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Chapter 4

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University of Waterloo
BIOL 342
Christine Dupont

Chapter 4 – Readings CHEMICAL SYNTHESIS, AMPLIFICATION, AND SEQUENCING OF DNA We are now able to chemically synthesize a DNA molecule, amplify it with PCR and obtain the sequence of it as well. CHEMICAL SYNTHESIS OF DNA - The process is easy, inexpensive, and has contributed to molecular cloning significantly Chemically synthesized DNA is used for: - assembling whole genes - amplifying specific DNA sequences - introducing mutations into cloned genes - screening gene libraries - sequencing DNA Chemical synthesis of DNA doesn’t follow the biological direction of DNA synthesis..  each incoming nucleotide is coupled to the 5’ hydroxyl (normal replication, nucleotides added to the 3’ end) Phosphoramidite Method - this method of Chemical DNA synthesis is the procedure of choice This is a multistep process, addition of NUCLEOSIDES not nucleotides - initial nucleoside is attached to a spacer molecule by its 3’ OH - this spacer molecule is covalently attached to a support CPG controlled pore glass bead - a DMT group is attached to the 5’ of the first nucleoside to PREVENT the 5’OH from reacting NONSPECIFICALLY prior to the addition of the 2 nucleosid
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