| Methods and apparati using single polymer analysis -> Monitor Keywords |
|
|
  Methods and apparati using single polymer analysisUSPTO Application #: 20080103296Title: Methods and apparati using single polymer analysis Abstract: The invention relates to methods for analyzing and characterizing single polymers such as nucleic acid molecules. In preferred embodiments, the single molecules are analyzed using single molecule detection and analysis systems. (end of abstract) Agent: Wolf Greenfield & Sacks, P.C. - Boston, MA, US Inventors: Xiaojian David Zhao, Jeffrey D. Randall, Bijit Kundu, Jessica Kesty, Steven R. Gullans, Eugene Y. Chan, Martin Fuchs, Jenny E. Rooke USPTO Applicaton #: 20080103296 - Class: 536 221 (USPTO) The Patent Description & Claims data below is from USPTO Patent Application 20080103296. Brief Patent Description - Full Patent Description - Patent Application Claims
RELATED APPLICATIONS [0001]This application is a continuation of U.S. Non-Provisional Application having Ser. No. 10/773,084, filed Feb. 5, 2004, and entitled "METHODS AND APPARATI USING SINGLE POLYMER ANALYSIS" which is allowed, which is a continuation-in-part of U.S. Non-Provisional Application having Ser. No. 10/448,264, filed on May 28, 2003, and entitled "METHODS AND APPARATI USING SINGLE POLYMER ANALYSIS" which is pending, which claims priority to U.S. Provisional Application having Ser. No. 60/383,968, filed on May 28, 2002, and entitled "METHODS AND APPARATI USING SINGLE POLYMER ANALYSIS", and U.S. Provisional Applications having Ser. Nos. 60/437,892, 60/441,334 and 60/441,337, filed Jan. 3, 2003, Jan. 20, 2003 and Jan. 21, 2003, respectively, and entitled "ACCURATE AND SENSITIVE DIRECT mRNA QUANTIFICATION FROM TOTAL RNA SAMPLES BY SINGLE MOLECULE COUNTING", the entire contents of all of which are herein incorporated by reference. FIELD OF THE INVENTION [0002]The invention relates to methods and apparati for analyzing single polymers such as single nucleic acid molecules. BACKGROUND OF THE INVENTION [0003]The polymerase chain reaction, cloning, and other amplification methods have been the cornerstones of genetic analysis. Technologies that are deriving from these methods have led to the genomics revolution that we see today. The sequencing of the human genome published in 2001 has been made possible because of the ability to clone and amplify DNA. Likewise, there are many other methods of analyzing DNA that are dependent on these technologies. [0004]Single molecule detection, as defined in this application, is the detection of one fluorophore or one molecule. Single molecule detection has only been recently possible through the use of advanced optical detection methods. These methods include CCD fluorescence detection such as by Sase et al., 1995. Other methods that have achieved single molecule sensitivity include fluorescence correlation spectroscopy (Eigen and Rigler, 1994; Kinjo and Rigler, 1995), far-field confocal microscopy (Nie et al., 1994), cryogenic fluorescence spectroscopy (Kartha et al., 19995), single molecule photon burst counting (Haab and Mathies, 1995; Castro and Shera, 1995), two-photon excited fluorescence (Mertz, 1995), and electrochemical detection (Fan and Bard, 1995). These methods have not been applied extensively to the study of genetics because of difficulty in their implementation. Accordingly, most of these detection methodologies have not gained the attention of geneticists and molecular biologists. SUMMARY OF THE INVENTION [0005]The merging of single molecule detection and analysis and tagging chemistries that offer unique advantages in a single molecule detection setting is a breakthrough for molecular biology and genetic analysis. To this end, the invention relates to methods that exploit the ability to detect and thus analyze single molecules such as single nucleic acid molecules. Often times in molecular biology, it is necessary to amplify molecules such as nucleic acid molecules in order to conduct any analysis. That is because until recently most hardware used for genetic analysis was not capable of detecting single molecules. With the advent of detection systems with increased sensitivity, it is now possible to study molecules without prior amplification. This new approach is advantageous since the amplification process is known to introduce artifacts (e.g., sequence errors) into the amplified product that were not present in the parent molecule. Using prior art methods that included an amplification step, the information derived from an amplified product may be an amplification artifact rather than an inherent feature of the parent molecule, and in most instances it is difficult to distinguish between these two. [0006]The analyses described herein can be performed using single molecule detection and analysis systems. One such system is the Gene Engine.TM. which has been described in greater detail in published PCT Patent Applications WO98/35012, WO00/09757 and WO01/13088, published on Aug. 13, 1998, Feb. 24, 2000 and Feb. 22, 2001 respectively, and in U.S. Pat. No. 6,355,420 B1 issued on Mar. 12, 2002, the entire contents of which are incorporated herein. [0007]Accordingly, the invention provides in one aspect a method for analyzing a single nucleic acid molecule comprising exposing a single nucleic acid molecule to at least two distinguishable detectable labels for a time sufficient to allow the detectable labels to bind to the single nucleic acid molecule, and analyzing the single nucleic acid molecule for a coincident event using a single molecule detection system, wherein the coincident event indicates that the at least two distinguishable detectable labels are bound to the single nucleic acid molecule. [0008]The single nucleic acid molecule may be a DNA molecule or an RNA molecule, although it is not so limited. Preferably, it is denatured to a single stranded form in order to facilitate hybridization with a unit specific marker, or a primer, or a newly synthesized nucleic acid molecule, as the case may be. Although the single nucleic acid molecule may be linearized or stretched prior to analysis, this is not necessary as the single molecule detection system is capable of analyzing both stretched and compacted nucleic acids. This is particularly the case when coincident events are detected since these events simply require the presence or absence of at least two labels, but are not necessarily dependent upon the relative positioning of the labels (provided they are sufficient proximal to each other in some instances to enable energy transfer from one label to another). [0009]The distinguishable detectable labels may be present on different unit specific markers (i.e., a dual labeled probe) or on the same unit specific marker (i.e., a singly labeled probe). The at least two distinguishable detectable labels encompass two, three, four, five, or more labels. In some important embodiments, only two labels are required. [0010]The method may further comprise exposing the single nucleic acid molecule to a third detectable label that binds specifically to a mismatch between the single nucleic acid molecule and a unit specific marker, and wherein a coincident event between the first, second and third detectable labels is indicative of the mismatch. In this case, the coincident event encompasses the presence of first, second and third detectable labels on the hybrid formed by the single nucleic acid molecule and a unit specific marker. [0011]The method may further comprise exposing the single nucleic acid molecule and detectable labels to a chemical or enzymatic single stranded cleavage reaction prior to analyzing the single nucleic acid molecule. In these embodiments, the cleavage reaction can accomplish several things including but not limited to cleaving the single nucleic acid molecule and the unit specific marker at the location of a mismatch, digesting the unbound probes whether they be DNA or RNA in nature, and digesting single nucleic acid molecules that did not hybridize to a probe. Chemical and enzymatic cleavage methods are known in the art. For instance, the enzymatic single stranded cleavage reaction may use a single stranded RNA nuclease, a single stranded DNA nuclease, or a combination thereof. Various single stranded RNA nucleases are known in the art including but not limited to RNase I. Similarly, various single stranded DNA nuclease are known in the art including but not limited to S1 nuclease. [0012]In some embodiments, the hybridization and/or reaction mixture is cleaned prior to analyzing the single nucleic acid molecule. As used herein "cleaning" refers to the process of removing one or more of the following: unbound probes, unhybridized nucleic acid molecules, unbound or unincorporated labels (such as unincorporated nucleotides), and cleaved products following exposure to a chemical or enzymatic cleavage reaction. This cleaning step can be accomplished in a number of ways including but not limited to column purification. Column purification generally involves capture of small molecules within a column with flow-through of larger molecules (such as the target hybridized nucleic acid molecules). In other embodiments, a cleavage reaction and a column purification are used in combination to remove unwanted molecules. It is to be understood however that the method can be performed without removal of these molecules prior to analysis, particularly since coincident detection can distinguish between desired hybridization events and artifacts. Thus, in some embodiment, the unbound detectable labels are not removed prior to analysis using the single molecule detection system. [0013]The method preferably reads out a coincident event. The coincident event may take many forms including but not limited to a color coincident event. It can also be a binding coincident event, in which the binding of two unit specific markers is determined. It can further be the coincident existence of two or more detectable labels on a target molecule (including but not limited to the existence of a donor FRET fluorophore and an acceptor FRET fluorophore). The coincident event may also be the proximal binding of a first detectable label that is a donor FRET fluorophore and a second detectable label that is an acceptor FRET fluorophore. In this latter embodiment, a positive signal is a signal from the acceptor FRET fluorophore upon laser excitation of the donor FRET fluorophore. This latter embodiment requires a single molecule detection and analysis system that comprises one detector and one laser since a positive signal from the FRET pair is generate by only one laser and is emission from only one fluorophore. [0014]In certain embodiments, the method involves the use of at least one unit specific marker to which is attached one of the distinguishable detectable labels. In these and other embodiments, the method may further comprise exposing the single nucleic acid molecule to the labeled unit specific marker in the presence of a polymerase and labeled nucleotides. Preferably, the unit specific marker and nucleotides are differentially labeled. In this case, it is possible to synthesize a new nucleic acid molecule extending from the unit specific marker (i.e., the unit specific marker acts as a primer for the newly synthesize nucleic acid molecule). The newly synthesized nucleic acid molecules is therefore complementary to the single nucleic acid molecule which acts as a template for the newly synthesized strand. In these embodiments, the detectable labels are incorporated into the newly synthesized strand. [0015]The method can be further used to determine the length of the single nucleic acid molecule based on the signal intensity emitted by the newly synthesized strand. In these embodiments, the method is a method of determining integrity of a nucleic acid sample (such as an RNA sample) from which the single nucleic acid molecule derived. That is, it can be used to determine the level of degradation in, for example, the RNA sample as a propensity of short RNA molecule is indicative of degradation of the sample, while long RNA molecules are not. The method therefore may involve determining the signal intensity from the hybrid of the single nucleic acid molecule and the newly synthesized nucleic acid molecule (or alternatively of the newly synthesized nucleic acid molecule alone) as a measure of the length of the newly synthesized nucleic acid molecule (and thus of the template single nucleic acid molecule). The signal intensity is proportional to the length, therefore a greater intensity will indicate longer single nucleic acid molecules while lower intensity will indicate short and thus degraded single nucleic acid molecules. [0016]In some embodiments, the unit specific marker and nucleotides are labeled with a FRET fluorophore pair. In embodiments which involve hybridization of two unit specific markers, then they can similarly be labeled with corresponding FRET fluorophores. That is, one unit specific marker is labeled with a donor FRET fluorophore and the other is labeled with an acceptor FRET fluorophore. Alternatively, the unit specific marker is labeled with either a donor or an acceptor fluorophore and the nucleotides are labeled with an acceptor or a donor fluorophore respectively. [0017]In another embodiment, one detectable label is attached to a unit specific marker and is a first FRET fluorophore, and the other detectable label is incorporated into a newly synthesized nucleic acid molecule hybridized to the single nucleic acid molecule and is the donor or acceptor of the first FRET fluorophore. That is, if the first FRET fluorophore is a donor fluorophore, then the newly synthesize nucleic acid molecule has incorporated into it an acceptor fluorophore, and vice versa. [0018]The choice of polymerase will depend upon the nature of the template and the newly synthesized nucleic acid molecule. In one embodiment, the polymerase is a DNA polymerase. In another embodiment, the polymerase is a reverse transcriptase. [0019]In important embodiments, the single nucleic acid molecule is present in a nanoliter volume. That is, it is only necessary to load a nanoliter volume into the single molecule detection and analysis system. In still other important embodiments, the single nucleic acid molecule is present at a frequency of 1 in 1,000,000 molecules or 1 in 2,000,000 molecules in a nucleic acid sample (such as an RNA sample). Accordingly, the method can be used to detect and analyze nucleic acid molecules that are extremely rare. [0020]In important embodiments, the detectable labels are present on a unit specific marker that is a DNA, RNA, PNA, LNA or a combination thereof. In this and other aspects of the invention, RNAi molecules can be similarly used. In other embodiments, the detectable labels are provided as molecular beacon probes. The detectable label may also be attached to a nucleic acid molecule hybridized to a universal linker attached to a unit specific marker. Continue reading... Full patent description for Methods and apparati using single polymer analysis Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Methods and apparati using single polymer analysis patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. Start now! - Receive info on patent apps like Methods and apparati using single polymer analysis or other areas of interest. ### Previous Patent Application: Process for the preparation of sucrose-6-ester by esterification in the presence of solid superacid catalyst Next Patent Application: Methods and devices for removal of organic molecules from biological mixtures using a hydrophilic solid support in a hydrophobic matrix Industry Class: Organic compounds -- part of the class 532-570 series ### FreshPatents.com Support Thank you for viewing the Methods and apparati using single polymer analysis patent info. IP-related news and info Results in 1.36833 seconds Other interesting Feshpatents.com categories: Novartis , Pfizer , Philips , Polaroid , Procter & Gamble , |
||
