Thursday, July 8, 2010

Understanding of DNA Molecules

The structure of the large DNA molecule, which was known to be the main material of the chromosomes, remained a mystery until Watson and Crick proposed the double helix structure in 1953.6 Chromosomal (genomic) DNA consists of two complementary polyanionic chains made up of long sequences of four different nucleic bases. Since the four bases are complementary, the double stranded DNA molecule is capable of exact self-replication from either strand. The diameter of the double helix is about 2 nm, while the length of the DNA polymer can be enormous, i.e., several centimeters in a putatively “stretched out”-state.

In a typical human cell, DNA molecules with a total length of 1 meter have to be packed into a nucleus of about 5 m in diameter. The compaction is mediated by the so-called nucleosomes, which contribute about 50% of the total mass of the chromosomes. Nucleosomes are formed by 4 to 5 different types of histones; small, basic proteins with a high proportion of positively charged amino acids (25% lysine or arginine). Histones, which bind tightly to the sugar-phosphate backbone of DNA, also have important regulatory functions. Compacted DNA is not active, meaning it can be neither replicated nor transcribed into RNA and finally into proteins. The histones control the compaction and the compaction-reversal through a regulated process that is gene or sequence specific. The exact biochemical basis of this regulation strategy is still unknown, but the essential role of histones in life is supported by the fact that their amino acid sequence is among the best conserved throughout evolution. Apparently, even minor changes in the histone structure have dire consequences for the organism in question.

Plasmid DNA is an independent type of DNA, which occurs naturally in many microorganisms in addition to the genomic DNA of the respective organism. Plasmids are comparatively small (typically 5–10 kba), circular DNA molecules that can multiply independently from the genomic (chromosomal) DNA. They occur naturally in the supercoiled (major percentage) and the open circular form. Linearized fragments of plasmid DNA can be obtained by “digestion” of the plasmid with restriction endonucleases, i.e., enzymes that cut the DNA at specific base pair sequences. For various reasons, plasmid molecules are the preferred tools for genetic engineering. Plasmids can easily be amplified in bacteria. They are separated from the larger chromosomal bacterial DNA by a denaturation/renaturation process, where the chromosomal DNA forms an insoluble precipitate, because it renatures more slowly. Purified plasmids can be transferred into eukaryotic cells either in their natural, supercoiled form or as linearized molecules.

Tags: Bio Technology, Bio Genetics, Gene Delivery