Solid phase methods for amplifying multiple nucleic acids

Details Solid phase methods for amplifying multiple nucleic acids Solid phase methods for amplifying multiple nucleic acids

1999-06-04

2001-10-09

Whisenant, Ethan (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C536S023100, C536S024300

Reexamination Certificate

active

06300070

ABSTRACT:

BACKGROUND OF THE INVENTION
Recent developments in microarray technology have made it possible to contemplate simultaneously analyzing many hundreds of thousands of individual genetic elements within a single nucleic acid sample. There already exists technology in which probe arrays containing several hundred thousand oligonucleotides are present on a single glass chip (1 cm
2
). (Wang, D. G., et al.,
Science,
280:1077-1082 (1998)). However, this increase in synthesis capability has exceeded the capacity of polymerase chain reaction (PCR) amplification technology to provide hybridization targets. For example, a microarray containing probes for 10
4
randomly distributed human single nucleotide polymorphisms (SNPs) could be used to generate a detailed genomic map of a single individual in a single hybridization experiment. Currently, it is extremely difficult to amplify more than 100 independent loci in a single PCR reaction. (Wang, D. G., et al.,
Science,
280:1077-1082 (1998)). Therefore, using current PCR technology, at least 100 individual PCR reactions, each reaction amplifying 100 distinct loci, must be performed and pooled to take full advantage of a 10
4
loci SNP typing chip.
Similar difficulties are anticipated for other multiplex genotyping technologies, such as mass spectrometry. (Hall et al.
Nature Biotechnology,
16:1352-1365 (1998), and Fu et al.,
Nature Biotechnology,
16:381 (1998)). For this reason, there is a need for new methods that enable massively multiplex nucleic acid amplification. Ideally, such methods would make it possible to produce 10
3
to 10
4
different products in a single reaction. Coupled with microarray or multiplex mass spectrometry typing methods, such multiplex amplification methods would make it possible to rapidly generate high density, whole genome SNP maps for large numbers of individuals. This would immediately accelerate genetic research in many areas including genetic analysis of complex traits (e.g., asthma, high blood pressure, and various forms of heart disease), human genetic disease research, pharmacogenomics and cancer biology. Improved multiplex amplification methods would also greatly facilitate analysis of gene expression.
SUMMARY OF THE INVENTION
The present invention discloses methods for amplifying target nucleic acid molecules using a solid-phase amplification method. One such method is described in U.S. Pat. No. 5,641,658, the teachings of which are incorporated by reference herein in its entirety. This single-stage solid-phase amplification method is referred to herein as “bridge amplification.”
The present invention encompasses a multi-stage bridge amplification method which uses a recovered single-stranded amplification nucleic acid molecule to initiate a second stage of bridge amplification. Subsequent stages of bridge amplification follow where each subsequent stage of bridge amplification is initiated with a single-stranded amplification nucleic acid molecule produced in the previous stage of bridge amplification. This multi-stage method is recursive, and therefore provides for an iterative process whereby a single target molecule can be amplified over a hundred thousand-fold. This iterative process significantly increases the amplification power of bridge amplification.
More specifically, described herein is a solid-phase, multi-stage method for amplifying one, or more, target nucleic acid molecules comprising two or more stages of bridge amplification. In the present method, one or more single-stranded nucleic acid molecules are produced in the first stage of bridge amplification which are used to initiate a second stage of bridge amplification, and single-stranded nucleic acid molecules produced in the second stage of bridge amplification are used to initiate the third stage of bridge amplification, and so forth, through multi-stages of bridge amplification to produce amplified target molecules.
The first stage of bridge amplification involves one, or more, target nucleic acid molecules mixed under conditions of hybridization with a solid support comprising immobilized oligonucleotide primers which are specific for the target molecules. For example, a sample (i.e., test sample) can contain a single type of target molecule and the solid support can comprise a pair of immobilized primers specific for that type of target molecule. Alternatively, the sample can contain multiple target molecules and the solid support will comprise multiple pairs of immobilized primers wherein each pair of primers are specific for one of the target molecules. The target molecules hybridize with their specific immobilized primers. The hybridization complexes that form are then subjected to amplification via thermocycle reactions, thus forming double-stranded amplification nucleic acid molecules. Amplification comprises approximately from about five to about fifty thermocycles, each thermocycle comprising denaturation, primer annealing and polymerization reactions (primer extension) carried out under conditions appropriate for each reaction. Typically, amplification comprises about thirty-five thermocycles.
The double-stranded amplification nucleic acid molecules are cleaved and denatured, thereby releasing single-stranded amplification nucleic acid molecules. These newly released single-stranded amplification nucleic acid molecules are then contacted with a fresh solid support comprising specific immobilized primers and initiate a second stage of bridge amplification. The stages of bridge amplification can be repeated until the desired amplification of the target molecule is achieved. The amplified target nucleic acid molecules can then be analyzed on the solid support, or they can be cleaved from the support for analysis by solution phase or solid phase methods.
The oligonucleotide primers of the present invention are immobilized to a solid support. These primers are specific for a given target nucleic acid molecule. Preferably, the primers are single-stranded DNA molecules. In one embodiment of the invention, a set of primers (e.g., a set of primers comprises a first and a second primer) specific for amplifying a target molecule is immobilized to a solid support. The first primer is complementary to a nucleotide sequence region contained within the target molecule, for example, the 3′ terminal end. The second primer is complementary to the 3′ terminal end of the complementary nucleic acid strand of the target molecule. There are multiple sets of primers specific for various target molecules attached to the same solid support. Preferably, at least one member of a primer set contains a cleavable moiety. More preferably, the two primers in each primer set have different cleavable moieties. For example, one member of a primer set can comprise a restriction site within its nucleotide sequence.
Preferably, the target molecule is a DNA molecule. Other nucleic acid molecules are within the scope of this invention, for example, RNA. The target nucleic acid molecule (or simply, target or target molecule) can originate from plant or animal tissue. Preferably, the target molecule contains one nucleotide sequence region that can hybridize to a first immobilized primer. The target molecule can be in a double-stranded or single-stranded form. If the presented target molecule is in a double-stranded form, then it is treated so as to render it into a single-stranded form.
The solid support can be beads, particles, sheets, dipsticks, rods, membranes, filters, fibers (e.g., optical and glass), and the like. Preferably, the solid support is a bead. The material composition of the solid support includes, but is not limited to, plastic, nylon, glass, silica, metal, metal alloy, polyacrylamide, polyacrylate, crosslinked-dextran and combinations thereof. Preferably, the solid support is capable of being modified by the attachment of oligonucleotide primers.
Bridge amplification begins with a hybridization complex formed between a target molecule and a first oligonucleotide primer. (See FIG.
1
A). Preferably, the target molecule hybrid

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