Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-07-27
2004-11-02
Fredman, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C436S094000, C436S173000, C530S350000, C250S282000
Reexamination Certificate
active
06811977
ABSTRACT:
FIELD OF THE INVENTION
The invention relates generally to methods for detecting and measuring the level of nucleic acids, and specifically to detection and measurement methods using mass spectrometry.
BACKGROUND
Although a variety of methods to detect and measure nucleic acids have been developed, no method provides a highly accurate means of detecting many genes in a biological sample. The simplest method in common practice for detection of mRNA transcripts is the northern blot. Northern blot analysis can be used to detect a small number of transcripts of interest, however quantitation of the level of a specific transcript using northern blot analysis is difficult and often inaccurate. RT-PCR can be used on its own or in an intermediate-scale method known as the rapid analysis of gene expression (RAGE). RT-PCR and RAGE suffer from biases resulting from the PCR priming and amplification process and are further limited by the use of measurement of band intensity on gels to quantify gene levels. In addition, the methods are not well-suited to a high level multiplexing, i.e., measuring many genes at once in a single sample.
In order to address the poor quantitation of previous methods, newer methods based on sequencing have been suggested. One such method, serial analysis of gene expression (SAGE), allows the quantitative and simultaneous analysis of a large number of transcripts. In SAGE, the cDNA library constructed from all the transcripts in a cell, i.e., the transcriptome, is concatenated into large chunks and then sequenced. The sequencing data is computationally converted into quantitative levels of gene expression via the frequency of occurrence of sequences representing a given gene transcript. This method can also be used for gene discovery, but it cannot be targeted to specific subsets of genes of interest (e.g., all known oncogenes or all known tumor suppressors or all known G-coupled membrane receptors). SAGE is accurate, but it is slow and relatively expensive since it requires a large amount of sequencing for each sample to be studied.
One new method, called massively parallel signature sequencing (MPSS), is a high throughput method making use of specially litigated adapters and signature sequencing. It is sequencing-based and suffers many of the same disadvantages as SAGE, but it is more efficient because separation of cDNA fragments is not required.
The most common methods in practice for large-scale analysis of the transcriptome employ DNA microarrays, either robotically spotted or microfabricated. Such arrays permit biologists to monitor 5-10,000 genes per experiment in most implementations, for example, by using a DNA chip, provided that the experiment begins with large amounts of starting sample. Thus, RNA extraction must be from sizable amounts of tissue, cell or embryo cultures, or that the initial RNA sample must be amplified via various PCR-based strategies. It would be highly desirable to eliminate amplification steps and to instead make measurements of RNA presence and levels directly.
Methods using microarrays suffer various limitations. First, microarrays rely on hybridization to cDNAs or oligonucleotides that are bound to the surface of a solid support. The kinetics and physics of such interactions are poorly understood and difficult to optimize in comparison to hybridization interactions in liquid phase. For example, inconsistencies can be introduced during the creation of DNA samples that are deposited or synthesized on the solid matrix. Furthermore, diffusion parameters and the limited accessibility of DNA fixed onto the chip to test samples all conspire to make quantitation and reproducibility difficult. Second, microarrays require a relatively large amount of input RNA to achieve high sensitivity, particularly when rare genes are assessed. Third, microarrays have a limited dynamic range. Cells express RNA significantly over four or five orders of magnitude, and microarrays are only capable of working within one or two orders of magnitude. These issues of input material severely constrain their applications in biology where the investigator wants to assay the transcriptome in small groups of cells or individual cells. This arises often in developmental biology, neurobiology and increasingly in other biological fields. Arrays typically require material from 10
6
cells or more per 10,000 genes measured. Fourth, microarrays are technically difficult to fabricate, and have a high per-experiment cost. Finally, quantitatively accuracy is limited since microarrays meant to deliver identical results differ by greater than 200%, which is greater than biologically significant differences in gene expression. Sensitive computer algorithms used to evaluate microarray data do not rectify this problem since they perform poorly with high or variable noise levels in the data.
There is thus a need for a highly sensitive method for detecting nucleic acids in biological samples. The present invention meets that need and more.
SUMMARY OF THE INVENTION
In one embodiment, the invention provides a method of detecting a specific nucleic acid in a sample. The method includes contacting the nucleic acid with a first oligonucleotide linked to a selector tag and a second oligonucleotide linked to a detector tag, in a reaction mixture under conditions that allow the first and second oligonucleotides to specifically hybridize with the nucleic acid. The first and second oligonucleotides hybridized in such a way that the first oligonucleotide is located immediately adjacent to the second oligonucleotide to form adjacently hybridized first and second oligonucleotides. Next, the adjacently hybridized first and second oligonucleotides are ligated to form a ligated oligonucleotide, and the detector tag associated with the ligated oligonucleotide is identified.
Another embodiment of the invention provides a method of detecting a plurality of specific nucleic acids in a sample. The method includes contacting each specific nucleic acid with an oligonucleotide pair in a reaction mixture under conditions that allow the oligonucleotide pair to specifically hybridize to the nucleic acid such that the oligonucleotide pair members are located immediately adjacent to each other thereby forming an adjacently hybridized oligonucleotide pair. Each oligonucleotide pair comprises a first oligonucleotide linked to a selector tag and a second oligonucleotide linked to a detector tag. Each adjacently hybridized oligonucleotide pair is ligated to form one or more ligated oligonucleotides; and the one or more detector tags associated with the one or more ligated oligonucleotides is identified.
Still another embodiment of the invention provides a method of detecting a nucleic acid in a sample. The method includes amplifying the nucleic acid with a primer pair to form a dual-tagged amplification product in a reaction mixture. The primer pair is a first oligonucleotide linked to a selector tag and a second oligonucleotide linked to a detector tag. Following amplification, the detector tag associated with the dual-tagged amplification product is identified.
Yet another embodiment of the invention provides a method of detecting a nucleic acid in a sample. The method includes contacting the nucleic acid with an oligonucleotide linked to a detector tag under conditions that allow the oligonucleotide to specifically hybridize to the nucleic acid to form a mixture of hybridized oligonucleotide and unhybridized oligonucleotide. A next step includes separating the hybridized oligonucleotide from the unhybridized oligonucleotide; and identifying the detector tag, thereby detecting the nucleic acid.
Another embodiment of the invention provides a kit containing an oligonucleotide primer pair and an agent that binds to the selector tag. The primer pair includes a first selector oligonucleotide linked to a selector tag and a second selector oligonucleotide linked to a detector tag.
Still another embodiment of the invention provides a kit containing a first selector oligonucleotide linked to a selector tag, a second
Davis Mark E.
Kirshenbaum Kent
Murphy John F.
Tirrell David A.
Wold Barbara J.
California Institute of Technology
Fredman Jeffrey
Gray Cary Ware & Freidenrich LLP
LandOfFree
Rapid, quantitative method for the mass spectrometric... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Rapid, quantitative method for the mass spectrometric..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Rapid, quantitative method for the mass spectrometric... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3348413