The following are the properties of DNA replication. Understanding these properties is helpful in understanding the mechanisms used for manipulating DNA. (1) Semi-conservative (discussed last lecture, demonstrated with the Meselson-Stahl experiment) (2) Usually bidirectional from a single point of origin (discussed last lecture), (3) Multiple origins are found on eukaryotic chromosomes. Some regions such as heterochromatin replicate later than others. (4) Requires 5" nucleoside triphosphates as precursors. (5) Requires a template strand to copy off of. (6) Requires a primer. This is composed of RNA and is synthesized by the Primase enzyme. (7) Only grows at the 3’ end of the chain. A hydroxyl there attacks the nucleoside triphosphate, releasing two phosphates and leaving the third as a phosphate bond. (8) Discontinuous on one strand, continuous on the other. On the discontinuoys strand, small "Okazaki" fragments are synthesized as intermediates in replication. These are joined by DNA ligase enzyme. (9) Multiple proteins are involved. These include: primase, DNA polymerases I and III, DNA ligase, topoisomerase, helicase, plus single strand binding proteins.
With this information, we can study and manipulate DNA in various ways. DNA hybridization involves pairing of single strands of DNA from different sources. This is done after denaturing the DNA with heat, and putting the single stranded DNA at an appropriate salt concentration and temperature. Restriction enzymes cut the DNA at defined base sequences. A sticky end is produced, which is useful in cloning of DNA (e.g., if a DNA is inserted into a plasmid). The Southern blot is a way to transfer DNA from a gel onto a filter. The resulting filter is probed (DNA hybridization) with labeled DNA to determine where on the gel that a particular fragment migrated. This is the basis of detecting genetic polymorphisms using the RFLP (restriction fragment length polymorphism) method.
The discovery of the PCR (polymerase chain rection) method by Kary Mullis was a major advance in molecular biology. This method allows a large amount of a specific sequence to be quickly produced in the laboratory. It involves adding primers to specific, short (ca. 20 base) sequences, DNA polymerase enzyme and precursors 9dATP, dGTP, dCTP, dTTP) to a sample containing the sequence to be amplified. The materials are then sequentially cycled through a denaturation step (95 degrees C), a renaturation step (50-60C) in which the primers hybridize to the DNA to be amplified, and an extension step (ca. 70C) in which the DNA is synthesized. These steps are repeated a number of times in that order (denaturation, renaturation, extension) to produce many copies of a specific sequence. The use of DNA polymerases from hot springs bacteria (Taq polymerase) allows the enzyme to survive the denaturation step. Otherwise new polymerase must be added after each denaturation cycle.