5. Cytogenetic methods
Cytogenetics is a section of genetics that studies the visible carriers of genetic information: mitotic, meiotic and polytene chromosomes, interphase nuclei, to a lesser degree - the mitochondria and chloroplasts. Therefore, cytogenetic methods are, first of all, the methods of chromosomes studying – their number counting, structure and behavior during cell division description, and the relationship between the change in chromosome structure to the features variability.
Human chromosomes can be studied in peripheral blood lymphocytes, bone marrow cells, tumors and others. Abnormal chromosome set or structure indicates the presence of certain diseases, such as trisomy for the twenty-first chromosome is evidence of Down syndrome.
Cytogenetic methods are based on the cytologic analysis of genetic structures, comparing genetic phenomena with the structure and behavior of chromosomes and their parts (analysis of chromosomal and genomic mutations, building of chromosome cytological maps, cytochemical study of gene activity, and so on).
For chromosomes and other carriers of genetic information structure study light and electron microscopy are used.
Molecular cytogenetic method
The method of molecular fluorescence in situ hybridization (FISH) is based on the ability of chromosomal DNA to form stable hybrid molecules with known by nucleotide composition nucleic acids fragments (probes) directly on the preparations of fixed cells, chromosomes and interphase nuclei. Result of hybridization is detected due to fluorescent labels.
This method allows proceeding from the study of chromosomes morphology to the DNA sequences analysis.
The main scope of fluorescence in situ hybridization (FISH): clinical cytogenetics, prenatal and pre-implantation diagnostics, oncology, oncogenetics, hematology.
6. Biochemical methods
Enzyme activity determination allows detecting metabolic disorders (e.g. hereditary disease phenylketonuria).
Studying of subcellular fractions chemical composition by chromatography, gel electrophoresis, qualitative reactions show proteins and amino acids composition.
Autoradiography, or tracer method, based on the radioactive isotopes introduction into the cells: tritium 3Н, carbon 14С, sulfur 35S, phosphorus 32Р, iodine 131I and others. In a certain intervals from the treated material samples are prepared. Then the surface of these samples is blanketing with thin layer of photo emulsion with silver halide. If there is radioactive isotope in a certain parts of the cell glow appears. Glow localization indicates areas of the cells with intensive biochemical reactions. For example, with tritium in the medium it is possible to visualize DNA replication.
Dideoxy method of DNA sequencing (or Sanger method) developed by Fred Sanger. This method acts by interrupting the ability of the polymerase to add new nucleotides at specific points along the DNA chain.
Chemically modified nucleotide, called a dideoxynucleotide (ddNTP), replaces one of the nucleotides in the chemical mixture. A ddNTP is missing the 3′-hydroxyl group. Because of this, the DNA polymerase is unable to synthesize a phosphodiester bond between a dideoxynucleotide and the next nucleotide in the chain. This ceases DNA replication at this point. So, this method is sometimes referred to as chain termination.
A researcher prepares four identical reaction tubes. Each tube contains a single stranded copy of the DNA for sequencing and a primer for replication reaction beginning. In each tube the researcher places normal A, G, T, and C nucleotides, and a small quantity of dideoxy A (ddATP) in the first tube, dideoxy G (ddGTP) in the second tube, dideoxy T (ddTTP) in the third tube, dideoxy G (ddCTP) in the fourth tube. Once the polymerase begins replication, it will occasionally incorporate one of the ddNTPs into the strand, thus stopping replication. In the tube containing ddATP there will be an assortment of DNA fragments, varying in length, each with a ddATP at the end of the fragment.
The four reactions are then loaded into a polyacrylamide gel and exposed to an electrical current to separate the fragments by size. Smaller fragments will migrate further in the gel than large fragments. In order to detect the results of the reaction, the dideoxynucleotides are labeled with radioactive phosphorous (32P). The gel is then placed with a piece of photographic film and developed after 24 hours of exposure.
The Results of a Dideoxy Sequencing Reaction
(Courtesy of Ricochet Productions)
To read the results, start with the band at the very bottom of the gel. This represents the smallest fragment. Since it is in the C column, the reaction must have been terminated with the incorporation of a dideoxy C nucleotide into the strand at that point. The next smallest fragment is located under the T column, indicating that the next nucleotide in the sequence is a thymine. The results are read in a ladder-like configuration up the gel. The sequence indicated is CTTCAGGTACAGTAA.
Southern blotting
Developed by Edwin Mellor Southern (1938–) in 1975, Southern blotting is a procedure that uses the processes of gel electrophoresis and DNA probes to obtain more information about a gene, namely the size of the fragment that it resides on after genomic DNA is exposed to a specific restriction enzyme.
When genomic DNA is cut using a restriction enzyme, the result is fragments of varying length. When this cut DNA is separated using gel electrophoresis, and subsequently stained using ethidium bromide, the result is not a series of distinct bands, but rather a smear (мазок) of DNA fragments. The Southern blot transfers the fragmented DNA in the agarose gel to a nitrocellulose membrane using either pressure or a vacuum so that it can be probed using a DNA probe that has been labeled with 32P, a radioactive phosphorous isotope. A DNA probe is prepared using a short stretch of nucleotides (oligonucleotides). The probe may be generated from a cloned and sequenced similar gene in another species, or from a cloned gene within the same gene family.
There are several important steps in the Southern blot procedure. Samples are placed in an agarose gel, which typically contains a high concentration of agarose, to provide adequate separation of the fragments. Once the gel electrophoresis process is complete, the gel is exposed to a sodium hydroxide solution that denatures the double helix structure of the DNA into a single-stranded molecule. Single-stranded DNA (ssDNA) is necessary to allow binding of the nucleic acid probe later in the procedure. The gel is then transferred to a piece of nitrocellulose paper using either pressure or a vacuum.
Once the transfer is complete, the membrane is placed in a special oven to permanently bind the DNA fragments to the membrane. The probe is then placed in a solution and applied to the prepared membrane.
The environmental conditions under which the probe is applied to the membrane will determine how effective the probe will be in binding to its target DNA sequences.
Low stringent (строгий) conditions, which typically consist of high salt concentrations and low temperatures, will allow the probe to bind to DNA fragments that are not quite a perfect match for the probe. This is especially useful if the researcher is looking for anything similar to the probe, or is using a nucleotide sequence in the probe which is derived from an organism that is evolutionarily distant from the species being studied.
High stringency involves high temperatures and low salt concentrations. Under these conditions the probe will only bind to those fragments on the DNA that are complementary to the nucleotide sequence of the probe.
The excess probe is then washed off and the nitrocellulose membrane is placed with a piece of photographic film. When developed, the film will provide an indication of those areas of the membrane where the probe bound to a DNA fragment.
Southern blots are also useful in determining whether a gene exists in multiple copies in the genome.
There are several variations of the Southern blot.
The Northern blot (originally called a “reverse-Southern” blot) separates RNA using gel electrophoresis and then probes the membrane with an RNA (sometimes DNA) probe. Northern blots are useful in studying patterns of gene expression, although they have been widely replaced by the use of microarrays.
The Western blot is used to study the proteins in a cell or tissue. Western blot uses antibodies to indicate the position of a protein on the membrane.
Biophysical Methods:
electron paramagnetic resonance (EPR)
nuclear magnetic resonance (NMR)
scanning calorimetry,
various types of spectroscopy (e.g. spectrophotometry, mass spectroscopy, fluorescence spectroscopy).
Biotechnological methods:
methods of cell engineering, genetic engineering