DNA Synthesis Guided Discussion

Introduction The goal of this discussion is for the students to lead a description of DNA synthesis. This discussion can later be extended to describe the process of sequencing by synthesis within the MiSeq DNA sequencer. Visual aids (see images included below) may be helpful in describing the process of DNA synthesis and sequencing by synthesis. (Images for the template strand and primers are currently not included in this document.)

DNA synthesis First review the DNA synthesis reaction: One strand of DNA is copied to make a second strand. The sequence of the new strand will be the “reverse complement” of the original sequence.

Ask the Students: What components are required for DNA synthesis? What does DNA polymerase need to make a new strand of DNA? Answers should include the following components.

Component Role

DNA polymerase Enzyme that synthesizes DNA

Template DNA that will be copied in the DNA synthesis reaction Short piece of single-stranded DNA that is complementary to the template Primer strand Nucleotides Building blocks of DNA Enzyme that unwinds double-stranded DNA to form single-stranded DNA Helicase, etc. (Helicase and other accessory proteins involved in DNA replication may be (optional) considered optional for the purpose of this discussion. Among the enzymes in replication, DNA polymerase is the most important player.)

Walk through the DNA synthesis: 1. Primer binds to complementary region of template DNA 2. DNA polymerase binds to primer 3. Nucleotides randomly float into the active site of the DNA polymerase 4. When complementary nucleotide moves into position, DNA polymerase attaches it to the growing strand 5. Once the correct base has been added, DNA polymerase moves to the next position Additional points:  Polymerase can’t begin synthesis until it is bound to primer  Polymerase can only add one nucleotide at a time.  Polymerase only adds the complementary base (mistakes are possible but rare)  This process happens FAST – consider how quickly your cells need to divide. (There are 3 billion bases in the human genome, so even the division of one single cell involves quite a bit of DNA synthesis!)

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DNA Polymerase doi: 10.2210/rcsb_pdb/mom_2000_3

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