biophsical technique

BIOPHYSICAL TECHNIQUES


Scientist using a stereo microscope outfitted with a digital imaging pick-up
•CIRCULAR DICHROISM, a method for detecting chiral groups in molecules, especially to determine the secondary structure of proteins
•DUAL POLARISATION INTERFEROMETRY, an analytical technique used to measure the real-time conformation and activity of a wide range of biomolecules and their interactions.
•ELECTRON MICROSCOPY, to gain high-resolution images of subcellular structures
•FLUORESCENCE SPECTROSCOPY, which can be used to detect structural rearrangements, as well as interactions of biomolecules
•FORCE SPECTROSCOPY probes the mechanical properties of individual molecules or macromolecular assemblies using small flexible cantilevers, focused laser light, or magnetic fields.
•GEL ELECTROPHORESIS, which is used to determine the mass, the charge and the interactions of biological molecules
•ISOTHERMAL TITRATION CALORIMETRY or ITC which measure the heat effects caused by interactions
•MASS SPECTROMETRY is a technique that gives the molecular mass with great accuracy.
•MICROSCOPY, for example using laser instruments for scanning and transmission.
•OPTICAL TWEEZERS AND MAGNETIC TWEEZERS allow for the manipulation of single molecules, providing information about DNA and its interaction with proteins and molecular motors, such as Helicase and RNA polymerase.
•NMR SPECTROSCOPY, giving information about the exact structure of biological molecules, as well as on dynamics
•SINGLE MOLECULE SPECTROSCOPY is a general term applied to a class of techniques that are sensitive enough to detect single molecules and often incorporates fluorescence detection.
•SMALL ANGLE X-RAY SCATTERING (SAXS) is a technique that gives a rough low resolution molecular structure.
•SPECTROPHOTOMETRY, the measurement of the transmission of light through different solutions or substances at different wavelengths of light. Colorimetry is an example of this.
•ULTRACENTRIFUGATION, which gives information on the shape and mass of molecules various chromatography technique, which are used for the purification and analysis of biological molecules
•X-RAY CRYSTALLOGRAPHY, another method to gain access to the exact structure of molecules with atomic resolution.





Stringency control

•Stringency can be regarded as the specificity with which a particular target sequence is detected by hybridization to a probe.
•Thus, at high stringency, only completely complementary sequences will be bound, whereas low-stringency conditions will allow hybridization to partially matched sequences.
•Stringency is most commonly controlled by the temperature and salt concentration .
•stringent conditions -(lower salt or higher temperature) until the desired result is obtained.

•The melting temperature (Tm) of a probe–target hybrid can be calculated to provide a starting-point for the determination of correct stringency.

• The Tm is the temperature at which the probe and target are 50% dissociated. For probes longer than 100 base pairs:

Tm = 81.5°C + 16.6 log M + 0.41 (% G + C)

where M = ionic strength of buffer in moles/litre.
•With long probes, the hybridization is usually carried out at Tm − 25°C.
•When the probe is used to detect partially matched sequences, the hybridization temperature is reduced by 1°C for every 1% sequence divergence between probe and target.

•Oligonucleotides can give a more rapid hybridization rate than long probes as they can be used at a higher molarity.

•Also, in situations where target is in excess to the probe, for example dot blots, the hybridization rate is diffusion-limited and longer probes diffuse more slowly than oligonucleotides.

•It is standard practice to use oligonucleotides to analyse putative mutants following a site-directed mutagenesis experiment where the difference between parental and mutant progeny is often only a single base-pair change

•The availability of the exact sequence of oligonucleotides allows conditions for hybridization and stringency washing to be tightly controlled so that the probe will only remain hybridized when it is 100% homologous to the target.
•Stringency is commonly controlled by adjusting the temperature of the wash buffer. The ‘Wallace rule’ is used to determine the appropriate stringency wash temperature:

Tm = 4 × (number of GC base pairs) + 2 × (number of AT base pairs)

.In filter hybridizations with oligonucleotide probes, the hybridization step is usually performed at 5°C below Tm for perfectly matched sequences. For every mismatched base pair, a further 5°C reduction is necessary to maintain hybrid stability.
The design of oligonucleotides for hybridization experiments is critical to maximize hybridization specificity. Consideration should be given to:
•probe length – the longer the oligonucleotide, the less chance there is of it binding to sequences other than the desired target sequence under conditions of high stringency;
•oligonucleotide composition – the GC content will influence the stability of the resultant hybrid and hence the determination of the appropriate stringency washing conditions. Also the presence of any non-complementary bases will have an effect on the hybridization conditions.

How many clones are required?
Let n be the size of the genome relative to a single cloned fragment. Thus, for the human genome (2.8 × 106 kb) and an average cloned frag- ment size of 20kb, n = 1.4 × 105. The number of independent recombinants required in the library must be greater than n, because sampling variation will lead to the inclusion of some sequences several times and the exclusion of other sequences in a library of just n recombinants. Clarke and Carbon (1976) have derived a formula that relates the probability (P) of including any DNA sequence in a random library of N independent recombinants:
Therefore, to achieve a 95% probability (P = 0.95) of including any particular sequence in a random human genomic DNA library of 20kb fragment size:
Notice that a considerably higher number of recombinants is required to achieve a 99% probability, for here------