Capture of single stranded nucleic acids

Details Capture of single stranded nucleic acids Capture of single stranded nucleic acids

1997-05-30

2001-03-20

Zitomer, Stephanie (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C536S022100, C536S023100, C536S024300

Reexamination Certificate

active

06203978

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to solid phase supports or substrates for use in the capture of single stranded nucleic acids. The invention also relates to a method of capturing single stranded nucleic acids using such supports or substrates. In particular, but not exclusively, the invention relates to the use of magnetic solid phase supports for capturing PCR (polymerase chain reaction) primers.
BACKGROUND OF THE INVENTION
PCR and other amplification methods offer the best option for sensitive, reliable, cheap and easy detection, characterization, quantitation and discrimination of nucleic acid sequences. A few copies of template target nucleic acid can be replicated in vitro or in situ to provide enough product nucleic acid for analysis. However, the end product often has to be separated and purified from the reaction mixture before its identification and characterization is possible, and whilst amplification can be rapid, identification and characterization can be slow and laborious. The characterization and identification of the nucleic acid amplification product represents a considerable bottleneck in the processing of samples particularly in diagnostic applications of amplification methodologies. For this reason quicker and more convenient methods are desirable for amplification product detection and characterization to improve high throughput screening of biological samples.
With respect to amplification product detection, it is known that PCR products, either modified or unmodified, can be identified and characterized spectrophotometrically. However, determination of the amplification product can be difficult or impossible in the presence of modified or unmodified excess primers present in the reaction mixture at completion of amplification. It is necessary to be able to discriminate between these two species (i.e. amplification products and primers) to permit product detection and characterization, on line, at the end of the amplification process.
In view of the above it is known that single stranded nucleic acid sequences complementary to other nucleic acids can be used to ‘capture’, by hybridization, such other sequences and that these capture sequences can be derivatized to a support. Such a support (hereinafter known as ‘the support’) could be a magnetizable or non-magnetizable particle or bead, porous or non-porous membrane, microtitre plate, paper or surface or substrate material that may be contacted with a fluid medium.
Techniques currently exist which make use of these principles. It is possible to detect and characterize a PCR product by its denaturation and subsequent ‘capture’ to a complementary single stranded oligonucleotide immobilized to a support. The support is frequently represented by a microtitre plate; the single stranded capture sequences are bound to the wall of the wells of the plate and are used to hybridize to a denatured, single stranded amplification product. Excess primers are removed from the system by washing the bound product in the microtitre plate wells, and the amplification product is subsequently detected and characterized by spectrophotometric means. However these techniques are frequently time consuming and complex and risk introducing contamination and artefacts into the process.
An alternative approach would be to remove excess primers from the amplification reaction mixture subsequent to amplification and determine the amplification product directly in solution spectrophotometrically. This would have the benefit of being a potentially faster simpler and more efficient process for amplification product/primer separation and detection and characterization. In the present invention such benefits and advantages over known prior art procedures are provided by a substrate/support where separation, detection and characterization of amplification products are analyzed spectrophotometrically in solution.
Surprisingly we have observed that a magnetizable solid phase support (MSPS) derivatized with single stranded nucleic acids having random sequences of nucleotide bases can anneal to and thereby capture single stranded nucleic acids as effectively as capture sequences having nucleotide sequences complementary to those to be captured. This discovery is applicable to the capture of all single stranded nucleic acid species including PCR primers. Essentially this discovery indicates that complementarity between single stranded nucleic acid capture sequences and the sequence to be captured is unnecessary to effect the latter's capture. We have surprisingly observed that a mixture of totally random sequences, present as oligonucleotides, derivatized to a support can function as well as specific, complementary capture sequences in the removal of single stranded nucleic acids from solution, including primers from PCR reaction mixtures.
SUMMARY OF THE INVENTION
In the present invention, these purposes, as well as others which will be apparent, are achieved generally by providing a solid phase substrate or support derivatized with single stranded nucleic acids having random sequences of nucleotide bases and related method of manufacture. The substrate/support is preferably magnetizable, but any structure capable of attaching the random sequences of nucleotide bases can be used in the invention.
The random sequences of nucleotide bases are prepared by automated synthesis and are preferably 20 to 30 nucleotides long, most preferably 25. However, in accordance with the invention, both shorter and longer sequences also work, for example, random capture sequences as short as 5 nucleotides and as long as 175 nucleotides, which is the upper limit for automated synthesis, can be used. Random sequences longer than 175 bases, made by ligation, can also be used. Prior to attachment to the substrate these sequences are further modified with materials to facilitate addition to the support, such materials include biotin or a primary amine group. The substrate is also modified to promote such addition. To permit conjugation with the biotinylated nucleic acid sequences the support is derivatized with streptavidin. The substrate is also derivatized with carboxymethyl groups which are conjugated to the primary amine group. Both modifications provide means of attachment of the random sequences to the substrate/support which is then used to capture single stranded nucleic acids in amplification processes.
The invention also provides a method of capturing one or more single stranded nucleic acids by addition of the solid phase substrate derivatized with single stranded nucleic acids having random sequences of nucleotide bases to a fluid medium containing both single stranded and double stranded nucleic acids. The single stranded nucleic acids anneal to the nucleic acids of the substrate to result in the captured sequences.
In the embodiment where the substrate is magnetizable, the substrate with the captured sequences is removed by a magnet from the fluid medium to form a medium free of the substrate. This medium is then assayed for the presence of double stranded nucleic acids which have not annealed to the nucleic acids of the substrate.
The captured sequences are single stranded nucleic acids selected from the group consisting of oligonucleotides, polynucleotides and nucleic acid bases. Preferably, the captured sequences are primers, particularly polymerase chain reaction primers, used in nucleic acid amplification reactions.
In addition the primers and their amplification products produced can be made fluorescent by attaching a label to the 5′-terminus of the primer or attached to a nucleotide base of the primer. The labels used in the invention process include fluorochrome or a primary amine group and are preferably in the form of its N-hydroxysuccinimide ester or its isothiocynate derivative.
Encompassing the various aspects of the invention a method for assaying double stranded nucleic acids is provided. The process includes treatment of a fluid medium containing single and double stranded nucleic acids with the magnetizable

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