Semiconservative DNA replication Each strand of DNA acts as a template for synthesis of a new strand Daughter DNA contains one parental and one newly synthesized strand DNA Replication Bidirectional DNA replication in E. coli New strands of DNA are synthesized at the two replication forks where replisomes are located DNA Polymerase E. coli contains three DNA polymerases DNA polymerase I - repairs DNA and participates in DNA synthesis of one strand DNA polymerase II - role in DNA repair DNA polymerase III - the major DNA replication enzyme, responsible for chain elongation Synthesis of the new strand is always 5 3 Diagram of the replication fork Lagging-Strand Synthesis is Discontinuous Leading strand is synthesized as one continuous polynucleotide (beginning at origin and ending at the termination site) Lagging strand is synthesized discontinuously in short pieces (Okazaki fragments) Pieces of the lagging strand are then joined by a separate reaction Diagram of lagging-strand synthesis DNA pol III requires short stretch of RNA as a Primer before it can add new nucleotides DNA pol I removes these from the lagging strand DNA ligase joins the Okazaki fragments Sequencing DNA Using Dideoxynucleotides Sanger method uses 2,3-dideoxynucleoside triphosphates (ddNTPs) which are incorporated at the 3 end of a growing chain in place of a dNTP Since ddNTPs lack a 3-hydroxyl group, subsequent nucleotide addition cannot take place Small amounts of ddNTPs terminate replication of some chains at each step, leaving a set of fragments of different lengths The Polymerase Chain Reaction Amplifies Selected DNA Sequences The polymerase chain reaction (PCR) is used for amplifying a small amount of DNA Also can increase the proportion of a particular DNA sequence in a mixed DNA population PCR technique is illustrated on the next 3 slides three cycles of the PCR reaction) PCR needs Template DNA DNA to be copied Taq polymerase a heat stable DNA polymerase Nucleotides the monomers needed to make the DNA polymer RNA primer needed for polymerase to start making DNA Buffer Thermocyler alternates temperature PCR Procedure Mix all together Heat to denature DNA separate strands Cool to allow RNA primers to attach Adjust temperature to optimum for replication Repeat increase number of DNA molecules by a factor of 2 for each cycle Make copies (extend primers) Starting DNA Template 5 3 5 3 Add primers (anneal) 5 3 5 Forward primer Reverse primer DNA Amplification with the Polymerase Chain Reaction (PCR) Separate strands (denature) 5 3 In 32 cycles at 100% efficiency, 1.07 billion copies of targeted DNA region are created PCR Copies DNA Exponentially through Multiple Thermal Cycles Original DNA target region Thermal cycle