Genome sequencing: do you need the whole DNA or cut it?

An important step has been taken towards a universal genetic census
Alexey Timoshenko, GZT.RU

Scientists have made an important step towards the treatment of hereditary diseases and gene therapy. Studies of individual DNA differences have been carried out with previously inaccessible accuracy.

Geneticists whose results are presented in the journal Proceedings of the National Academy of Sciences (Brian Teague et al., High-resolution human genome structure by single-molecule analysis) have demonstrated a new method for analyzing the human genome. Using the method of so-called optical mapping of DNA, they were able to identify significant areas that differ in different people, and to do it quickly and cheaply.

Identifying individual differences across the entire DNA, as the authors of the study write, is like being able to view images in Google Earth. Instead of snatching out individual details, a full scan allows you to see the whole picture.

How does it work?How exactly did the researchers manage to achieve faster and cheaper scanning?

To answer this question, we must first say that the traditional way of reading DNA is based on two processes:

• cutting a DNA molecule into many pieces about a thousand nucleotide pairs long each
• reproduction of each fragment by means of the so-called polymerase chain reaction

By comparing many fragments and determining the sequence of nucleotide pairs in each of them, scientists assemble the entire genome – but the smaller the individual pieces, the more difficult the task has to be solved. If we assume that the entire set of analyzed DNA molecules is like a book, then the traditional way of sequencing (reading) it will be like reconstructing a text from fragments of the same shape with separate sentences or even words.

And this, according to Brian Teague, one of the authors of the study and a scientist from the University of Wisconsin, is the main drawback of conventional technology. Continuing the book analogy, we can say that geneticists receive in addition to unique pieces of text (for example, "...ien, mon prince. Genes et Lucques ne sont plus que des apana..." – definitely a fragment of the first paragraph of "War and Peace") areas that cannot be unambiguously placed in the overall mosaic (where, for example, will the fragment "At the table" fall? Only in the first volume of Tolstoy's work there are five suitable places!). If the pieces were bigger, like"...yei, who served at the table, seemed to have a feeling..." – there were no such problems. And it is even better, of course, to read a book, and not a bunch of sheets cut from it.

 
Schematic diagram of optical mapping of DNA:
with the help of fluorescent probes , it is possible to determine,
in what sequence should the DNA be collected from the pieces.
Source: Fong Chun Chan, Kendric WangDavid Schwartz's group at the University of Wisconsin took the first step towards fast DNA reading back in 1993.

Then scientists created a technology that allowed them to first cut DNA into relatively large (a million pairs of nucleotides against a thousand!) areas, mark each of them with a fluorescent dye and then analyze their sequence. It turned out faster, but it was still not enough for serious work with the human genome with more or less adequate time and money.

Ten years later, in 2003, it was possible to abandon the "tearing of the book into pieces" – a method was created that made it possible to do with one DNA molecule, which was only cut, its strands were pulled apart and then the sequence of nucleotides was determined using fluorescent labels.

 
The scheme of the method that allows for the analysis of the genome
do without cutting the DNA molecule into many small pieces.
Source: Fong Chun Chan and Kendric Wang

In 2004, the method was tested when reading the DNA of bacteria, in 2007 it was used to study the genome of rice, in 2009 maize was analyzed – and then it was the turn of man.

What is the result?According to Schwartz, their new development is equivalent to the transition from telescopes to observing the sky with binoculars or the naked eye.

This may seem like a paradoxical comparison, but the scientist explains that not always a close look at one detail allows you to get all the necessary information. And just as astronomers, along with powerful instruments like the Hubble orbital telescope, use wide-angle cameras (for example, the WISE asteroid search system), geneticists are interested not only in finding local differences at the level of individual nucleotides. Knowing how DNA molecules differ at the level of large repeating sites may be equally important.

In addition, Schwartz claims that in the near future (for several years) it will be possible to read a personal genome in an hour, and the cost of sequencing will decrease to $ 1,000 per person – and this will transfer the procedure, so far available only to selected scientific institutes, to the category of routine (although expensive, about the same amount is a complex tomographic examination) medical operations.

So at the turn of the century, the reading of the genome was rapidly becoming cheaper.
Source: Human Genome Project InformationThe above-described technique can be used to study previously inaccessible features of personal genetic differences that make up fragments of thousands, hundreds of thousands and millions of nucleotides (single "letters" of DNA), which can change places with each other, repeat, change length or be arranged in reverse order in chromosomes.

The role of such DNA variations is not fully understood. However, according to some studies, they can have a significant impact on the work of genes responsible for the synthesis of protein molecules.

Until now, when studying differences in individual genomes, scientists have focused on the so–called single nucleotide polymorphisms - substitutions of individual DNA "letters" in a genome consisting of about 3 billion nucleotides. This approach was due to the fact that existing DNA sequencing techniques based on the "cutting" of chromosomes into short fragments do not allow us to study the variability affecting repeats, deletions and movements of large DNA fragments. In addition, some of the genetic information remains unread.

When analyzing four personal genomes, Schwartz and colleagues found more than 4,000 differences in large sections of chromosomes. According to scientists, variability at this level can explain not only the uniqueness of each of the billions of people living on Earth, but also identify the genetic causes of certain diseases or developmental abnormalities, and develop methods for their treatment.

Portal "Eternal youth" http://vechnayamolodost.ru02.06.2010