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  Section: Biotechnology Methods » Molecular Biology
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Molecular Biology
  The Central Dogma
  Protein Synthesis in Cell Free Systems
  Polytene Chromosomes of Dipterans
  Salivary Gland Preparation (Squash Technique)
  Extraction of Chromatin
  Chromatin Electrophoresis
  Extraction and Electrophoresis of Histones
  Karyotype Analysis
  In Situ Hybridization
  Culturing Peripheral Blood Lymphocytes
  Microslide Preparation of Metaphases for In-Situ Hybridization
  Staining Chromosomes (G-Banding)
  Nucleic Acids
  Extraction of DNA from Bovine Spleen
  Purification of DNA
  Characterization of DNA
  DNA-Dische Diphenylamine Determination
  Melting Point Determination
  CsCl-Density Separation of DNA
  Phenol Extraction of rRNA (Rat liver)
  Spectrophotometric Analysis of rRNA
  Determination of Amount of RNA by the Orcinol Method
  Sucrose Density Fractionation
  Nucleotide Composition of RNA
  Isolation of Genomic DNA—DNA Extraction Procedure
  Isolation of Genomic DNA from Bacterial Cells
  Preparation of Genomic DNA from Bacteria
  Extraction of Genomic DNA from Plant Source
  Extraction of DNA from Goat Liver
  Isolation of Cotton Genomic DNA from Leaf Tissue
  Arabidopsis Thaliana DNA Isolation
  Plant DNA Extraction
  Phenol/Chloroform Extraction of DNA
  Ethanol Precipitation of DNA
  Isolation of Mitochondrial DNA
  Isolation of Chloroplast DNA
  DNA Extraction of Rhizobium (CsCl Method)
  Isolation of Plasmids
  RNA Isolation
  Preparation of Vanadyl-Ribonucleoside Complexes that Inhibit Ribonuclease Activity
  RNA Extraction Method for Cotton
  Isolation of RNA from Bacteroids
  Isolation of RNA from Free-Living Rhizobia
  Estimation of DNA purity and Quantification
  Fungal DNA Isolation
  Methylene Blue DNA Staining
  Blotting Techniques—Southern, Northern, Western Blotting
  Preparing the Probe
  Southern Blotting (First Method)
  Southern Blotting (Second Method)
  Western Blotting
  Western Blot Analysis of Epitoped-tagged Proteins using the Chemifluorescent Detection Method for Alkaline Phosphatase-conjugated Antibodies
  Southern Blot
  Southern Analysis of Mouse Toe/Tail DNA
  Northern Blotting
  Restriction Digestion Methods—Restriction Enzyme Digests
  Restriction Digestion of Plasmid, Cosmid, and Phage DNAs
  Manual Method of Restriction Digestion of Human DNA
  Preparation of High-Molecular-Weight Human DNA Restriction Fragments in Agarose Plugs
  Restriction Enzyme Digestion of DNA
  Electroelution of DNA Fragments from Agarose into Dialysis Tubing
  Isolation of Restriction Fragments from Agarose Gels by Collection onto DEAE Cellulose
  Ligation of Insert DNA to Vector DNA
  PCR Methods (Polymerase Chain Reaction)
  Polymerase Chain Reaction
  DNA Amplification by the PCR Method

The interphase chromosomes of eukaryotic cells are complex molecular structures composed primarily of a DNA core and a protein matrix complexed into a long thread-like structure. This basic chromosome thread is then coiled through several layers of organization and ultimately gives rise to a structure that can be visualized with a light microscope.

Chemically, the interphase nucleus is composed of a substance known as “chromatin”, which is further subdivided into euchromatin and heterochromatin. The distinction between these subdivisions is based on quantitative distribution of the basic chromosome fiber, with a higher concentration found in heterochromatin. Heterochromatin, therefore, will stain more intensely than euchromatin, since the fiber is packed tighter within a given volume.

Proteins extracted from chromatin have been classified as either basic or acidic in nature. The basic proteins are referred to as “histones” and the acidic as “nonhistone proteins”. Histones play an integral part in the structural integrity of a eucaryotic chromosome. They are organized into specific complexes, known as nucleosomes, and around which the DNA molecule is coiled. Acidic proteins within the nucleus compose many of the DNA replication and RNA transcription enzymes and regulatory molecules. They vary in size from small peptides of a few amino acids to large duplicase and replicase enzymes (respectively, DNA and RNA polymerases).

Transcription of DNA on the chromosome fiber results in the presence of a host of RNA species found within the nucleus of the cell. When the RNA is transcribed from the “nucleolar organizer” region of a genome and complexed with ribosomal proteins, granules are formed, which collectively produce a “nucleolus,” visible at the light microscope level of resolution. When transcribed from other portions of the genome, the RNA is either in the form of pretransfer RNA, or heterogeneous nuclear RNA (hnRNA). The precursor tRNA must be methylated and altered before becoming functional within the ytoplasm, and the hnRNA will also be significantly modified to form functional mRNA in the cytoplasm.

Thus, a chemical analysis of chromosomes will yield DNA, RNA, and both acidic and basic proteins. It is possible to extract these compounds from an interphase nucleus (i.e., from chromatin) or to physically isolate metaphase chromosomes and then extract the components. For the former, the nuclear envelope will be a contaminating factor, as will the nucleolus. For Isolated chromosomes, many of the regulatory molecules may be lost, since the chromosomes are essentially nonfunctional during this condensation period.


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