international project "Human Genome" 1989-2004 cost $ 300 million, decoding the Venter genome using a patented technique was rumored to cost about $ 1 million. Compared to these amounts, the $500,000 spent on a Chinese and a Nigerian is nothing, especially when you consider that only $100,000 was spent on consumables – reagents and the like.

Applied Biosystems Corporation, the main competitor of Illumina, promises that the next generation of its sequencing machines will reduce the cost of determining the DNA sequence to $ 10 thousand per human genome, and some are counting on $ 5 thousand. And we intend to do it faster than in a day.

However, the damned disappointment of meeting with a dream, it seems, cannot be avoided in the case of the genome. The more accessible the individual genome becomes, the more scientists begin to wonder: "Why is it needed?".

The results of hundreds and thousands of studies, which almost every day in recent years have revealed the next "genes" of cancer, Alzheimer's disease, diabetes and many other congenital diseases, very rarely have real predictive power. So the practical benefits of them are questionable.

Well, you found out that your risk of getting stomach cancer is 50% higher than that of your neighbor. So what? Won't you eat salty and sweet and someday die of a heart attack in a restaurant - out of frustration that you can't have the most delicious dish?

However, similar considerations can be expressed about almost any branch of technical and medical progress. According to many scientists, although the era of individual genomics is near, it is still unclear what it will look like. You can fantasize as much as you want, but it's easier to wait. Judging by the latest issue of Nature, it's not long to wait.

Portal "Eternal youth" www.vechnayamolodost.ru
07.11.2008