Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1997-07-11
2001-08-14
Jones, W. Gary (Department: 1656)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S091200, C435S402000, C536S022100, C536S023100, C536S024200
Reexamination Certificate
active
06274351
ABSTRACT:
The present invention relates to a process for the solid-phase amplification of nucleic acids, as well as to a reagent kit which is useful for carrying out the process.
The present invention also relates to a process for immobilizing a primer on a solid phase.
Amplification on a solid phase consists of the elongation, during a PCR reaction or other types of amplification, such as LCR, SDA, etc., of a primer which is prebound to a solid support. Such a technique makes it possible to obtain an amplification product one specific strand of which is covalently attached to the solid phase, without using any steps other than PCR. This makes it possible to carry out detection before denaturation on double-stranded DNA, or after denaturation on a specific strand of the amplification product by combining PCR and detection without changing support.
Methods for the solid-phase amplification of nucleic acids have been described in WO 89/11546, AU 47144/89 and WO 93/09250.
This type of amplification on a support may be very useful for all the molecular biology diagnostic applications, in particular for detecting infectious targets or genomic targets. This technique makes it possible to reduce detection times as well as the risks of errors, since the sample is not transferred from one well to another but the entire experiment takes place on the same support. Moreover, for all the applications requiring only a single specific DNA strand, such as cloning or sequencing, this technique may allow a real saving in time and great ease of use avoiding many intermediate steps.
The primer involved in the PCR, which is bound at its 5′ end to the solid support, must form part of the strand which it is desired to elongate on the solid support. The 3′ end of the primer must be free, unmodified and homologous with the target, in order to allow its elongation by a polymerase.
However, a major drawback of solid-phase amplification is the low yield of elongation on the solid phase.
SUMMARY OF THE INVENTION
In order to decrease the solid support/oligonucleotides steric interactions and to improve the accessibility of Tag polymerase to the hybrid formed by the bound primer and the complementary amplification product, a connecting or “linker” arm is placed at the 5′ end of the bound primer. This linker arm is also referred to as a “spacer arm” since it serves to physically distance the 3′ end of the bound primer from the solid support so as not to hinder the cooperation of the various amplification reagents with the primer.
It has been discovered, according to the present invention, that one of the parameters influencing the yield of elongation lies in the mode of binding of the primer to the solid support. The nucleotides of the primer themselves are liable to be involved in covalent bonding with the support under the coupling conditions used between the “linker” and the solid support, this contributing towards the low yield of elongation of the primer. More precisely, it has been discovered that, more than the size of the linker arm, it is the reactivity or the multiplicity of the potential binding sites of the linker arm on the support which increases the yield of elongation of the primer.
The subject of the present invention is a process for the solid-phase amplification of nucleic acids, in which a primer immobilized on a functionalized heat-resistant solid support is used, characterized in that the said primer is immobilized on the solid support by means of a covalent bond between the solid support and a functional group of a polyfunctional molecule, the said molecule itself being linked to the 5′ end of the said primer.
More precisely, the said primer is immobilized on the solid support by means of a connecting (or “linker”) arm which consists of a residue of the said polyfunctional molecule placed between the solid support and the 5′ end of the primer and establishing a covalent bond between a functional group of the solid support and a first functional group of the said polyfunctional molecule, on the one hand, and between the 5′ end of the primer and a second functional group of the said polyfunctional molecule, on the other hand.
By increasing the number of functional groups in the linker arm, the elongation yield is increased. It is thought that this is due to the fact that the probability is thereby increased that the binding to the solid support takes place by means of the linker arm, that is to say without the primer itself actually being involved.
REFERENCES:
patent: 4882127 (1989-11-01), Rosenthal et al.
patent: 5451453 (1995-09-01), Gagnon et al.
patent: 6060288 (2000-05-01), Adams et al.
patent: 480408 (1992-04-01), None
patent: WO 9001546 (1990-02-01), None
patent: WO 9100868 (1991-01-01), None
patent: 91/08307 (1991-06-01), None
patent: WO 9303052 (1993-02-01), None
patent: WO 9304199 (1993-03-01), None
patent: WO 9309250 (1993-05-01), None
patent: WO 9313220 (1993-07-01), None
patent: WO 9315228 (1993-08-01), None
GENSET
Jones W. Gary
Saliwanchik Lloyd & Saliwanchik
Tung Joyce
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