Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
2000-01-21
2003-09-02
Nguyen, Nam (Department: 1753)
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
C204S452000, C204S603000, C204S612000
Reexamination Certificate
active
06613210
ABSTRACT:
DESCRIPTION TECHNICAL FIELD
The present invention relates to the genical fields of molecular imaging and genetic sequencing.
BACKGROUND OF THE INVENTION
Current methods of nucleic acid sequencing and mapping, and protein and tissue imgaging, are based on radioactive, bio- and chemiluminescent emitters, photographic plates, and some electronic techniques. None of these have in practice been found to be entirely satisfactory.
Fluorescent imaging, radio labelling and bio- and chemiluminescent markers used with film and/or emulsion are expensive, very slow, limited and difficult to interface to computers. The techniques involved are difficult, and require hazardous handling and disposal procedures requiring substantial technical expertise. The materials used are often difficult and expensive to obtain, and have short shelf lives. Photographic imaging, which is frequently used, takes days or sometimes weeks or months to accomplish, and is limited by virtue of its small dynamic range and relatively poor linearity of response.
Of the electronic techniques, phosphor imaging/multiwire proportional chamber (MWPC) and the microchannelplate/MWPC approaches are unpopular with many molecular biologists because of their limited linearity and cost.
It may be helpful by way of background to set out in some detail the current state of the art in nucleic acid imaging. There are two separate methodologies which are currently in common use, details of which are set out below.
First, there are those involving the use of a photographic film to visualise chemiluminescent or radioactive labels and secondly, those using a light emitter, such as ethidium bromide, which chemically binds to nucleic acids and emits orange light under UV stimulation. The first imaging technology is usually used in nucleic acid sequencing. This process involves the chemical labelling of a component of the sequencing reaction, usually the primer, with a radio-isotope or chemiluminescent marker, or tag. Subsequent to the sequencing reaction and electrophoresis, this tag is used to image the position of the nucleic acid bands by the exposure of normal photographic film to the dried electrophoresis gel. This is a lengthy process, taking from 24 to 72 hours, depending on the sequencing technique used. Many of the radioactive markers used in nucleic acid sequencing are extremely hazardous and introduce extra complications to the sequencing process therefore any imaging system which removes the necessity for these markers would be extremely advantageous.
The second technology, that of light emitters, is generally used for DNA restriction analysis and plasmid construction planning. This technique relies on the imaging of nucleic acids in simple agarose gels using the carcinogenic chemical ethidium bromide which emits orange light after UV stimulation to visualise the size and estimate the concentration of nucleic acid present. Ethidium bromide is a highly undesirable component of this technique with dangerous accumulative medical consequences. Its removal from the imaging technique would be very advantageous in every application of agarose gel analysis.
Nucleic acid sequencing, as opposed to spatial imaging, normally makes use of a rather different process. This process involves the chemical labelling of a component of the sequencing reaction, usually the primer, with a radioisotope or bio or chemiluminescent marker, or tag. Subsequent to the sequencing reaction and electrophoresis, this tag is used to image the position of the nucleic acid bands by exposing photographic film to the dried electrophoresis gel. This is a lengthy process, taking from 24 to 72 hours depending on the sequencing technique used.
DNA restriction enzyme analysis (DNA mapping) and vector construction is a fundamental aspect of molecular biology. These mapping techniques also rely on the photographic imaging of nucleic acids fragments in simple agarose gels using ethidium bromide. This marker emits orange light after UV stimulation, to visualise size and estimate concentration of nucleic acids. Ethidium bromide is an undesirable component of this technique with dangerous cumulative carcinogenic consequences. Its removal from the imaging technique would be very advantageous in every such application of agarose gel analysis.
The imaging of peptides and proteins is often the end destination of many genetic engineering processes. However, it also forms a huge portion of general biological, biochemical and medical research. Indeed, it is hard to think of a bioscience area that is not dependent, at least in some way, on protein analysis. The analysis is again normally based on electrophoretic techniques, being dependent on the addition of a marker. Generally these are radioactive although chemiluminescence and specialised chemical stains are also used.
The field of tissue imaging is hugely important in the areas of drug development, and medical and molecular diagnostics. Traditionally, â-emitting markers may be used to image the distribution of a drug within a tissue sample. This process typically takes weeks or even months, and requires the use of potentially hazardous substances.
It will be understood that all of the present methods mentioned above require the use of either radioisotopes or other hazardous substances in addition, at least some of the techniques listed require the use of expensive and inconvenient electrophoresis gels. To summarise, the major problems are as follows:
1. Training. Health & Safety standards require all workers in contact with any form of radioactivity to have extensive training in the handling, use and disposal of radioactivity.
2. Use. The incorporation of radioactivity into a system is often a complex and time-consuming process. The worker must take extreme precautions, for example with the isotope
32
P, which is commonly used in DNA sequencing, has to be shielded from the worker by perspex, which makes an already complex experiment much harder. Subsequent to the initial experiment, the further manipulation of the already fragile electrophoresis gel is complicated by the radioactive present. Radioactivity can be replaced by chemi- or bio- luminescent imagers, but these, and while these are safer they are still complicated to use.
3. Time. All these imaging systems rely on the use of autoradiography to visual the nucleic acids or proteins. This is achieved by drying the gel onto a piece of filter paper and exposing it to a piece of photographic film. The film must be exposed for anything between 24 hrs to 3 months. Therefore it can take from days to months to even see if the experiment worked. This is a major problem in molecular biology and increases the length of research projects significantly.
4. Expense. The various components of these experiments are expensive. Radioactivity has a limited shelf-life because of its natural decay, and it is also expensive, with
35
S costing about L250 for 20 sequencing reactions. The film used is also very expensive as some of it measures 35×45 cm.
5. Disposal. These processes generate large volumes of solid and liquid waste, all of which must be disposed of legally and responsibly. This is also very expensive and troublesome.
SUMMARY OF THE INVENTION
According to a first aspect of the present invention there is provided a method of imaging molecules within a biological sample comprising shining a UV light onto the sample, and detecting the position of molecules of a selected class by the molecular UV absorption of molecules of that class.
According to a second aspect of the present invention there is provided a molecular imaging device for imaging molecules with a biological sample, comprising a UV light source arranged to shine onto a sample to be investigated and a UV detector arranged to detect the position of molecules of a selected class by the molecular UV absorption of molecules of that class.
According to yet a further aspect of the present invention there is provided an electrophoresis apparatus comprising an electrophoresis material onto which samples to be analysed are loade
Hassard John Francis
Hassard Stuart
Mainwood Alison Mary
Imperial College of Science Technology and Medicine
Nguyen Nam
Noguerola Alexander
Wallenstein & Wagner Ltd.
LandOfFree
Molecular imaging does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Molecular imaging, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Molecular imaging will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3111293