Proportional amplification of nucleic acids

Details Proportional amplification of nucleic acids Proportional amplification of nucleic acids

1999-04-05

2003-06-24

Whisenant, Ethan (Department: 1637)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C435S091510

Reexamination Certificate

active

06582906

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates generally to the proportional amplification of nucleic acids. The methods of the present invention can facilitate the amplification of minute sample amounts of nucleic acids in a manner that may preserve the relative abundance of the individual nucleic acid species, or portions thereof, existing in the original sample.
BACKGROUND OF THE INVENTION
The isolation, characterization and manipulation of nucleic acids has numerous present or potential applications, including those in the basic research, diagnostic and forensic fields. Valuable information about gene expression in in vivo, in situ, and in vitro systems can be obtained by monitoring the abundance of the mRNA encoded by those genes. Methods involving the synthesis of cDNA from mRNA have also enhanced the study of gene expression, for example, by facilitating gene cloning and the production of desired recombinant proteins.
With existing methods for the study or use of MRNA and cDNA, one problematic scenario can arise where the sample size is small, or the relative abundance of an individual MRNA or cDNA species in a sample is low. In such situations, where the availability or accessibility of the desired mRNA or cDNA is compromised (or their amounts are otherwise limited), the lower limits of monitoring or manipulation systems may be exceeded, thus leaving the desired MRNA or cDNA undetected, unrecoverable or unworkable. Therefore, the amplification of such MRNA and cDNA is an important molecular biology methodology, with particular significance in facilitating the detection and study of a broader range of mRNA molecules, and the isolation and manipulation of mRNA available in only minute quantities.
Although methods exist for the amplification of nucleic acids, they generally suffer from a phenomenon known as biased amplification. In these cases, the amplified population does not proportionally represent the population of nucleic acid species existing in the original sample. This drawback may preclude meaningful or reliable conclusions regarding the absolute amount or relative abundance of a desired nucleic acid species in the tested sample.
One common problem encountered by past amplification methods is the preference for the amplification of shorter nucleic acid templates. The enzymes responsible for the production of complements or copies of the nucleic acid templates (e.g., DNA and RNA polymerases, or reverse transcriptases) achieve such synthesis through a sequential, oriented process, whether 5′ to 3′ or 3′ to 5′. The probability that such an enzyme will complete a copying event thus may be greater with nucleic acid templates of shorter length. Accordingly, in a sample population containing nucleic acid templates of variable lengths, longer templates may be less likely than shorter templates to be amplified in complete, full-length form. This can result in a bias in the amplified population in favor of nucleotide sequences proximal to the 3′ poly(A) tail of MRNA, for example, a phenomenon known as 3′-sequence bias.
The synthesis of longer templates can also be difficult or less efficient due to interference from secondary and tertiary structure in the template. For example, with respect to nucleic acid amplification based on polymerase chain reaction (PCR) methodologies, longer templates in a sample may be under-represented in the amplified product if respective primers cannot anneal to begin another round of copying because the first round did not proceed to completion. Other potential sources of bias can reflect relative differences between longer and shorter templates. For example, longer templates may (i) not denature sufficiently, or (ii) have a greater likelihood of mismatches, and thus error propagation through amplification, but (iii) have an ability to anneal more easily.
Known nucleic acid amplification methods have not successfully overcome such problems with biased amplification. U.S. Pat. No. 5,716,785, for example, describes an amplification system involving the production of antisense RNA from cDNA synthesized from a MRNA population. The methodology set forth in the '785 patent also results in 3′-sequence biased amplification.
The foregoing shows a need for methods and products involving the amplification of nucleic acids in a manner to facilitate the preservation of the relative abundance of the individual nucleic acid species existing in the original sample.
SUMMARY OF THE INVENTION
An objective of the present invention is therefore the proportional amplification of nucleic acids.
In accomplishing these and other objectives, the present invention preferably provides methods for the proportional amplification of nucleic acids that may comprise creating fragments of a single-stranded DNA population, synthesizing double-stranded DNA from the fragments of a single-stranded DNA population, and producing multiple copies of sense RNA from the double-stranded DNA. In another preferred embodiment, the present invention provides methods for the proportional amplification of nucleic acid that may comprise creating fragments of a double-stranded DNA population, and synthesizing multiple copies of the fragments of a double-stranded DNA population. In yet another preferred embodiment, the present invention provides methods for the proportional amplification of nucleic acid that may comprise synthesizing multiple copies of a double-stranded DNA population, and creating fragments of the multiple copies of a double-stranded DNA population.
In addition, the present invention preferably provides methods for the proportional amplification of nucleic acid that may further comprise labeling the multiple copies of the fragments of a double-stranded DNA population, or producing multiple copies of RNA from the multiple copies of the fragments of a double-stranded DNA population, or producing multiple copies of RNA from the fragments of the multiple copies of a double-stranded DNA population.
In a preferred embodiment, the present invention provides methods in which the single-stranded or double-stranded DNA population may be produced from a nucleic acid population selected from the group consisting of one or more of the following: genomic DNA, cDNA, total RNA, poly(A)
+
RNA, and oligonucleotides. In a preferred embodiment, the poly(A)
+
RNA may be mRNA.
The present invention also preferably provides methods, which may further comprise making fragments of the RNA or DNA obtained by the described proportional amplification methods, contacting the fragments with a solid support comprising nucleic acid probes, and detecting the presence or absence of hybridization of the fragments to the nucleic acid probes on the solid support. In a preferred embodiment, the solid support, which may comprise nucleic acid probes, can be selected from the group consisting of a nucleic acid probe array, a membrane blot, a microwell, a bead, and a sample tube.
In another embodiment, the present invention preferably provides methods in which the described steps are repeated once or multiple times. For example, in a preferred embodiment, the present invention may further comprise creating an additional set of single-stranded DNA from the multiple copies of sense RNA, synthesizing an additional set of double-stranded DNA from the additional set of single-stranded DNA, and producing an additional set of multiple copies of sense RNA from the additional set of double-stranded DNA.
In a preferred embodiment, the fragments of a single-stranded DNA population of the present invention may be from about 30 nucleotides to about 3,000 nucleotides in length. In another, these fragments may be from about 30 nucleotides to about 750 nucleotides in length. In yet another, these fragments may be from about 30 nucleotides to about 150 nucleotides in length.
The present invention may preferably provide methods wherein the RNA may be isolated from an eukaryotic cell or tissue, mammalian cell or tissue, or human cell or tissue. In a preferred embodiment, t

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