Molecular probes hybridization techniques

Armed with the common techniques of molecular biology and immu-nocytochemistry, an investigator is in a good position to apply in situ hybridization to EM for localization of nucleic acids at the ultrastructural level. McFadden (9) has a review on such use of in situ hybridization techniques. In the review, McFadden has included details of some of his laboratory protocols needed in in situ hybridization from fixation and labeling to probe labeling to the hybridization steps for localization of specific RNAs at the EM level. His protocol for hybridization is outlined below ... 

In situ hybridization (ISH) consists of the application of hybridization techniques to intact cells which demonstrate genetic information within a morphologic context. This technology takes advantage of the hybridization properties of nucleic acids and offers a distinct technique to directly analyze sequence information in intact tissues. In essence, it combines cytogenetic techniques with molecular biology to probe gene alterations at molecular levels. Development of... 

Chan, A., Coupling different molecular techniques onto a porous microarray for diagnostic applications Detection of deletions and duplications in Duch-enne muscular dystrophy (DMD) using multiplexed amplifiable probe hybridization (MAPH) on PamChip , First International Conference on Microarrays for Diagnostics, San Diego, CA, March 21-22, 2002. 

The answer is c. (Murray, pp 812-828. Scriver, pp 3-45. Sack, pp 57-76. Wilson, pp 123-149.) Fluorescent in situ hybridization (FISH) analysis is a technique in which molecular probes that are specific for individual chromosomes or chromosomal regions are used to identify these regions. FISH probes frequently identify chromosomal regions that are submicro-scopic and therefore may be useful when standard karyotypic analysis is normal. In this case, the fact that only one signal is present, despite the fact that there are two number 22 chromosomes, indicates that a submicro-scopic deletion has occurred. The parental chromosome of origin cannot be determined using this technique unless that parent also carries a similar deletion and his or her chromosomes are evaluated. 

We have seen that NMR can provide detailed molecular structure information. It is possible to join together or couple an HPLC instrument with an NMR spectrometer. The HPLC performs the separation of a complex mixture and the NMR spectrometer takes a spectrum of each separated component to identify its structure. We now have a new instrument, an HPLC-NMR instmment. We call a coupled instrument like this a hyphenated instmment. The coupling of two instruments to make a new technique with more capabilities than either instmment alone provides results in a hyphenated technique or hybrid technique. HPLC-NMR is made possible with a specially designed flow probe instead of the standard static probe. For example, Bruker Instruments (www.bruker-biospin.com) has a flow probe for proton and C NMR with a cell volume of 120 p,L. Complex mixtures of unknown alkaloids extracted from plants have been separated and their stmctures completely characterized by HPLC-NMR using a variety of 2D NMR... 

DNA is subsequently analyzed by various characterization techniques, such as molecular weight determination, electrophoresis, and hybridization to known probes. Using these methods, the presence of DNA unique to a particular species can be confirmed. Alternatively, with appropriate primers, a mixture of DNA fragments, which are uniquely characteristic of an individual or organism, will result in a fingerprint that can be used for identification. 

Mycobacteria of the Mycobacterium avium complex are implicated in disseminated bacterial infections in AIDS patients. RFLP studies followed by hybridization with radiolabeled probe specific for an insertion sequence in M. avium (IS 1311) have been useful for typing M. avium stains (R2). A variety of molecular techniques are available for the diagnosis of Chlamydia trachomatis infection. In addition to PCR, a method based on the ligase chain reaction has also been found to be sensitive to the detection of C. trachomatis infection in urine specimens collected from male and female subjects (VI). The differentiation between low-risk genotypes of human papilloma virus (HPV 6 or 11) from genotypes of high... 

Molecular Self-Assembly. Reductive techniques, such as those used in the microelectronics industry, can produce structural features smaller than about 200 nm. The use of proximal probes and other nanomanipulative techniques can be considered to be a hybrid of the reductive lithographic techniques and die synthetic strategies of assembling functional nanostructures atom by atom, or molecule by molecule. The organization of nanostructures and devices by the self-assembly of the component atoms and molecules, a ubiquitous phenomenon in biological systems, forms die noncovalent synthetic approach to nanotechnology. 

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