Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-03-02
2003-08-12
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200
Reexamination Certificate
active
06605435
ABSTRACT:
The present invention relates to a method for characterizing all of the DNA fragments in a sample, said fragments being small in size and often being damaged and/or in trace amounts, in particular in finished or transformed products. The invention is based on the pre-amplification of all of the DNA fragments before an optional specific amplification and, therefore, the method is particularly suitable for the analysis, by molecular hybridization, in particular on DNA chips, of all of the DNA originating from any type of sample, possibly highly complex and/or having undergone denaturing treatments.
Various methods for characterizing a sample which is complex from a biochemical point of view, based on the identification of nucleotide sequences, are used in particular in the agro-foods domain. These methods can be applied to raw materials, such as a simple grain, however, they show certain limitations with regard to the analysis of a product containing many elements forming part of its composition and/or having undergone treatments which denature the molecules to be characterized. It is therefore necessary to enrich and/or to purify the material contained in a sample of interest if it contains treated or conditioned constituents originating from various origins, as is the case for finished products.
Once the nucleic acid material has been obtained, to a degree of purity sufficient to allow its analysis, it must be subjected to one or more tests intended to characterize it by molecular hybridization. However, these methods are generally restricted to analytical laboratories, because of either the use of radioactivity (although methods such as luminescence are tending to replace radioactivity), or the number of samples able to be analyzed simultaneously being too small. In certain cases, these techniques cannot be used because of a lack of sensitivity, this lack being essentially due to the low amount, and/or to the poor quality, of the nucleic acid material recovered from the sample.
The most commonly used technique is the PCR (Polymerase Chain Reaction). This method makes it possible to amplify specifically, in the course of many reaction cycles (of the order of 25 to 45), a nucleic acid included between two primers specific for known nucleotide sequences. These primers are oligonucleotides of the order of 15 to 40 bases, the sequence of which matches perfectly with the flanking sequences of the sequence to be amplified. It is conventional, using one nucleic acid sample, to amplify only one sequence.
“Multiplex” PCRs have been described in [Apostolakos (1993) Anal Biochem, 213, 277-284]. Under specific conditions, it is possible, in the same reaction tube, to amplify several sequences simultaneously using several pairs of primers. The number of pairs of primers rarely exceeds 3. Specifically, above this number, the amplifications lose their specificity (appearance of unexpected amplification products) or one or more amplifications does (do) not function, or barely function(s), although an example of the multiplex amplification of 9 sequences has been described [Edwards (1994) PCR Methods Applic., 3, S65-S75].
Other techniques, more or less derived from PCR, have been developed
LCR (Ligase Chain Reaction), based on the use of a heat-stable DNA ligase [Barany (1991) Proc. Natl Acad Sci USA, 88, 189-193].
Gap-LCR is derived from LCR.
ERA (End Run Amplification) is developed by Beckman Instruments, its derivative being GERA (Gap-ERA) [Adams (1994) Novel amplification technologies for DNA/RNA-based diagnostics meeting, San Francisco, Calif., USA].
CPR (Cycling Probe Reaction), which uses a DNA-RNADNA chimera and ribonuclease H [Duck (1990) BioTechniques, 9, 142-147], and is developed by the company ID Biochemical Corporation.
SDA (Strand Displacement Amplification) [Walker (1992) Nucleic Acids Res., 20, 1691-1696], patented by the company Becton Dickinson, which allows multiplex analysis [Walker (1994) Nucleic Acids Res., 22, 2670-2677]. However, it is difficult to analyze more than 3 sequences simultaneously by this method.
TAS (Transcription-based Amplification) [Kwoh (1989) Proc. Natl Acad Sci. USA, 86, 1173-1177], uses reverse transcriptase and T7 polymerase. Self Sustained Sequence Replication is related to TAS [Gingeras (1990) Ann. Biol Clin., 48, 498-501].
NASBA (Nucleic Acid Sequence-Based Amplification) is quite similar to 3 SR [Kievits (1991) J Virol Methods, 35, 273-286].
Finally, the properties of the Q&bgr; replicase (RNA dependent-RNA polymerase isolated from the Q&bgr; bacteriophage) were brought to light before PCR [Haruna (1965) Proc Natl. Acad. Sci USA, 54, 579-587], and this enzyme was used in amplification techniques, from 1983 [Miele (1983) J Mol. Biol, 171, 281 295].
In view of the documents cited above, it appears that it is not possible to characterize hundreds, and even more so thousands, of nucleotide sequences contained in a solution of DNA, in a restricted number of steps.
It is, however, possible to amplify, in a limited number of steps and using a considerable number of primers (greater than the number used in multiplex), virtually all of the DNA contained in an extract.
One of the approaches might be the AFLP (Amplified Restriction Fragment Polymorphism or Amplified Fragment Length Polymorphism) technique, which consists in using restriction enzymes to digest the DNA at specific sites, and then linkers which are attached specifically to these cleavage sites, and which also provide a DNA sequence sufficient to then allow the hybridization of primers. In a method sold, for example, by the company Gibco-BRL, the EcoRI and MseI enzymes are used, and then 8 linkers/primers for each cleavage site are used for the amplification step.
This method is, however, restricted to the analysis of DNA of quite good quality (generally directly extracted from a tissue or from an organism). Specifically, in order for the amplifications to take place, the two linkers must be present at the ends of the digested DNA, and therefore the DNA must have been digested at these sites. In the case of DNA derived from a transformed product, the size of this DNA is of the order of a few hundred base pairs (200 to 400). The probability of the presence of an EcoRI site (EcoRI recognizing a site composed of the 6-base pair palindrome; GAATTC) is ¼
6
, i.e. one potential site per 4096 base pairs.
Restriction enzymes which recognize the most common sites (4 base pairs), such as MseI, will, on the other hand, statistically cleave the DNA every 256 base pairs. Since it is necessary to cleave the DNA twice in order to generate the two PCR priming sites, the probability of generating these two sites on a fragment of a few hundred base pairs is low, and the amplification products do not reflect all of the starting DNA.
Short repeated sequences, dispersed throughout the length of the genome, termed “microsatellites”, have been used to amplify, with the aid of primers complementary to these microsatellite sequences, the sequences included between them [Zietkeiwicz (1994) Genomics, 20, 176 183; Weising (1995) PCR Methods Applic., 4, 249-255]. This type of amplification makes it possible especially to carry out genetic typing, “fingerprinting”, based on a qualitative analysis of the amplification products [Thomas (1993) Theor Appl Genet 86, 985-990]. A large proportion of the genome can thus be amplified, but not all of it, in particular because certain microsatellite sequences are too far apart to allow PCR amplification.
Three methods have been described, claiming the nonspecific amplification of all the nucleotide sequences of a sample [Ludecke (1989) Nature, 338, 348-350 or Kinzler (1989) Nucleic Acids Res, 17, 3645-3653, Zhang (1992) Proc Natl Acad; USA, 89, 5847-5851; and Grothues (1993) Nucleic Acids Res, 21, 1321-1322, and U.S. Pat. No. 5,731,171].
The principle of this latter method, which is drawn from the two others and which claims a
Chaubron Franck
Martin Anne-Céline
Provot Christian
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Genolife
Horlick Kenneth R.
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