Direct nucleic acid amplification kit, reagent and method

Abstract

The present invention relates to compositions, methods and kits which can be used to amplify nucleic acids with the advantage of decreasing user time and possible contamination. The dried reagent composition of the invention can be used for easy processing and amplification of nucleic acid samples.

Description

FIELD OF INVENTION[0001]The present invention relates to the field of nucleic acid amplification, particularly to the use of a polymerase chain reaction to amplify nucleic acids. The invention provides methods and kits which can be used to amplify nucleic acids by lyophilizing or freeze-drying nucleic acid amplification reagents for easy amplification of nucleic acid samples. The invention has applications in the easy processing of nucleic acids and is particularly useful in genotyping, diagnostics and forensics.BACKGROUND OF THE INVENTION[0002]The polymerase chain reaction (PCR) is a common tool used in molecular biology for amplifying nucleic acids. U.S. Pat. No. 4,683,202 (Mullis, Cetus Corporation) describes a process for amplifying any desired specific nucleic acid sequence contained in a nucleic acid or mixture thereof.[0003]U.S. Pat. No. 5,705,345 (Lundin et al.) describes a method of nucleic acid preparation whereby the sample containing cells is lysed to release nucleic aci...

AI Technical Summary

Benefits of technology

The present invention provides a way to make nucleic acid amplification easier and faster. By incorporating all the necessary reagents in a dried format, the number of steps is reduced, saving time and making it easier to use. This approach can be used in a high-throughput setting with automated sample processing. The dried reagent mixture is easy to dissolve in water, making it a convenient format for genetic interrogations. This invention also minimizes operator error, reduces sample mix-up, and improves sample processing for carrying out PCR reactions. The dried reagent mixture can be pre-dispensed into reaction vessels, ensuring reproducible and accurate amplification reactions.

Problems solved by technology

However the incorporation of cyclodextrin with oligonucleotides was for the cellular uptake of oligonucleotides and not for the amplification of nucleotides in a PCR reaction.

Current methods for DNA amplification involve a DNA purification procedure which often involves several steps which increases the chance of contamination.

This is a tedious process and prior art methods have a number of clear disadvantages in terms of cost, complexity and in particular, user time.

The purification of nucleic acids on such spin columns includes a number of complex and tedious steps.

However there are numerous technical challenges involved in producing a lyophilised or freeze-dried reagent for PCR analysis.

All of these procedures have limitations and drawbacks including consistency and reliability.

With dry-blending technology (Muzzio et al 2002, Powder Technology 124, 1-7), it is often difficult to obtain homogeneous blends of chemicals due to their different densities.

Furthermore, homogeneity is especially difficult to achieve when very small amounts of ingredients are mixed with large amounts of other ingredients.

Even if homogeneity is achieved, it is difficult to reproducibly dispense small amounts of the blended biological chemicals.

With spray-drying, however, it is difficult to dispense precise amounts of blended chemicals.

However, the agglomerated particles are generally less soluble than the original spray-dried particles or powders.

Also, these procedures sometimes use fluorocarbon cryogenic solutions which can be hazardous to the environment.

Using this procedure, it is difficult to obtain uniformly sized particles and to produce a uniform coating.

One drawback to the freeze-drying is the use of fluorocarbon refrigerants which are difficult to dispose of and the freeze-drying process may be imprecise and also difficult to regulate.

Indeed, regular freeze drying of reagents may not provide an entire solution for particularly labile reagents used in molecular biology processes.

Furthermore, degradation of the product during the freeze drying process is common and a freeze dried product is not always perfectly stable during storage.

Process control is critical and can be difficult to regulate (Tang & Pakil, 2004, Design of Freeze-Drying Processes for Pharmaceuticals: Practical Advice; Pharmaceutical Research, 21, 191-200).

Some problems with air drying processes are that the dried product is not in a readily dispensable form.

Also, the biological reagents must be stable at or above the temperature of the drying process and it is a difficult process to control accurately.

One drawback of this technology is that the reagent spheres are fragile and tend to disintegrate.

One drawback of the aforementioned references is that normally the stabilized and glassified biological materials are ground into powders, compounded into tablets, or maintained in a thin glassy film in a container like a microtitre plate.

However, this process has resulted only in limited success.

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