Deoxyribonucleic Acid and Related Products - Manual
Deoxyribonucleic acid (DNA) is a double stranded, helical nucleic acid molecule that consists of nucleotide monomers, each composed of a deoxyribose sugar, a phosphate group, and one of four nitrogenous bases: adenine, cytosine, guanine, and thymine.
In 1869 Friedrich Miescher discovered a mixture of compounds in the nucleus of a cell. He termed this mixture nuclein (Miescher 1871). The major component of nuclein is DNA. As the chromosome theory of inheritance was gradually accepted, scientists agreed that chromosomes consisted of some sort of polymer, but did not understand whether it was DNA, RNA, or protein. Building upon experiments performed by Frederick Griffith where he transferred a genetic trait from one bacterium to another (Griffith 1934), Avery, MacLeod, and McCarty demonstrated DNA to be the genetic material in 1944 (Avery et al. 1944).
In 1952, Hershey and Chase confirmed DNA as the genetic material with work on a T2 phage whose structure had recently been shown by electron microscopy. In 1953, James Watson and Francis Crick built models of the DNA molecule based on data gathered from x-ray diffraction experiments by Rosalind Franklin and Maurice Wilkins for which Watson, Crick, and Wilkins jointly received the Nobel Prize in 1962. Watson and Crick proposed DNA is a double helix and that the bases of each strand are on the inside of the helix, with the base of one strand pairing to the base of the other strand. The two strands being complementary allows DNA to replicate semiconservatively--one strand of the parental double helix is conserved in each daughter strand. In 1958, Meselson and Stahl proved DNA replication in bacteria is semiconservative.
In 1969, Hershey shared the Nobel Prize in Physiology or Medicine for his work on determining the properties of DNA. In the 1970s restriction enzymes were discovered. It became clear that the discovery of restriction enzymes and the new era of recombinant DNA technology was transforming biology. In 1973, Stanley Cohen, Herbert Boyer, and Paul Berg created the first genetically engineered organisms (Cohen et al. 1973, and Jackson et al. 1972). In 1978, Werner Arber, Daniel Nathans, and Hamilton Smith shared the Nobel Prize in Physiology or Medicine for the discovery of restriction enzymes.
The late 1970s and 1980s brought the principles and techniques of DNA testing. The first genetic test using DNA was restriction fragment length polymorphism (RFLP, pronounced “rif-lip”) and required large blood samples (Jeffreys et al. 1987). Polymerase Chain Reaction (PCR) was developed in the 1980s by Kary Mullis (Mullis 1990). It became the standard test for DNA testing in the 1990s, enabling researchers to quickly produce millions of copies of a specific DNA sequence, bypassing the need for bacteria and large quantities of DNA (Chakraborty et al. 1992). In 1989, Francis Collins and Lap-Chee Tsui sequenced the first human gene containing the defective gene that causes cystic fibrosis (CFTR) (Riordan et al. 1989). In 2001, the Human Genome Project and Celera Genomics released the first draft of human genome. The thirteen-year Human Genome Project was successfully completed in 2003. DNA polymorphisms began to be widely used to reconstruct human evolutionary history, and greater understanding of the DNA damage sensing, signaling, and the interplay between protein phosphorylation and ubiquitin pathway has shown important implications for aging and cancer.
DNA Structure and Function:
Each chromosome contains one long molecule of DNA, with hundreds or thousands of genes arranged along its length. The DNA molecule is a double helix, with sugar-phosphate bonds on the outside, and base pairs on the inside. The bases pair as such: adenine (A) with thymine (T), and guanine (G) with cytosine (C). DNA replicates semiconservatively and requires an RNA primer. When the parental strands separate, each serves as the template for making a new, complementary strand. E. coli DNA replication is semidiscontinuous; one strand is replicated continuously and the other is replicated discontinuously. This discontinuous replication forms Okazaki fragments. E. coli proofreading is carried out by DNA polymerase III, which will only use a base-paired nucleotide as a primer, stalling DNA replication if a misincorporation is present. During this stall, 3’ to 5’ exonuclease of the DNA polymerase III holoenzyme can remove the misincorporated base. Mammalian cells have five polymerases: alpha, beta, delta, epsilon, and gamma. alpha and delta participate in replication of both DNA strands, and beta and epsilon function in DNA repair. gamma is believed to replicate mitochondrial DNA (Weaver 2008).
Worthington offers a variety of DNA including:
- DNA from calf thymus prepared and purified by a method developed at Worthington to have lower protein and RNA contamination than most other commercial preparations. This highly polymerized DNA is an excellent substrate for deoxyribonuclease. A sodium salt, it must be converted to magnesium form to be susceptible to DNase.
- DNA, cellulose
- DNA, salmon testes
- DNA, E. coli
- DNA, lambda, E. coli W8850 (lambda C1857-57)
- DNA, lambda, BstEII fragments
- DNA, lambda, EcoRI fragments
- DNA, lambda, HindIII fragments
Technical Note: One base pair is approximately 680 Daltons. One A260 unit is equivalent to 50 µg DNA products.