Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1996-02-12
1999-02-02
Horlick, Kenneth R.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
436 94, C12Q 168, G01N 3348
Patent
active
058663282
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
The present invention relates to a fast method for the determination of a sequence of a nucleic acid, DNA or RNA, which is useful, in particular, for the sequencing of an unknown DNA or RNA or alternatively for the detection of a specific DNA or RNA sequence for diagnosis.
DNA sequencing is a major objective of molecular biology, and is at the heart of the human genome sequencing project.
Furthermore, demonstration of the presence of a specific DNA sequence in a physiological sample constitutes, at the present time, the major line of development of diagnostic methods. After the immunological technique, attention is now being turned to diagnostic methods that demonstrate modifications of the DNA itself in order to prevent, in particular, antibiotic resistance (see Nature, 1992, 358, p. 591), genetic abnormalities, the risks of cancer associated with genetic modifications and viral infections, for example infections associated with HIV or with hepatitis viruses.
These diagnostic methods comprise, at the present time, the so-called "direct hybridization" methods, which will detect the presence of this sequence by direct hybridization of a probe with the sample. The method is cumbersome and imprecise, in particular because it necessitates a detection by gel and exposure on light-sensitive film or radioactive counting.
Methods employing an amplification, in particular the PCR method, which, before the demonstration of the sequence by hybridization, will first amplify the corresponding portion of the DNA using, for example, primers and a polymerase, are excessively sensitive to contamination.
These methods have, in addition, the drawback of being indirect recognition systems, because the desired sequence is recognized only via the use of A probe.
SUMMARY OF THE INVENTION
The method according to the present invention makes it possible to demonstrate the hybridization or the nucleotide sequence should this be necessary. Hence, there cannot be any bias or ambiguity. This considerable progress is due to the fact that, for the first time, it is almost as fast to demonstrate the nucleotide structure of the desired sequence directly as to demonstrate it indirectly, for example using a probe.
It is even possible, as a result of the method of the invention, to demonstrate directly the hybridization between two complementary sequences without resorting to complete sequencing of this sequence.
Nucleic acid sequencing is nowadays carried out chiefly by the so-called Sanger method well known to biologists. In this technique, the DNA to be sequenced is first cut into small segments (of approximately 5 kb), which are then cloned into plasmids inserted into a bacterial strain. The plasmids are amplified in this strain and extracted. The inserted DNA is then copied from a plasmid promoter. This transcription is interrupted after the insertion of a random number of nucleotides. This consequently gives rise to a population of all the possible interrupted copies of the initial DNA. This population is separated by gel electrophoresis according to the molecular weight of the numerous copies. The position of the nucleotides along the DNA is read directly on the gel for each DNA fragment.
This process is very laborious. For a DNA of approximately 15,000 base pairs (15 kbp), the final step (after extraction of the plasmids) alone takes a week. Consequently, it is commonly accepted that the sequencing of a DNA comes to approximately 5 francs per base pair at the date of filing of the present application.
The method according to the present invention, based on physical techniques and electronic treatments, differs from the current approaches, which are chemical or biochemical. Its advantages are numerous: sequencing of very long genes (greater than 10 kbp) without having to segment them into small pieces, as is the case in the present methods. approximately 15,000 bases to be sequenced in a week. The method enables approximately 100 of them to be sequenced in a second, that is to say 60 million in a week. t
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Steven B. Smith et al., "Direct Mechanical Measurements of the Elasticity of Single DNA Molecules by Using Magnetic Beads", Science, vol. 258, Nov. 1992, pp. 1122-1126.
Hansma et al., Science 256, 1180-1184 (1992).
Bensimon Aaron
Bensimon David
Chiffaudel Arnaud
Croquette Vincent
Centre National de la Recherche Scientifique
Horlick Kenneth R.
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