Chemistry: molecular biology and microbiology – Apparatus – Including measuring or testing
Reexamination Certificate
2000-06-02
2003-04-08
Siew, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Apparatus
Including measuring or testing
C435S006120, C435S091100, C435S091200, C435S286500, C435S402000, C536S022100, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330, C436S518000
Reexamination Certificate
active
06544777
ABSTRACT:
BACKGROUND OF THE INVENTION
Rapid, accurate diagnosis of acutely ill patients is critical for their survival. Typically, thousands of dollars in diagnostic tests are performed within 24 hours of a patient's admission to hospital. Accurate diagnosis of a treatable condition allows appropriate therapy to be started and unnecessary, potentially harmful medications to be stopped. On the other hand, some diagnostic tests require days to complete, some are invasive or even dangerous to perform, and all contribute to the upward spiral of medical costs. Selection and interpretation of appropriate tests in the appropriate order is therefore a highly valued skill, necessary to the physical health of the patient and the financial health of the care provider.
Requiring fast, accurate responses for an ever-expanding list of diagnostic questions, clinical laboratories turn more and more frequently to answers from molecular genetics. This rapidly evolving discipline comprises the study of gene structure and function at the molecular level. The most straightforward diagnostic application of this approach is to search clinical specimens for the presence of a particular gene or a particular allele (one variety of a particular gene). It is possible to use this direct approach to diagnose genetically transmitted diseases such as Huntington's chorea (by detecting the disease-causing allele), or to diagnose occult infections with agents such as
Bartonella henselae,
the agent of cat-scratch disease (by detecting genes specific for that organism). Gene detection tests such as these have already found a welcome place for themselves within the vast arsenal of tests offered by reference laboratories. In some cases (notably the detection of herpes simplex virus in cerebrospinal fluid or
Chlamydia trachomatis
in genital specimens) amplification and detection of genes have become the front-line standard diagnostic tests for conditions difficult to diagnose by other means. These tests require <24 hours from specimen to final result, and replace less sensitive methods with a turnaround time of several days or even weeks. Gene detection tests of this kind remain expensive, however, and have to be tailor-made for one or two organisms at a time. They are not useful for diagnosing disease caused by certain organisms such as bacteria of the genus Staphylococcus, which is normally present on the skin but which can also cause life-threatening disease.
A less obvious application of molecular genetics to clinical diagnosis requires analysis of gene transcription rather than the presence or absence of a particular gene. Disease-associated genes are present in all living things, including human hosts and parasites of all kinds (worms, protozoa, fungi, bacteria and viruses). In some cases, the mere presence of genetic material in a human specimen is enough to signify disease—the presence of genes specific for human immunodeficiency virus, for example, or trisomy 21 for a diagnosis of Down's syndrome. In other cases, however, a “pathological” gene may be present, but clinically silent for a variety of reasons. Examples include the defective hemoglobin gene which causes sickle cell anemia when two copies are present, but minimal disease when one copy is transcribed along with the normal hemoglobin allele, and no disease at all when the sickle cell allele is present but not transcribed. Moreover, genes for certain components of the immune system are present in every cell, but are only transcribed —that is, copied from DNA into RNA—when the host organism is diseased. The presence of these genes is universal, but transcription of them usually indicates a disease state.
In addition to associations between diseases and transcription of particular genes, we find that various combinations of gene transcription are required for specific pathologic outcomes. An example is found in certain lymphomas derived from B lymphocytes. Transcription of the myc gene when the bcl gene is not transcribed in these cells leads to limited proliferation followed by self-destruction of the proliferating cells. If the myc gene is transcribed in the presence of bcl transcription, however, the cell's proliferation is unrestrained, and malignancy may result. The number of two-fold gene interactions is large enough to daunt even the most stout-hearted diagnostic molecular geneticist. When one contemplates the possibility of three-fold, four-fold or more complicated gene interactions, however, it becomes quite impossible to analyze all the possible interactions using methods which detect transcription of only one gene at a time.
Identification of transcription products typically involves five steps: RNA extraction, amplification, hybridization, labeling, and detection, with labeling usually performed during the hybridization or amplification steps. The researcher disrupts the sample in the presence of enzymes which inhibit degradation of RNA. Organic solvents remove protein and lipids, while differential acid and salt concentrations enrich RNA in and deplete DNA from the sample. One can amplify the extracted RNA by reverse transcription (making DNA from an RNA template) followed by the polymerase chain reaction (PCR—which makes a double-stranded DNA product), or by transcription-mediated amplification (TMA—which makes a single-stranded RNA product). At this stage a “label” may be incorporated into the amplified product. Labels are small molecules bound to the components of nucleic acids. Ideally, the label does not interfere with nucleic acid chemistry. The label allows detection in one of four ways. It can emit radiation, it can serve as substrate for an enzyme, which makes a colored product, it can emit light itself (luminescence or fluorescence), or the label can serve as antigen for an antibody bound to a larger molecule which has one of the first three functions. The resulting product, with or without label, is then hybridized to a nucleic acid “probe” of known sequence. In general, either the probe is bound to a fixed surface and the amplified target is labeled, or the amplified target is bound to a fixed surface and the probe is labeled. Following hybridization, the probe produces radiation, light or color reaction. In measuring this, one identifies the presence of nucleic acid complementary to the probe in the target amplified from the original specimen.
To better understand the transcription process, and more specifically hybridization, an individual must understand the roles of nucleic acids in the process. Nucleic acids are chains of subunit molecules called nucleotides, which can be assembled in any order. The length of a chain is denoted by a number followed by the suffix ‘-mer’, hence, dimer, trimer, tetramer, and so on to decamer, with longer chains denoted by “11 -mer”, ‘24-mer’ etc. DNA is made of the nucleotides deoxyadenosine (A), deoxycytosine (C), deoxyguanosine (G) and deoxythymidine (T) while RNA is made of the nucleotides adenosine (a), cytosine (c), guanosine (g) and uracil (u). Nucleic acid sequences are written as strings of letters—for example ACGT, a DNA tetramer. Nucleic acids “hybridize’, or form double-stranded molecules, in a defined pattern. A or a lines up opposite T or u, and C or c line up opposite G or g. Nucleotides which line up opposite each other according to this scheme are called ‘complements’. The RNA complement to the above-mentioned DNA sequence ACGT would be ugca, while the DNA complement would be TGCA. Hybridization is most specific—that is, a nucleic acid hybridizes solely to its complement—when the temperature is high, the salt concentration low, and the nucleic acid long, typically ≧15 nucleotides.
Single strands of nucleic acids make stable duplexes by hydrogen bonding with strands of complementary bases. Hybridization is the process of forming these duplexes from complementary single-strands of nucleic acids. Both deoxyribonucleic acids (DNA) and ribonucleic acids (RNA) may be hybridized.
Thus a probe sequence, for example DNA, may be immobilized on a solid surface (the
Hibbs Jonathan
Schrenzel Jacques
Patterson Thuente Skaar & Christensen P.A.
Siew Jeffrey
LandOfFree
Non-cognate hybridization system (NCHS) does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Non-cognate hybridization system (NCHS), we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Non-cognate hybridization system (NCHS) will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3031200