Outline of Recombinant DNA technology. Application of UV spectroscopy in recombinant DNA technology

Transcription

1 NIKHIL.K.POTDUKHE

2 Outline of UV spectrophotometer Outline of Recombinant DNA technology Application of UV spectroscopy in recombinant DNA technology References

3 Lambert law: When a beam of light is allowed to pass through a transparent medium, the rate of decrease of intensity with the thickness of medium is directly proportional to the intensity of the light. Beer law: The intensity of beam of monochromatic light decreases exponentially with the increase in concentration of the absorbing substance arithmetically.

4 A = abc or A = ξbc A: absorbance b : Sample Path length C : Sample Concentration a : Absorbance Constant ξ : Molecular Absorbance Constant

5 Deviations in absorptivity coefficients At high concentrations (>0.01M) due to electrostatic interactions between molecules in close proximity Scattering of light due to particulates in the sample Fluorescence or Phosphorescence of the sample Changes in refractive index at high analysis concentration Shifts in chemical equilibria as a function of concentration

6 The recorder assembly The spectrometer itself this houses the lamps, mirrors, prisms and detector. The spectrometer splits the beam of radiation into two and passes one through a sample and one through a reference solution (that is always made up of the solvent in which you have dissolved the sample). The detector measures the difference between the sample and reference readings and communicates this to the recorder. The samples are dissolved in a solvent which is transparent to UV light and put into sample cells called cuvettes. The cells themselves also have to be transparent to UV light and are accurately made in all dimensions. They are normally designed to allow the radiation to pass through the sample over a distance of 1cm

7 Cont.. Spectrometric instruments have a common set of general features. Here we look at specific features for the UV/Visible experiment. Sources: D2 lamp, W filament (halogen lamp), and Xe arc lamp. Wavelength Selectors: Filters and Monochromators. Sample Containers: Fused silica, quartz, and glass. Detectors: Phototube, PMT, photodiode, photodiode array,ccd array

8 Strike a low voltage DC arc in a lamp filled with D2. Gives continuum emission from 160 to 400 nm.

9 Reflecting focusing assembly Lamp Condenser Lens A heated W filament, gives off blackbody radiation. Add a small amount of a halogen gas. Sublimated W reacts with halogen to form tungsten halide; does not deposit on quartz cover (no blackening) but does redeposit on filament (extends life).

10 Tube filled with Xe (or sometimes a mixture of Hg and Xe), invented in 1940, commercialized in 1961 by Osram. Pass a low voltage DC current to excite Xe. The broad spectral output closely resembles natural daylight, and is often used in projection systems (e.g. 15 kw IMAX systems) 150 Watt Xe lamp

11 A filter is device that allows the required wavelength to pass but absorb of other wavelength wholly or partially. Filters are of two type Absorption filter It work by selective absorption of unwanted light. Absorption filter is solid sheet of glass colored by pigment which is dispersed in glass. Interference filter - It work by reflection of unwanted light. A semitransparent metal film is deposited on a plate of glass. Then it is coated with dielectric material. When ray of light incident on it part of light reflects back whereas remaining light is transmitted

12 A monochromator is an optical device that transmits a mechanically selectable narrow band of wavelengths of light or other radiation chosen from a wider range of wavelengths available at the input. Accepts polychromatic input light from a lamp and outputs monochromatic light. Components : Entrance slit, Dispersion device, Exit slit Entrance slit provides narrow source light to avoid overlapping of monochromatic light. Exit slit select narrow band of dispersed spectrum for observation of detector.

13 Diffraction grating is an optical component with a regular pattern, which splits (diffracts) light into several beams traveling in different directions. Grating consist of large number of parallel lines ruled on highly polished surface such as alumina. The directions of these beams depend on the spacing of the grating and the wavelength of the light so that the grating acts as a dispersive element.

14 Prism is a transparent optical element with flat, polished surfaces that refract light. The prism disperse the light radiation in to individual colors or wavelength. Prisms are typically made out of glass, but can be made from any material that is transparent to the wavelengths for which they are designed. The resolution depends upon the size and refractive index. Prisms have higher dispersion in the UV region.

15 Successful spectroscopy requires that all materials in the beam path other than the analyte should be as transparent to the radiation as possible. Also, the geometries of all components in the system should be such as to maximize the signal and minimize the scattered light. Polystyrene nm Methacrylate nm Glass nm Suprasil Quartz nm Keep the cuvette clean. Don t clean with paper products (Kim-wipe); use optical paper. Store dry. Don t get finger prints on them. Store carefully and gently.

16 Detectors : Phototube Photomultiplier tube Photodiode Channeltron

17 Phototube: Detector is composed of : Photo cathode- It is coated with elements of high atomic volume like Potassium or silver oxide. Collector anode Photo cathode liberates electron towards anode when light incident on it

18 Photomultiplier tube: It is most sensitive of all detector. In this detector multiplication of photoelectron by secondary emission of electron achieved by using photo diode and series of anode (dynodes) Up to 10 dynodes are used, maintained at V higher than preceding one It can detect very week signal. light dynodes anode electrons photocathode voltage divider network high voltage

19 Photodiode: A photodiode is formed by sandwiching an undoped layer of Si between a heavily doped p-layer and a heavily doped n-layer. Photons whose wavelength is between 400 nm and 1100 nm can be absorbed in the intrinsic layer, producing an electron-hole pair. The bias potential sweeps these carriers to the opposite regions, producing a current in the external circuit. Photodiodes are more sensitive than phototubes, but far less sensitive than PMT s, since they only generate ~1 electron-hole pair per photon. On the other hand they are about the size of a transistor and require no high voltage support.

20 A continuous dynode chain is built into a single unit. Excellent and widely used electron multiplier. If the front end is a photo emissive surface then you have a compact PMT. Channeltrons require high vacuum to operate.

21 Mechanism:

22 Cont. A beam of light from a visible and/or UV light source is separated into its component wavelengths by a prism or diffraction grating. Each monochromatic (single wavelength) beam in turn is split into two equal intensity beams by a half-mirrored device. One beam, the sample beam, passes through a small transparent container (cuvette) containing a solution of the compound being studied in a transparent solvent. The other beam, the reference (colored blue), passes through an identical cuvette containing only the solvent. The intensities of these light beams are then measured by electronic detectors and compared. The ultraviolet (UV) region scanned is normally from 200 to 400 nm, and the visible portion is from 400 to 800 nm.

23 Single beam spectrophotometer: This consist of tungsten lamp as source of light. This light radiation focused on slit by using concave mirror. This light passes through simple absorption filter where the only required wavelength of light passed through it and passed through it and falls on the sample cell where the solution to be analyzed is present. The sample or standard solution absorbs apart of the radiation and rest is transmitted. The intensity of transmitted light is determined by photovoltaic cell.

24 Double beam spectrophotometer: It is similar to that of single beam instrument. Here the light beam after passing through filter is split into sample beam and reference beam by using beam splitter. These beam pass through sample and reference solution and fall onto detector separately The final read out is in absorbance or transmittance, obtained after electronic manipulation of 2 detectors.

25 DNA is the keeper of the all the information needed to recreate an organism. All DNA is made up of a base consisting of sugar, phosphate and one nitrogen base. There are four nitrogen bases, adenine (A), thymine (T), guanine (G), and cytosine (C). The nitrogen bases are found in pairs, with A & T and G & C paired together. The sequence of the nitrogen bases can be arranged in an infinite ways, and their structure is known as the famous "double helix".

26 The sugar used in DNA is deoxyribose. The four nitrogen bases are the same for all organisms. The sequence and number of bases is what creates diversity. DNA does not actually make the organism, it only makes proteins. The DNA is transcribed into mrna and mrna is translated into protein, and the protein then forms the organism.

27 Recombinant DNA is the general name for taking a piece of one DNA and combining it with another strand of DNA. Thus, the name recombinant! Recombinant DNA is also sometimes referred to as "chimera."

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29 Determining the Concentration of Double-Stranded DNA Determining the Concentration of Single-Stranded DNA Molecules Molecular Weight of DNA Oligonucleotide Quantitation To determine the purity of Nucleic acid

30 Quantitation is typically performed by taking absorbance measurements at 260 nm, 280 nm, and 320 nm. Absorbance at 260 nm is used to specifically detect the nucleic acid component of a solution. Absorbance at 280 nm is used to detect the presence of protein (since tryptophan residues absorb at this wavelength). Absorbance at 320 nm is used to detect any insoluble light-scattering components. A spectrophotometer capable of providing a scan from 200 to 320 nm will yield maximum relevant information

31 Each of the four nucleotide bases has a slightly different absorption spectrum, and the spectrum of DNA is the average of them.

32 To determine the concentration of single-stranded DNA (ssdna) as a µg/ml amount, the following conversion factor is used: 1 OD of ssdna - 33µg /ml

33 The average molecular weight of a DNA base is approximately 330 Daltons (or 330 grams/mole). The average molecular weight of a DNA base pair is twice this, approximately 660 Daltons (or 660 grams/mole). These values can be used to calculate how much DNA is present in any biological source. For small singlestranded DNA molecules, such as synthetic Oligonucleotide, the molecular weight of the individual nucleotides can be added to determine the strand's total molecular weight (MW) according to the following formula: MW = (n A x 335.2) + (n c x 311.2) + (n c x 351.2) + (n T x 326.2) + P where n x is the number of nucleotides of A, C, G, or T in the Oligonucleotide and P is equal to for dephosphorylated (lacking an end phosphate group) or 40.0 for phosphorylated Oligonucleotide.

34 An amount of an Oligonucleotide express in terms of optical density (OD) units. An OD unit is the amount of Oligonucleotide dissolved in 1.0 ml giving an A260 of 1.00 in a cuvette with a 1-cm light path length. It is calculated by the equation: OD units = (A260 ) x (Oligonucleotide volume) x (dilution factor)

35 Certain of the subunits of nucleic acids (purines) have an absorbance maximum slightly below 260 nm while others (pyrimidines) have a maximum slightly above 260 nm. An A260/A280 ratio of 2.0 is characteristic of pure RNA An A260/A280 ratio of 1.8 is characteristic of pure DNA An A260/A280 of 0.6 is characteristic of pure protein A ratio of less than 1.7 means there is probably a contaminant in the solution, usually either protein or phenol. DNA Purity (A260/A280) = (A260 reading A320 reading) (A280 reading A320

36 William Kemp(1991), Organic Spectroscopy,3rd edition, MACMILAN, Canada. Kori S.S., Halkai M.A. (2000), Pharmaceutical Biotechnology, First Edition, Vallabh Prakashan, Efficient Offset Printers, Delhi. Robert M. Silverstein, Francis x. Webster, David J. Kiemle (2005), Spectrometric Identification of Organic Compound, 7th Edition, John Wiley and Sons, USA

37 Questions?

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